Thermostability is the capacity of an organism to survive and thrive at high temperatures and is a characteristic with many biotechnological applications, such as food processing. We are seeking to understand the mechanisms of thermostability in viruses by using an RNA virus, phi-6 Cystovirus. We are evolving the phi-6 virus to become thermostable by exposing it to a target temperature of 61°C over many generations (wild type viruses grow at 25°C). In order to see if exposure to different intermediate temperatures affects the genetic pathway to thermostability, we will expose the phi-6 virus to different temperature regimes prior to reaching the target temperature. The temperature treatments are sudden, where the virus is exposed to the target temperature throughout the entire course of the experiment, moderate, where the virus is exposed to the target temperature halfway through the experiment, and gradual, where the virus is exposed to the target temperature on the final day of the experiment. The genome of phi-6 has already been characterized, which enables us to compare the genome of the evolved, thermostable phi-6 viruses to the ancestor and allows us to study the genetic basis of thermostability. In future studies, we hope to use these thermostable viruses to learn about costs and benefits of maintaining thermostability.

While the current, classical, and experimental treatment for ovarian cancer focuses on destroying malignant cells, these cells are actually moving targets for therapies. The tumor “stroma” contains dominantly tumor-associated macrophages (TAMs). TAMs produce factors that support tumor growth and block immune responses against the tumor. Our strategy to eliminate TAMs is based on a new adenovirus vector that expresses suicide gene specifically in TAMs. Our adenovirus vector will transduce TAMs in cell culture and mouse tumor models. We will use a microRNA based system to avoid suicide gene expression in myeloid cells other than TAMs (such as white blood cells). To generate that system, we used mouse tumor models and isolated macrophages from blood and tumors and delineated their micoRNA expression profile using microarrays with >1,000 known microRNAs. We selected the microRNA with the highest expression level white blood cells and a low level in TAMs (mmu-miR-106a). As a negative control, we chose a microRNA that was high in TAMs (mmu-miR-210). My task was to insert 4 target sites with 100% homology to the selected microRNAs into the 3’UTR of a GFP gene, which was under the control of an EF1a promoter. The GFP expression cassette, together with a cassette containing the mCherry gene under the control of the PGK promoter, were inserted into adenovirus (Ad5/11) vectors. We demonstrated in vitro and in vivo, after intravenous injection into mice, that transduction efficiency of white blood cells and TAMs was comparable for the Ad5/11-miR106 and control vector (based on mCherry expression). However GFP expression from Ad5/11-miR106 was absent in white blood cells due to miRNA-mediated degradation of GFP mRNA. My future plans are to substitute the GFP gene with a suicide gene and to demonstrate that corresponding Ad5/11 vectors selectively kill TAMs in vivo, which in turn should slow down tumor progression.

 Blindness is a condition that impacts people across the globe. Some of these people are born blind due to a genetic defect, while others become blind through an accident or as the result of disease. Thus dissecting the underlying molecular causes of blindness is a critical component in improving the lives of many people. To study causes of blindness, we are analyzing disease causing mutations in the model organism Danio Rerio, or zebrafish. The recessive ZVM10 mutation causes very early degeneration of both rod and cone retinal photoreceptors, causing complete blindness. To study this mutation we have determined the identity of the mutated gene. I helped in this endeavor by first determining the position of the mutation within the zebrafish genome (i.e. map the mutation). This was done by mating the ZVM10 AB fish with a polymorphic fish strain. The crossovers between the different DNAs were traced, and used to identify the area homozygous for the AB allele. By continually reducing this region we specified a small group of genes that potentially were mutated. I then helped sequence these genes. A single base pair mutation that introduces a premature stop codon was identified in the gene encoding N-ethylmaleimide-sensitive attachment protein, beta (NAPB). I am currently working on a reliable genotyping protocol based on this mutation to sort mutated from non-mutated fish. The mechanism of photoreceptor degeneration in this mutant will further be studied by examining the role of this gene in wild type cells. The identification of this mutation will permit the identification of people who are blind due to this genetic mutation, through genetic screening of the patient. Finally, if this gene is also involved in human blindness, it will become a candidate for rescuing blindness through gene therapy.

Leishmania, a kinetoplastid parasite, causes a wide range of diseases with asymptomatic to lethal pathologies accompanied by widespread human and animal disease, death, and economic loss. Current drugs targeting Leishmania are inadequate. Leishmania exhibits an unusual DNA modification – a glucose is added to certain thymine nucleotides – mainly in the telomeric repeat regions of the genomic DNA, but also in strand switch regions (SSRs) between polycistronic transcription units (PTUs), where transcription initiation and termination occur. This DNA modification converts about 1% of thymine (T) in the nuclear DNA to β-glucosylhydroxymethyluracil (base J). Previous studies show that J plays an important role in transcription termination, and removing J entirely causes Leishmania to die. Using episomal plasmids, it has also been shown that the primary DNA sequence is sufficient to direct J insertion and that an enzyme called JBP2 is required. My project focuses on determining the mechanism of J insertion in Leishmania by using Single Molecule Real Time (SMRT) sequencing to identify the positions of J in SSR and telomeric repeat sequences inserted into plasmids grown episomally in Leishmania, and to search for a DNA sequence motif common to J sites that may be the insertion signal for recruiting the enzymes that modify T to J. We found that the J insertion motif in both SSR and telomeric repeat sequences appears to involve a 14 basepair spacing of Thymine on opposing strands, with GC-rich sequence in between. Additional experiments will further define the J insertion motif using additional SSR sequences and mutated telomeric repeats sequences, and will address whether the insertion sequence is specific to JBP2. Furthering our understanding of the role of J in Leishmania transcription may provide new targets for disease therapies and also help explain the origins of transcription regulation in other eukaryotes.

Ubiquitin (Ub) is a small post-translational signaling protein with roles in DNA double stranded break (DSB) repair, apoptosis, and protein degradation.  The pathway for attaching Ub to a substrate involves sequential interactions of three enzymes: Ub activating enzymes (E1), Ub conjugating enzymes (E2), and Ub ligases (E3).  Substrate proteins can be poly-ubiquitinated and the nature of the poly-Ub chain determines the fate of the substrate.  One type of poly-Ub chains signal for a DSB repairing complex, as opposed to another type of poly-Ub chains that signal for proteasomal degradation.  RNF168 is an E3 involved in the DSB repair pathway. Whereas the E3 ligase binds its substrates, in ubiquitination mechanism it is the E2 that determines the type of Ub moieties formed. There are dozens of E2s found in the human cell; therefore, it is important to study and understand the E2:E3 pairwise interactions. Currently, the published investigations of RNF168 provide varying conclusions about RNF168’s relationship to specific E2s and with that its role in DSB repair.  In order to remove the ambiguity, I examined in vitro protein interactions between RNF168 and a library of human E2s using yeast-two hybrid experiments. The Klevit Lab has investigated the process of poly-ubiquitination with other E3s, BRCA1 and RNF8, and found involvement of different E2s at different stages of ubiquitination.  To further clarify the poly-ubiquitination mechanism of the RNF168:E2 pairs, I conducted similar activity assays to those done for BRCA1 and RNF8.  These in vitro assays consist of autoubiquitination reactions, where RNF168 itself is used as a proxy-substrate to determine different types of Ub product formation utilized with different E2s.  By yeast-two hybrid I have found that several E2s bound to RNF168. These interactions will be further confirmed and further tested with activity assays to determine the type of Ub modification by the different RNF168:E2 pairs.

Proteins perform a vast array of functions within all living organisms. These functions are heavily dependent on the protein’s 3-D structure. The loss of protein structure can lead to a wide variety of problems, one being the formation of insoluble protein aggregates, which can result from cellular stress. Aggregates can inhibit proper protein function, disrupt cellular homeostasis, and are implicated in many diseases. In order to combat aggregate formation, a family of proteins exists that interact with misfolded and aggregate prone proteins (clients). This family, known as the small heat shock proteins (sHSPs), delays the formation of insoluble aggregates in the cell. Their expression and activity as molecular chaperones have been seen to increase under stress conditions. However, how sHSPs delay aggregation is not well understood. We seek to better understand binding between sHSPs and their clients. Interactions between the sHSP αB crystallin and the model client Δ131Δ (a mutant of staphylococcal nuclease) have been characterized by Nuclear Magnetic Resonance (NMR). We hope to test the observations made from this simplified system in more functionally applicable assays. sHSPs not only interact with clients, but also amongst themselves. There are ten known human sHSPs, some of which have been shown to interact with each other. While this interaction is not well understood, we do know that sHSP monomers associate non-covalently to form dimers, which in turn form higher ordered oligomers. To begin understanding these interactions, we seek to investigate whether sHSPs can exchange their monomer subunits to form heterodimers. The ability to heterodimerize would suggest an even greater diversity of oligomer structure and function. We are interested in characterizing the properties of this proposed heterodimerization. Through site directed mutagenesis, experimental based assays, and gel electrophoresis, we seek to gain a detailed description of these proteins that play such a critical role in cellular health.

 A significant proportion of the 23 known human-specific duplicated genes are implicated in neurological processes. The Rho GTPase activating protein 11 (ARHGAP11) is one such gene that is also found in an area associated with schizophrenia, intellectual disability, and autism. We determined that the human-specific duplicated copy (ARHGAP11B) duplicated 5.3 million years ago (mya) shortly after the human-chimpanzee divergence (~ 6.5 mya). The human-specific duplicated copy is not found in all humans, and represents an incomplete version of ARHGAP11A, suggesting a potentially alternative function. The goal of my project was to identify sequence differences between A (ancestral) and B (duplicated) paralogs (i.e., genes related by duplication within the same species) and use these differences to assess gene variation and expression. By sequencing 64 different transcripts (i.e. protein coding regions) we found nine “paralog-specific variants” (or PSVs) allowing us to distinguish between A and B. We assessed the level of gene expression (how much a gene is “used”) for each paralog in human fetal brain samples and found that A was expressed approximately two times more than B. Interestingly, we found similar results from other tissue types including blood. Further, we have used these PSVs to identify potentially pathogenic mutations of ARHGAP11A from targeted large-scale sequencing data of a collection of children with intellectual disability. We are using these data to understand the function of different members of the ARHGAP11 gene family both on neurological development and potentially on the evolution of traits that make us uniquely human.

DNA replication is vital for cell division as it produces exact copies of all chromosomes for the two daughter cells.  During S-phase of the cell cycle, DNA replication begins at specific, defined locations within a chromosome known as origins of replication.  Interestingly, some origins activate in early S-phase while others activate late.  The biological significance of this temporal control of replication is largely unknown.  In multiple species, centromeric DNA, the site of recruitment for proteins required for separation of chromosomes during mitosis, has been found to be early replicating.  The Brewer/Raghuraman lab has recently shown that centromeres in the budding yeast Saccharomyces cerevisiae promote their own early replication by advancing the activation time of their neighboring origins.  The conservation of early centromere replication coupled with the Brewer/Raghuraman lab’s recent finding suggests that early centromere replication may be important for chromosome maintenance.  We set out to determine the consequences of a late replicating centromere by artificially delaying its replication time in S. cerevisae, through replacing its nearby origins with drug resistant markers.  To enhance sensitivity of the experiment, I impaired the activity of the spindle checkpoint that monitors correct chromosome segregation.  I also constructed a control chromosome with an early replicating centromere and the same markers.  I will then compare the degree of chromosomal instability, as measured by survival on drug plates, of the control cells to those that contain a late replicating centromere.  We expect the experimental group to show an increase in the number of cells that have lost the chromosome compared to the control cells.  Even if the effect is small we will perform long-term growth experiments to analyze how the cells cope with reduced genetic stability.  The results of this study will provide a better understanding of the biological importance of the temporal replication program.

Cleft lip with or without cleft palate is among the most common congenital birth defects, but its phenotypic presentation is highly variable. This variability not only impacts the clinical management of patients, but also poses many challenges for recurrence risk counseling. Population studies indicate a diversity of causative factors, including genetic and non-genetic factors. Although some genetic causes have been identified, further progress in this area will require more precise classification of subphenotypes. Numerous cleft lip/palate classification systems have been developed over the years, but there is a lack of consistency among these models. These discrepancies lead to problems regarding precision and interpretation when documenting clefts for research or treatment purposes. The Ontology of Craniofacial Development and Malformation (OCDM) project, part of the National Institute of Dental and Craniofacial Research’s FaceBase Program, aims to develop a framework of anatomical terminology to facilitate integration of diverse sources of research and clinical data, including genetic, embryological, and phenotypic information. The overarching goal is to maximize sharing of research data and promote collaboration to drive research into the causes and treatment of craniofacial disorders. To this end, we have comprehensively reviewed existing classification systems with respect to the methods of categorization of cleft types, including placement, severity, and other distinguishing features. Along with wide variation in terminology, there was also wide variation in the extent of content among systems. Each system categorized clefting differently, but none encompassed all other systems’ classifications, nor all cleft types observed in the clinic. It is predicted that integrating these systems through a common anatomical ontology will serve to facilitate standardization of cleft classifications and terminology. In addition, this foundation should enable further subclassification to ultimately benefit studies exploring relationships between causes and manifestations of orofacial clefting, as well as underpin advances in interventional therapies and surgical treatments.

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to lead to improved drugs. Most eukaryotic organisms synthesize sterols for essential biological functions that when manufactured incorrectly can lead to cell death. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediates of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T. cruzi in vitro and are now the center of research regarding anti T. cruzi drug discovery. We are developing novel azole compounds that target the sterol 14-demethylase homolog of T. cruzi to deprive the cell of its necessary sterols. However, further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of this research project is to analyze the ERG3 homologs of T. cruzi. The putative homologs have been cloned and sequenced. No significant mutations were found. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

Prostate cancer is the most common type of male malignancy in the Western world. Patient death often occurs when treatments which decrease circulating levels of male reproductive hormones are no longer effective. This lethal stage is termed castration-resistant prostate cancer (CRPC). Under normal conditions, androgens function through androgen receptors (ARs) to cause AR activation. The recently identified androgen receptor splice variants (AR-Vs), which lack part or all exons coding for the AR-ligand binding domain, are believed to cause the continuous AR activation found in androgen-independent conditions such as CRPC. Ubiquitin-conjugating enzyme E2 C (UBE2C) is an oncogene expressed in many types of solid tumors and has a function of marking proteins for degradation. Uncontrolled cell growth can be caused by overexpression of UBE2C which targets proteins that serve as cell-cycle check points to be degraded. Overexpression of UBE2C is reported to be positively correlated with AR-V expression. The transcription activation of UBE2C requires chromatin-looping through a phosphorylated transcription factor, mediator of RNA polymerase II transcription subunit 1 (p-MED1). My project investigates the relationship between AR-V and p-MED1 in mediating UBE2C gene expression. We used LnCaP and M12 human prostate cancer cell lines and transfected them with AR-Vs. Expression assays were carried out to measure the UBE2C gene expression levels and the presence of p-MED1 in response to AR-V expressions. We also performed functional assays such as co-immunoprecipitation to determine the functional interplay between AR-V and p-MED1 in mediating UBE2C transcription. We hypothesized that p-MED1 is required in AR-V-directed UBE2C expression. Our data suggest that, through a distinct transcriptome, the presence of p-MED1 as a mediator is required in AR-V-directed UBE2C expression and cancer cell proliferation. The importance of AR-V, p-MED1 and their mechanisms underlying CRPC progression might provide a new therapeutic approach for targeting AR-independent CRPC.

Abnormal perinatal environment, commonly manifested as low birth weight, has been associated with birth defects as well as chronic disease. In particular, studies have shown low birth weight to have a deleterious association with type II diabetes, hyperlipidemia, hypertension, cardiovascular disease, renal disease, and renal failure. Of specific interest are the mechanistic details concerning low birth weight subjects’ altered excretion. Maternal low protein diet (LPD) during gestation and lactation results in offspring with significantly lower birth weight. Using the maternal LPD rat model, we determined renal excretion of select endogenous compounds by collecting blood and urine at various times of age. Clearance of creatinine and uric acid are indicators of glomerular filtration rate and reabsorption, respectively. High Performance Liquid Chromatography assays were used to simultaneously quantify creatinine and uric acid in the urine and in the blood of the rat. It was found that creatinine clearance and uric acid clearance decreased in LPD rats in an age-dependent and sex-specific fashion. Specifically, there were observed significant decreases in both creatinine clearance and uric acid clearance in LPD male offspring at both age’s day 90 and day 120 old. No differences were observed in both creatinine clearance and uric acid clearance in LPD female offspring until age day 120 old indicating an age-dependent programming of females as opposed to males. The results strongly correlate with decreased and altered excretion observed in low birth weight subjects and suggest that perinatal LPD has a long-term programming of renal excretion. These findings could partially explicate the underlying mechanisms that lead to higher incidence of renal disease/dysfunction in perinatally growth restricted subjects. Furthermore, altered renal function could affect the renal handling of therapeutic agents and thus affecting the overall treatment outcomes in low birth weight subjects.

Pathologic enlargement of the esophagus, termed megaesophagus, results in a failure to complete peristalsis leading to vomiting, severe weight loss and potential death. Megaesophagus is characterized in a number of disease states including parasitic (Chaga’s disease), autoimmune (myasthenia gravis), and neuromuscular (muscular dystrophies), however, a detailed cellular and mechanistic understanding is lacking. To address this deficit, we performed a comprehensive examination of the onset, pathology and cellular composition of megaesophagus in the mouse model of Duchenne muscular dystrophy (DMD). DMD affects ~1/3500 male births and is a catastrophic and ultimately fatal muscle wasting disease resulting from a mutation in the gene encoding the integral skeletal muscle protein dystrophin. Although poorly defined in human DMD patients, megaesophagus is documented in canine and mouse models of DMD. Advancements in DMD therapies has increased patient lifespan and underscored a need to assess the impact of the disease in a range of organ systems, including the esophagus. We find that older dystrophic mice can develop severe megaesophagus, but that the timing of onset depends on the nature of dystrophin expression in muscle and non-muscle tissues and the genetic background of the mice. Further studies are in progress to delineate the precise dystrophin expression pattern in different cell types of the esophagus that contribute to pathology. Work presented here will be essential for future work aimed at identifying new therapeutic targets for DMD.

Since neuronal morphology is linked to neuronal function, the nervous system goes to great lengths to ensure that neurons achieve their proper shape, and failure to do so is associated with many cognitive disorders. However, the precise regulatory mechanisms underlying the coordinated expression of certain genes in a given cell type, at the right amount, and at the exact developmental time is one of the longstanding questions in developmental biology. MicroRNA biogenesis is a common mechanism by which cells regulate gene expression, often by “tuning” levels of target gene products, allowing for fine regulation of protein levels over a broad dynamic range. From a genetic screen for mutations in fruit flies that affect dendrite morphology in sensory neurons, we identified a novel Dicer-1 allele that causes stereotyped dendrite defects, including deregulated growth and defects in dendrite self-avoidance. Dicer-1 encodes a type III RNA endonuclease that is required for processing many types of small regulatory RNAs, including microRNAs. Time-lapse imaging and quantitative analysis of the dendrite patterning in Dicer-1 mutant sensory neurons reveals that there is a significant increase in the number and overall length of dendrite branches as well as an increase in dendrite-dendrite crossing-over, especially at late stages of larval development. Interestingly, mutation in a gene (pasha) that functions upstream of Dicer-1 to process a subset of miRNAs recapitulates the exuberant branching, but not the dendrite crossing phenotype of Dicer-1 mutants. We hypothesize that different classes of miRNAs regulate dendrite branching and dendrite self-avoidance and we plan to use microarray analysis of Dicer-1 and pasha mutants to identify the pathways regulated by these different classes of miRNAs.

Brain development is highly regulated by transcription factors that control often-variable levels of gene expression. Two transcription factors, Tbr1 and Tbr2, are expressed by neurons in the embryonic vertebrate brain during different stages. Abnormalities in Tbr1 and Tbr2 expression have been linked to autism, a developmental disorder characterized by impaired communication abilities with an incidence rate of 1.1% in the United States. Therefore, these transcription factors are of significant research interest. Tbr1 indicates fully differentiated postmitotic projection neurons that form a matrix through which newer neurons migrate. Tbr2 is a marker of intermediate progenitors, or unipotent neural cells, committed to a glutamatergic fate. In cortical development, neurons migrate from the brain interior toward the cortical plate and express Tbr1 and Tbr2 in well-documented time-dependent layers. However, less is known about Tbr1 and Tbr2 in the hypothalamus, also part of the forebrain. The adult hypothalamus performs diverse roles such as endocrine hormone secretion, hunger regulation, and circadian cycles. Because peak hypothalamic neurogenesis occurs during embryonic days (E) 12-14, I have examined mouse tissues from that period with techniques including immunohistochemical (IHC) fluorescent staining and Nissl staining in order to better identify the types of cells that express these transcription factors. IHC staining uses fluorescent antibodies to tag Tbr1 and Tbr2 as antigens, thus rendering cells that express the transcription factors highly visible under certain wavelengths of light. I also hope to gain information about the adult cells these progenitors eventually develop into. My results may enable the pursuit of research avenues such as migratory behavior of hypothalamic neurons, and a greater understanding of Tbr1 and Tbr2 may contribute to our current knowledge of autism associations.

Tribal communities often lack access to high-quality healthcare, prevention programs, cancer education, cancer screening tests, and cancer clinical trials. As a result, American Indians and Alaska Natives experience the worst cancer-related disparities and the poorest survival rates of all racial and ethnic groups in the U.S. Comic books have long been used as an educational tool to improve public health. In 2008, the Native Comic Book Project was launched as a youth-focused community education project of Native People for Cancer Control, a Community Network Program Center funded by the National Cancer Institute. The purpose of this ongoing project is to use comic book creation as a way to educate Native youth about cancer, especially methods of cancer prevention. Along with basic art skills, participants learn about traditional foods and wellness, non-ceremonial tobacco use, human papillomavirus, and obesity prevention. Modeled after Dr. Michael Bitz’s Comic Book Project, the Native Comic Book Project has been adapted for both urban and reservation-based youth by incorporating Native storytelling and traditional values. Its ultimate goal is to promote healthy decision-making for Native youth and their communities. We are currently conducting a formal evaluation of this project by using pre- and post-intervention assessments to measure knowledge, habits, and decision-making among youth participants regarding tobacco use, healthy eating, exercise, and human papillomavirus. The Native Comic Book Project has been implemented at 10 sites and has enrolled 55 participants. Data will be fully analyzed in Summer 2013.  Future planned activities for research and education include developing more youth-oriented health interventions.

New DNA sequencing technologies have increased the throughput of sequencing by several orders of magnitude, reaching upwards of 50 GB of data per day on a single instrument. However, advancing our understanding of how genetic variation impacts human health and disease depends on studies that survey very large numbers of individuals, and as such may only be economical by analyzing specific subsets of the genome. This is complicated by the fact that traditional methods of preparing DNA samples for sequencing do not allow selection for such candidate genes. One solution is the use of “capture by circularization” by molecular inversion probes (MIPs). MIPs allow dozens of genes to be targeted and amplified simultaneously; however, individual MIPs suffer from notoriously variable performance relative to one another, which results in uneven coverage of target regions. Prior work highlighted the potential for statistical analysis to improve MIP design and selection, inviting further testing. After performing a large scale sequencing run using an unbiased set of 12,000 MIPs, logistic regression was used to build a model capable of scoring MIPs prior to synthesis. New design software incorporating this model into the selection process was developed and tested by designing MIPs to over 60 new genes. Analysis of the resulting sequence data demonstrated the successful in silico identification of challenging genomic regions and a reduction in the number of gaps in coverage, which should support the use of MIPs in future large scale studies.

Cells process an enormous amount of information from their environment and translate this information into phenotypic behaviour that will dictate critical biologic activities. To do this, cells must collect, store, and respond to information both inside and outside the cell. This information is stored in the chemical composition of its cytoplasm, and these conditions will regulate complex biochemical pathways that relay the information from the cell’s exterior to the effector proteins that modulate behaviour. These pathways, known as signal transduction pathways, are largely regulated by phosphorylation. Protein kinases are the class of enzymes responsible catalyzing the phosphorylation of intermediary signaling enzymes (resulting in their activation), and thus, stimulate the transduction of an external stimulus to intracellular machinery. As cellular conditions change, protein kinases sensitive to these conditions will modulate their activity through a change in shape (conformation). Research shows that disregulating signaling pathways through aberrant kinase activity can lead to grievous disease, making them good drug targets for cancer treatments and other conditions. Selective pressure has lead to the 500+ kinases in the proteome to adopt well-conserved active and inactive conformations, making it difficult to understand how nature can distinguish between them. Due to low abundance of kinases, quantitative analysis of kinase conformations is difficult with existing methodology. To this end, we have developed a catch-and-release system that allows us to enrich and quantitatively distinguish between kinases of specific conformations. This project will focus on the development trifunctional probe that serves as a chemical handle to enrich kinases in the novel system. The new catch-and-release system employs improvements over existing biotin-steptavidin systems by taking advantage of bio-orthogonal chemistry and specific elution conditions that increase signal-to-noise ratios. Our probe marks significant progress towards a panel of probes that will profile kinases for diagnostic means and drug target identification.

Tubes are the foundation for many organs in all animals. Although tube formation is critical for development, relatively little is known about how cells coordinate their behaviors to form tubes. To investigate this process, we are studying the development of the dorsal appendages (DAs), two respiratory structures that are formed in Drosophila ovaries and protrude from the fly egg. We use a genetic approach to disrupt gene function, then study cell behaviors during tube formation. Previously, our lab showed that trimeric G proteins play a role in DA formation. Trimeric G proteins act as signal transducers and Gα, one subunit of the trimeric G-protein complex, is responsible for signal specificity. Although in situ hybridization showed that six of seven Drosophila Gαs are expressed in the ovary, functional studies demonstrated that Gα12 was most important. RNAi against Gα12 in a subset of DA-forming cells resulted in 90% of egg chambers exhibiting abnormal DAs. We hypothesize that concertina and its partial duplicate CG40005, the genes coding for Gα12, work together in DA-forming cells to coordinate cell behaviors. To test this hypothesis, we generated null mutations in cta and CG40005. We induced random deletions in the DNA via irradiation and screened for loss of a visible marker carried on a transposon near cta. Through complementation tests, we showed that 22 deletions extended into cta, or the neighboring essential gene, light. Although the genomic region contains many repeated sequences, I developed PCR tests and found 18 deletions disrupting cta, CG40005, and light, and one deleting only CG40005. Finally, rescue studies using large BAC clones generated by the P[acman] project will let us examine cell behaviors during DA formation in the absence of cta and CG40005. Since DA formation resembles neural-tube formation in vertebrates, our studies will give insight into tube-forming processes in other animals.

The nuclear transport pathway performs the fundamental function of moving cargo between the cytoplasm and nucleus in all eukaryotes. Nuclear transport is carried out through a highly conserved mechanism across all eukaryotes. Yet, in Drosophila, several components of the nuclear transport apparatus, including importins, evolve rapidly under positive selection. Genetic conflict with selfish elements has been suggested as a possible cause for this pattern of rapid evolution. Here, we present a comprehensive phylogenomic analysis of importin gene evolution in Drosophila. Importins are adapter molecules that directly mediate the transport of cargo into the nucleus. Our analysis reveals a recurrent pattern of gain and loss of importin paralogs across independent lineages of Drosophila. Interestingly, we discovered that almost all new copies of importins have acquired a testes-specific expression pattern since their birth through gene duplication. This pattern of repeated gains of testes-specific copies of importins suggests a function in suppressing genetic conflicts such as segregation distortion in the male germline. Segregation distorters such as SD in Drosophila melanogaster act by impairing nuclear transport in the testes. We are currently performing functional tests for the hypothesis that an increased dosage of these testes-specific importins may serve a role in suppressing segregation distortion in the male germline by restoring nuclear transport during spermatogenesis.

Organisms have evolved specific mechanisms to respond to molecular and environmental stress. These stress-response pathways are evolutionarily conserved and can modify lifespan in model organisms, making them popular targets of aging research. Using the nematode Caenorhabditis elegans (C. elegans), we have found the loss of important genes in stress-response pathways can cause a phenomenon called age-related vulval integrity defects (Avid). Worms with Avid show a small protrusion near their vulva that can eventually lead to expulsion of the intestine and premature death. While many labs have noted this phenotype, it is unknown if Avid is a natural part of C. elegans aging or if genetic and environmental factors induce this phenotype. Because of this, there is confusion as to whether worms with Avid should be censored in aging experiments. The goal of our project is to determine the mechanism of Avid and whether it is part of normal worm aging. Our initial work has identified several conditions and genetic pathways important for Avid, including temperature, food availability, the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. We have also identified that the reproductive period of the C. elegans lifecycle is influential on Avid frequency, despite the phenotype occurring post-reproductively. We are currently testing the hypothesis that Avid is caused by secretions from the bacteria worms eat that influence nematode physiology and lead to Avid. We hypothesize that the pathways that influence Avid frequency do so by altering the digestion of bacteria in the intestine or by modifying the worm’s immune response. To test our hypothesis we measured the quantity of bacteria ingested and digested by various worm strains at different temperatures and tested whether bacterial metabolites can cause Avid. We intend to combine our results and develop a more complete model of Avid.

In an effort to study evolution and mechanisms of adaptation, seven populations of S. cerevisiae were cultivated in chemostat under nutrient limitation. Whole genome sequencing and Single Nucleotide Polymorphisms (SNPs) analysis were performed on clones isolated at 200 generations. I sought to test the effect of each mutation on the cellular fitness thus to characterize beneficial and passengers mutations. By combining PCR and Sanger sequencing, I confirmed five mutations (4 SNPs, one indel) detected by whole genome sequencing on one clone. Using backcross-mating techniques with the ancestral strains, I was able to isolate each mutation and test their individual fitness. I found that 4 of the mutations are passenger mutations that do not impact the fitness of the cells. One of the mutations, a non-synonymous change in SGF73 leading to the truncation of the protein, increased the competitive fitness by 24%. Mutations leading to the truncation of SGF73 were also found in three other independent clones. The presence of similar mutations in four independent populations suggests that the loss of the protein is an important process of adaptation to sulfate limitation. I am now studying the dynamic and the spread of this mutation in the population over 200 generations. SGF73 is the human ortholog of ataxin-7 of the SAGA complex associated with spinocerebellar ataxia diseases and our findings may be important to study the role of this protein in the development of human disease.

It is well known that mammals contain several types of phospholipase A2. The cytosolic phospholipases A2 (cPLA2s) are one type, and are composed of six enzymes: cPLA2alpha, cPLA2beta, cPLA2gamma, cPLA2delta, cPLA2epsilon, and cPLA2zeta. There has been significant interest in the cPLA2alpha isoform because of the enzyme's 10 fold preference for the hydrolysis at the sn-2 position of the glycerol backbone in phospholipids, resulting in the liberation of arachidonic acid. Arachidonic acid serves as a precursor for several highly regulated inflammatory mediators that play an important role in asthma, atherosclerosis, and arthritis. To address these problems, inhibitors that target cPLA2alpha have been developed to serve as anti-inflammatory therapeutics. Wyeth pharmaceuticals has released a class of indole inhibitors that inhibit cPLA2zeta at <10 nM IC50. However, a recent study has shown that in cPLA2alpha-/-stimulated lung fibroblasts arachidonic acid production is lessened but still present. cPLA2zeta has been identified as the other enzyme involved in the release of arachidonic acid. To further our limited understanding of cPLA2zeta's role in the inflammatory process, we are developing selective and potent inhibitors of the cPLA2zeta isoform. Our synthetic strategy is to modify the scaffold of Wyeth's cPLA2alpha inhibitor. Analysis of the structure-activity relationships and 3D molecular modeling has revealed 4 different places on the scaffold to introduce substitutions. To date, we have generated and assayed a panel of inhibitors. These inhibitors display low nanomolar potency, but have only minimal selectivity for cPLA2zeta. Thus, our main focus is to increase the selectivity of our inhibitors towards cPLA2zeta in order to further our understanding of cPLA2zeta's role in arachidonic acid production.

The basic functional unit of the nervous system is the neuron, which contains two highly specialized compartments, the axon and dendrite. Dendrites received synaptic/sensory inputs, and the dendrite arborization pattern has a profound influence on the type and number of inputs a dendrite can receive. We are studying dendrite patterning in the Drosophila peripheral nervous system (PNS), with a particular focus on the role that non-neuronal signals play in shaping dendrite development. In the Drosophila PNS, sensory neurons grow on top of a monolayer of epithelial cells, which provide instructive cues to shape dendrite patterning. To date, there are plenty of tools available to genetically label and manipulate neurons, but few tools are available for genetic manipulation of epithelial cells. My goal is to design a reporter construct that will specifically label and facilitate targeted expression of transgenes in epithelial cells. To this end, I am assembling a modular plasmid construct which contains a minimal promoter, an enhancer sequence which we have found to be sufficient to direct epithelial expression, and the coding sequence of green fluorescent protein or other genes of interest. After assembling the construct, I will use germline transformation to generate transgenic flies and test the efficacy of the construct. If successful, this will represent the first epithelia-specific transcriptional reporter, and will provide a template for assembly of additional tools for studying dendrite-epithelia interactions.

Merkel Cell Carcinoma (MCC) is a rare, aggressive, neuroendocrine cancer of the skin. The disease is associated with a virus and is strongly linked to immune system function. Disease-specific mortality is >40% and few effective treatments are available for advanced disease. MCC metastases are typically treated by chemotherapy and/or radiation therapy (RT) delivered over >20 sessions. These options are usually successful at shrinking the targeted lesions, but these effects are often short-lived and/or associated with significant side effects. Mouse model data suggest that RT delivered in a single dose is more effective in promoting tumor immunity and that subsequent RT fractions suppress the function of lymphocytes that are recruited to the tumor. Therefore, we explored the use of single dose RT for MCC metastases. 28 patients with a total of 78 tumors were treated with a single dose of RT between 2010-2013. 86% of treated tumors showed a response that persisted for over 6 months. Overall, 45% of treated tumors had a complete radiological/clinical response and an additional 41% had partial responses (>50% reduction in size). These results are encouraging and show that single-dose RT is an effective palliative strategy with minimal side effects.

Everyday, people's lives are affected by the weather; whether sunshine or rain, weather is a part of daily life. Extreme weather events can bring normal activities to a halt. Weather events known as "blocking" involve a pause in the of the typical passage of storms in the jet stream, causing weather systems to stay in place for lengthened periods of time. Wintertime blocking events often cause extreme cold winter temperatures over land, and data show that blocking frequency is increasing. A potential cause for this is due to sea ice loss and the warming of the Atlantic Ocean. Our investigation is of the extraordinary January 2012 blocking event over Europe. The nature of this extreme event was tested using the Weather and Research Model applied to the domain including the Western Arctic Ocean, the North Atlantic Ocean, and Europe, with a resolution of 40 kilometers. The event was simulated over the period from January 15, 2012 to February 6, 2012, with horizontal boundaries and initial conditions constrained by atmospheric observations. In a series of model integrations, we manipulated the thickness of sea ice and sea surface temperature in high altitudes and found that there is a link between thinner sea ice and warmer ocean temperatures and an increased intensity of the blocking event over Europe. This discovery allows for a better understanding of the potential impacts of melting sea ice as the climate changes. 

We are using an animal model to explore the possibility that fetal ultrasound exposure in utero can be a contributing factor of autism. The Williams and Casanova Triple Hit Hypothesis suggests that autism is caused by a combination of genetic predisposition, a specific period of developmental vulnerability, and exposure to environmental factors during that time of vulnerability. We hypothesize that ultrasound is an environmental factor contributing to autism in those who are genetically predisposed. A previous study found that prenatal ultrasound exposure affects neuronal migration, indicating that ultrasound is not completely harmless to the developing brain. Our experiment will use an animal model of three strains of mice with varying degrees of predisposition to autistic-like behavior. Some pregnant mice will be anesthetized and exposed to a clinically relevant dose of ultrasound while control mice will be subjected to the same procedure with the ultrasound turned off. When the mice pups are 3 and 6 weeks of age, we will use a variety of behavioral tests to assay for autistic-like characteristics. Examples of autistic-like behaviors include reduced play, anxiety in open areas, little curiosity for a novel mouse, and affinity for darkness. Results of our study may relate ultrasound exposure to autism, which would lead to further animal studies and, eventually, recommendations on the use of ultrasound on pregnant women. A positive correlation between ultrasound and autism would suggest that pregnant women should be exposed to ultrasound only long enough to assay the fetus' health.

Neprilysin (NEP) is a plasma membrane metalloendopeptidase expressed in pancreatic islet β cells. In non-islet tissues, it has been shown to modulate ion flux. Whether NEP modulates potassium and/or calcium flux in β cells that are necessary for glucose-stimulated insulin secretion (GSIS) is unknown. Thus, the purpose of this study was to determine whether NEP acutely regulates GSIS and if so, where NEP acts in the secretion pathway. To do this, islets were isolated from 8-10 week old C57BL/6 mice. Following overnight recovery, islets were pre-incubated for 30 minutes in 2.8 mM glucose and then stimulated for 60 minutes with either 2.8 mM (basal) or 20 mM (stimulated) glucose in the absence or presence of the NEP inhibitor thiorphan (50 uM). To measure the effects of NEP inhibition on GSIS mediated by Ca2+ channel activity independent of K+ATP channel-induced depolarization, in some conditions a Ca2+ channel agonist (BayK-8644, 1 uM) was used in combination with a K+ATP channel opener (diazoxide, 500 uM). The results showed that acute inhibition of NEP significantly reduced 20 mM glucose-stimulated insulin secretion (128.1±16.0 vs. 80.5±11.4 pM/5 islets; n=11, p=0.03) without affecting basal insulin secretion (13.9±3.8 vs. 8.7±2.3 pM/5 islets; n=11, p=0.26). In addition, NEP inhibition did not alter insulin secretion in response to BayK-8644 plus diazoxide (33.0±6.1 vs. 38.2±8.1 pM/5 islets; n=6, p=0.62), suggesting NEP may be acting to modulate GSIS at a step that is proximal to the Ca2+ channel. It can be extrapolated from this data that NEP may play an important role in the insulin-secretion pathway and thus, acutely upregulating NEP may prove effective in treating type 2 diabetes. 

Ultrasonic neuromodulation is the process of stimulating different neural pathways in the brain through the use of transcranially delivered ultrasound in order to induce clinically relevant emotional, cognitive, physical or behavioral reactions. My research goal is to find out if it is possible to add anatomical specificity to current neuromodulation practice through the use of modulated focused ultrasound (mFU), where 'modulated' refers to adding complex temporal structure to the waveform to optimize neuromodulation. Since mFU has a small focus relative to the brain, the acoustic power from the ultrasound can be moved within the brain; previous experiments have used unfocused ultrasound which sends ultrasound through the entire brain. We hypothesize that we can map varying physical reactions through stimulation of different parts of the brain, with initial emphasis on the motor cortex because stimulation there produces readily observable results. With lightly anesthetized mice as our subjects, and always delivering ultrasound through the skin and skull, we began neuromodulation with unfocused transducers to transmit our ultrasound. This critical step was done to determine the optimal intensity, waveform shape and carrier frequency of ultrasound that created robust, observable effects. Next we translated these ultrasound parameters to our mFU transducer. The current stage of the process includes moving through different parts of each hemisphere and recording the associated motor response such as leg movements and tail flicks. We have already seen a change in activity between activating the front, middle, and rear regions of the brain, as well as dosing effects. Going forward, we intend to see if we could produce varied motor movements with greater spatial resolution using our focused ultrasound across a predefined grid atop the brain. Further studies will also change the applied stimulation intensity. Clinical implications for neuromodulation research include retraining brain function after injury and improving current therapeutics.

The PIRL genes are a family of nine genes discovered during the sequencing of the Arabidopsis thaliana genome, the first fully-sequenced plant genome. PIRLs are a previously uncharacterized class of plant leucine-rich repeat (LRR) proteins that are hypothesized to be important to plant development. As part of an ongoing systematic study to gain insight into PIRL functions, I examined Arabidopsis plants engineered to overexpress the PIRL9 gene. Plants containing an extra copy of PIRL9 designed to drive overexpression of the gene were identified and observed as they grew. Abnormal leaves were observed in most of these plants and stunted growth was quantified through measurements of leaf length and width, as well as primary stem height. Using qPCR, the amount of PIRL9 mRNA present in the different plants was quantified and linked to previous plant measurements. Data showed a correlation between severely stunted plant growth and overexpression of the PIRL9 gene. Significance of these results for PIRL9 function will be discussed, but further genetic and phenotypic analysis is needed to confirm and assess the effects of PIRL9 overexpression. 

Two strains of Botryococcus braunii algae were grown in three different sets of conditions to compare growth characteristics and lipid production. One species, UTEX LB 572, was a Race A Botryococcus braunii from the culture collection of the University of Texas in Austin. The second species, AC 762, was a Race B Botryococcus braunii from the culture collection of the University of Caen, France. UTEX 572 was found to produce two hydrocarbon chain lengths of C:16 and C:18 in a 2:3 ratio respectively. The AC 762 was found to produce hydrocarbons called botryococcenes of chain lengths between C:30 and C:36. The lipid chain lengths of both strains were evaluated by mass spectrometry. The UTEX 572 lipid content was quantified by analytical spectrometry after direct transesterification of the lipid content to biodiesel. The AC 762 lipid content was quantified by a photospectrometry technique developed by UC Berkeley researchers Eroglu and Melis (1985). Cells of both species were harvested during exponential growth phase from 400 mL of growth media and then freeze dried and analyzed gravimetrically. Nine flasks of each species were grown with 3000 lux average light intensity on a 12h:12h light:dark cycle at room temperature averaging 22° C. Three flasks of each species were grown with 1.5% CO2 in BG-11 media, three flasks of each species were grown in BG-11 media with air only and three control flasks were grown in respective carrier medias as those used in the algae collection of origin. Knowledge of the rates at which AC 762 and UTEX 572 produce hydrocarbons as well as the specific lengths and proportions of those hydrocarbons contributes to data needed for comparison of economic considerations in industrial applications.

Botulinum neurotoxin type A (BoNT/A) has been extensively used in human masticatory muscles for cosmetic reduction of large masseter muscles, relaxing the muscles and reducing pain in temporomandibular joint and muscle disorders, and decreasing loading on the mandible. Multiple applications of BoNT/A over time is a clinically routine practice to reach the treatment goal. However, it is unknown how the muscle responds to multiple neuromuscular blockages by BoNT/A, and whether or not the injected muscle shows functional recovery differently after each application. This study was designed to answer these questions. Adult New Zealand rabbits were injected 3 times either with BoNT/A (n=13) or saline (n=5) in one masseter at week 0, 12, and 24. Non-invasive surface electromyography (EMG) of bilateral masseter muscles was recorded during natural mastication 1 week before the first injection and every two weeks thereafter up to week 35. Jaw movement was video-taped and synchronized with EMG recording for identifying the chewing side. Body weight was tracked weekly to examine the effect of BoNT/A on general health. From each of these recordings, 15-20 consecutive chewing cycles from each chewing side were sampled and analyzed for chewing cycle length, muscle mean and integral activity, and durations. The preliminary results indicate that the recovery patterns of the 11-12 weeks after each BoNT/A injection differed significantly, and both the chewing frequency and muscle activity 11 weeks after the 3rd injection (week 35) were close to those of saline animals and the baseline recorded before injection. These findings provide new information about the consequences of multiple applications of BoNT/A on masticatory muscles, and may lead to the better understanding and use of BoNT/A in clinical dentistry.

Malaria is a parasitic infection due to Protozoa Plasmodium. This is a severe global disease with 250 million annual cases leading to 1-2 million deaths. The most virulent, P. falciparum, is responsible for severe clinical malaria and death. An array of anti-malarial treatments exist today, however parasites evolve continuously to develop resistance even to the newest frontline anti-malarial drugs. Previous studies in the Rathod laboratory have demonstrated that drug resistance can be generated in the lab and that some drugs can be utilized to characterize Accelerated Resistance to Multiple Drugs (ARMD) parasites, such as the laboratory strain Dd2, and non-ARMD parasites, such as the laboratory strain HB3. In the current study, we are using a cyclopropyl carboxamide (GSK 2645947) to select for resistant parasites. Cyclopropyl carboxamides are a chemically novel class of antimalarial agents identified recently and they prevent the growth of P. falciparum in the early trophozoite stages of the erythrocytic Plasmodium cycle. This project focuses on determining the conditions for the development of resistance against GSK 2645947 with Dd2 and HB3 by conducting a series of selection experiments utilizing different concentrations of GSK 2645947 and starting parasites populations. Unlike previous conditions which indicate that the Dd2 strain acquires resistance at a significantly higher frequency than the HB3 stain; the data from this study suggests that both Dd2 and HB3 strains respond similarly to GSK 2645947. Through this study, we can further understand the behavior of resistant P. falciparum parasites. 

Quantitative mass spectrometry (MS)-based proteomics is a powerful approach to measure relative expression changes across thousands of proteins in a complex mixture. Commonly, stable isotopes like ¹³C and ¹⁵N are incorporated into proteins and peptides by metabolic labeling (ML) or chemical labeling (CL) to produce “heavy” labeled forms. Mixtures of light and heavy peptides can be identified and quantified by MS. In ML, proteins are labeled in vivo by growing cells with isotope labeled metabolic precursors, while peptides are chemically labeled in vitro in CL. These two labeling approaches have distinct workflows and their own strengths and limitations. In a typical proteomics experiment, complex mixtures of peptides are separated on reversed phase liquid chromatography (LC) and sampled in real-time by the mass spectrometer for peptide identification and quantification. We propose to analyze mixtures of chemically and metabolically labeled peptides in single LC-MS runs, comparing each method’s quantitative accuracy and precision, and other factors, such as analytical losses from sample processing without errors introduced by stochastic sampling. We compare stable isotope labeling by amino acids in cell culture (SILAC) and reductive dimethylation CL by mixing equal amounts of peptides from each approach.Preliminary results indicate minimal differences between ML and CL in quantifying proteins (median of >2 peptide ratios). With individual peptides, however, CL showed 13% larger variance than the ML. We observed signal losses in CL samples compared to ML samples, and fewer quantified peptides and proteins in CL (6005 peptides, 897 proteins) than in ML (7448 peptides, 1059 proteins). We attribute the differences to the extra labeling step in CL, and will perform further analyses with ratio mixtures of heavy and light labeled peptides to clarify the source of variance. Our comparison of CL and ML will guide future optimization and applications of both quantitative proteomics labeling methods.

A major obstacle in the efficient delivery of genes using non-viral vectors is sequestration and subsequent degradation of the vector in the endosomal/lysosomal trafficking pathway. While incorporation of membrane-lytic peptides into gene delivery vectors has been shown to increase transgene expression by disrupting the endosomal vesicles, non-specific membrane-disruption caused by these modified vectors leads to significantly increased toxicity. One approach to mitigating this increased toxicity is to mask the membrane-lytic peptides inside a pH-responsive micellar core that destabilizes during vesicle acidification in the endosomal/lysosomal pathway, revealing the membrane-lytic segments only when internalized. Incorporating pH-responsive imidazole elements in the hydrophobic micelle core which become charged under acidifying conditions can disrupt the micellar structure by electrostatic repulsion, leading to display of the active peptide, membrane lysis, and increased DNA delivery to the cytoplasm. After synthesis of the pH-responsive motif a panel of representative DNA delivery polymers will be evaluated for pH-responsiveness, transfection efficiency, and cytotoxicity.

Those who have studied genetics have a keen understanding of the fact that incredibly small changes can have tremendous, and often tragic, consequences. In patients with Fragile X Syndrome (FXS), a repeating sequence of genetic information (CGG) in an X chromosomal region is repeated more often than in the general population. This results in a spectrum of conditions including intellectual disability, anxiety, and physical features that deviate from the norm. About one fifth of people who have FXS also have seizures; the underlying cause of seizures in this fraction of FXS individuals is unknown. We hypothesize that FXS individuals who have seizures will have specific genetic variants –duplications and deletions of genetic information –that are not seen in FXS individuals without seizures. The experimental group is composed of DNA samples of FXS patients with seizures, while the control group will be composed of DNA samples of FXS patients without seizures. In order to find genetic variants, I utilize a genomic technique called array-comparative genomic hybridization (array-CGH). In array-CGH, DNA samples are tagged with fluorescent dyes to contrast patient DNA (tagged with pink dye) from control DNA (tagged with blue dye). The ultimate product of this technique is a quantitative representation of any differences between patient and control DNA, which can be processed and analyzed by computer software. By comparing the genomes of these patients, we will determine if there are common genetic variants across the experimental group that are distinct from the control group. If such variants are found, the next step is to associate specific genes with the subset. Establishing an association between a gene and the FXS and seizure phenotype is essential because it deepens our understanding of the condition and provides a foundation for future therapeutic treatments.

A critical problem in the treatment of addiction is the tendency of addicted individuals to relapse to drugs-seeking with as many as 90% of addicts relapsing to use within one year. Exposure to stress is an important factor that precipitates relapse, though the exact mechanism is yet unknown. The Lateral Habenula (LHb) and the Rostromedial Tegmental Nucleus (RMTg), nuclei in the dorsal diencephalon, have recently become brain regions of interest in addiction research. Both regions have been shown to be important regulators of the midbrain dopaminergic systems, which are involved in relapse to cocaine seeking. However, very little is known about the precise role of these nuclei in stress-induced cocaine relapse. In our initial experiments, male, Long-Evans rats were handled for 3 days, and then injected with cocaine (10mg/kg) or exposed to intermittent footshock. Preliminary results demonstrate an increase in the expression of cFos, an early-detection transcription factor and marker for neuronal activity, in both the LHb and RMTg upon acute exposure to cocaine or foot shock. Studies are currently underway to determine the role of the LHb, and LHb projections to the RMTg on stress-induced cocaine reinstatement using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology in a rat self-administration reinstatement model.

Stem cell based therapeutics is a promising venture to regenerate a damaged heart after a myocardial infarction and to prevent the progression to heart failure. However, we currently do not have a clear understanding of the maturation processes of cardiomyocytes, and this has been one of the main obstacles limiting progress in the field. Here, we develop a device that simulates the mechanical conditions that human cardiomyocytes experience during their growth phase. The aim of the device is to apply a constant mechanical stress on cardiac tissue to test the effects of creep on cardiomyocyte size and alignment. Cardiac tissue used in this study is obtained by mixing a collagen solution with cardiomyocytes derived from the directed differentiation of human embryonic stem cells and pipetted into PDMS molds held in 6-well plates (6wp). Testing of initial designs incorporating a moving magnet below the 6wp coupled with a metallic base within the plate have resulted in too much friction, leading to uneven stretching of the cardiac tissue and the breakage of all constructs tested within 10 days. In response, a revised design is utilized, where a direct screw-driven platform suspended on top of the 6wp controls the mechanical stress applied to the cardiac tissue. This device effectively stretches the constructs to 110% of their initial length over a period of 14 days, without the breakage of any of the four constructs tested. Here we show that mechanical creep over an extended period of time affects cardiac morphology, where increased cardiomyocyte size and a greater degree of cell alignment within the cardiac tissue are observed with stretch versus non-stretched controls. This project provides insight on the mechanical cues involved in the maturation of cardiomyocytes, with the device offering a platform for in vitro modeling of human cardiac tissue.

Titanium dioxide (TiO₂) displays a variety of interesting properties and has been intensely investigated for applications in environmental cleanup, photocatalysis, dye-sensitized solar cells, and semiconductor fabrication among others. However, the underlying mechanisms which govern TiO₂ chemistry are not well understood. Here, the tendency for reduced TiO₂ nanoparticles to donate stored electrons to other reagents is being characterized. The reduced ("charged") nanoparticles are made by UV irradiation which excites an electron from the valence band to the higher energy conduction band of the TiO₂ nanoparticles. In the absence of oxygen and other oxidants, the charged TiO₂ nanoparticles are stable and can be easily monitored by their absorption of visible light and blue color. We are exploring the chemical reactions of the charged particles. If the optical spectrum changes after the addition of other reagents, that is an indication that the nanoparticles are reducing the reagents. Removal of the electrons from the nanoparticles has been previously accomplished using aqueous Cu²⁺, but not Zn²⁺ or Mn²⁺. I am repeating this result and extending it to other metal ions such as Cr³⁺ in order to estimate the standard reduction potential of the charged particles. In addition, I am studying optically and via NMR whether dimethyl sulfoxide (DMSO) is reduced to dimethyl sulfide by charged TiO₂ nanoparticles. Because one-electron reduction of DMSO results in extremely unfavorable intermediates we are probing whether the reaction by charged TiO₂ occurs by the direct transfer of two electrons from the TiO₂ nanoparticles to the DMSO. These studies will provide valuable insight into the electronic chemistry of TiO₂ and will aid in the application of TiO₂ to other systems which require the transfer of multiple electrons.

Solar technology is a potential way to help meet the growing demand for clean, renewable energy. Hybrid composites of inorganic quantum dots, with organic semiconducting polymers offer a potential means of producing low-cost, solution-processable photovoltaics. The synthesis of quantum dots typically involves the use of large surfactant molecules to facilitate particle growth and solubility. These native ligands act as electrical insulators that impede charge transport in photovoltaic devices. Therefore, it is necessary to exchange these large ligands with small molecules to achieve efficient charge carrier photogeneration and transport. In this research, we examine bulk heterojunction blends of low band gap PbS quantum dots with the conjugated polymer poly((4,8-bis(octyloxy)benzo(1,2-b:4,5-b')dithiophene-2,6-diyl)(2-((dodecyloxy)carbonyl)thieno(3,4-b)thiophenediyl)) (PTB1) treated with different ligands including cetyltrimethlammonium bromide (CTAB), tetrabutylammonium thiocyanate (TBAT), and 3-mercaptopropionic acid (MPA). We aim to elucidate how these different ligands influence the photovoltaic performance of PbS/PTB1blends by studying the differences in long-lived charge generation and recombination kinetics associated with the quantum dot ligand treatments using transient photovoltage (TPV) and photoinduced absorption (PIA) spectroscopy, respectively. We will also determine whether different ligand treatments influence film morphology using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). We hope to gain a better understanding of how quantum dot ligand treatments may be used to optimize the performance of hybrid photovoltaics.

Bacteria play important roles in marine nutrient cycles. However, most marine bacteria are uncultured and little is known about their specific functions in marine nutrient cycles. Environmental members of the gamma proteobacterial sulfur oxidizer (GSO) clade have the genetic potential to use energy from sulfur oxidation to assimilate carbon, suggesting that they contribute to carbon and sulfur cycling in the oceans. Here we report the isolation and initial characterization of an open ocean representative from the GSO clade. Our isolate, GSO-NP4, was purified and its identity confirmed by 16S rRNA gene analyses. This isolate grows to relatively low cell densities (2 x E5 cells/ml) and has a doubling time of approximately 19 hours. Six 1L cultures were required to obtain 3.1g of DNA for genome sequencing. Six to twelve additional 1L cultures were established to obtain enough DNA to sequence the complete genome of GSO-NP4. Genomic information of this isolate is essential for future studies to determine the roles of GSOs in marine carbon and sulfur cycles.

Francisella noatunensis is an emerging fish pathogen that causes disease and mortality for several economically important fish including tilapia and Atlantic cod. The Francisella Pathogenicity Island (FPI) is a region of the genome known to govern virulence for the genus. PdpA is one of the genes found in the FPI that has been implicated in the ability of Francisella tularensis, the human pathogen, to replicate in infected phagocytes. A recent study by our laboratory using zebrafish as a model host indicated that pdpA is important for the ability of F. noatunensis to cause mortality in infected zebrafish—thus the functional role of the pdpA protein appears to be conserved for the genus. Our goal is to compare the innate immune response of zebrafish during infection with either wild type or pdpA knockout strains of F. noatunensis to determine if the host immune response is altered. To accomplish this, we are monitoring the expression of key proinflammatory genes (IL1B and TNFA) using quantitative RTPCR during acute Francisella infection in zebrafish. The results from this study will provide additional information about the potential use of the F. noatunensis pdpA knockouts as live vaccine candidates for fish.

Malaria is a prominent parasitic disease that afflicts 300 to 500 million people and results in 1 to 2 million deaths each year, approximately 90% of which are young children under the age of 5 and pregnant women in sub-Saharan Africa. New therapeutic compounds for treating malaria are urgently needed due to the emergence of multidrug-resistant parasites including Plasmodium falciparum, the most fatal Malaria causing species that infects humans. A mass spectrometry-based screening of a natural product library yielded seven securinine-based compounds that were determined to bind to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). This enzyme catalyzes the formation of deoxyuridine monophosphate (dUMP) from deoxyuridine triphosphate (dUTP) which has important implications on DNA replication and thus makes dUTPase a good target enzyme for drug discovery. These seven compounds were tested using a standard biochemical assay and enzyme activity was measured through the detection of pyrophosphate, a product of the catalyzed reaction. Detection was done with two independent detection kits: indirectly through the detection of inorganic phosphate (pyrophosphatase and Malachite Green Kit) or directly through the detection of pyrophosphate (PPiLight Kit). In most drug screens, the goal is to discover possible drug-like molecules that inhibit the target enzyme, however, these securinine compounds were actually found to increase PfdUTPase activity. While it is unusual to pursue compounds that do not inhibit target enzymes, these compounds might still prove to be possible drug candidates by over activating PfdUTPase and killing the parasite in this manner. Whether this is a viable method of utilizing these compounds remains to be tested.

Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for viral replication. Recently, our lab conducted a global metabolic study to determine how vaccinia virus (VACV), a large double-stranded DNA virus, alters host cell metabolism. VACV is a model system to study poxvirus infections and was used as the live vaccine against smallpox. In our study, dual mass spectrometry platforms were used to analyze a broad range of metabolites from mock and VACV-infected fibroblast cells harvested at multiple time points within 24 hours of infection. One of the most notable metabolic alterations observed during VACV infection was increased glutamine utilization, or glutaminolysis. We have since shown that under conditions of glutamine starvation there is an approximate 90% decrease in VACV production in fibroblasts, thus revealing an essential role for glutamine during infection. Furthermore, this decrease in virus production could be rescued by the addition of tri-carboxylic acid (TCA) cycle intermediates, suggesting glutamine’s use as an anaplerotic, or replacement, substrate for the TCA cycle during infection. To determine at what stage(s) in the VACV life cycle glutamine is required, quantitative real-time RT-PCR and western blot analyses were performed to examine early and late viral transcript and protein levels. We have found that glutamine deprivation does not inhibit viral gene expression, but does significantly impact viral protein synthesis. Importantly, the addition of TCA cycle intermediates recovered viral protein levels, highlighting the need for glutamine carbon to maintain the TCA cycle at this stage in the VACV life cycle. This study reveals the robust metabolic program implemented by vaccinia virus during the course of infection and provides insight into the role metabolic reprogramming plays during viral replication. Ultimately, studies such as these may help to identify novel therapeutic methods to specifically target infected cells and inhibit viral replication.

Administration of Anti-Retroviral Therapy (ART) slows disease progression and reduces HIV-1 viremia to near undetectable levels. Poor adherence or withdrawal of therapy often results in rapid rebound of viremia with the potential for the establishment of drug resistant variants. HIV is known to establish latency in long-lived central memory T-cells (CM) and is often referred to as “the reservoir”. However, we now understand that there is both latent and actively replicating HIV-1 that comprises reservoir sites. Using sequence information and computational tools we can define reservoirs as cells or tissues harboring HIV-1 with genotypic features of reduced temporal structure, high diversity within the population, and low divergence from their most recent common ancestor (MRCA). This is because reservoirs are populated with virus seeded at both early and late periods of infection. If a reservoir is established in a site with reduced drug penetrance, “drug-sanctuaries”, continued viral replication and diversification could occur. This may result in the accumulation of drug resistant variants that lead to therapy failure. Here we hypothesize that tissue and cellular sites with phenotypes reflective of reservoirs may also be drug restricted providing a boundary for unrestricted viral replication. To test this hypothesis we sequenced single genome template derived amplicons (SGA) of env and pol regions of HIV-1. Presented is data from the autopsy of subject S104. S104 presented with multi-drug resistant genotypes in all tissues sampled. Bioinformatical analysis measuring viral genetic diversity within and divergence from the MRCA of the viral population of S104 demonstrated reservoir phenotypes in tissue virus isolated from lung. We continue to expand our characterization of reservoirs and compartments to clearly define sites that must be targeted to eradicate HIV-1 in the host.

Prostate cancer is the second most common cancer in American men, and it has been estimated that about 1 man in 6 will be diagnosed with prostate cancer during his lifetime. The prostate-specific antigen (PSA) serum test is widely used to screen men for prostate cancer, however the test has a false-positive rate of nearly 70% and leads to many unnecessary biopsies being performed. Anterior gradient 2 (AGR2) is a secreted protein produced by prostate cancer cells in primary tumors, and due to its cancer-specific property, AGR2 can potentially be used to develop a urine test for prostate cancer diagnosis. The expression of AGR2 in cancer patients were studied by frozen section immunohistochemistry, sandwich ELISA, tissue microarray, and statistical analysis. The results show that AGR2 can be a suitable biomarker used to develop a test in screening prostate cancer for the following reasons: (1) it is highly expressed in cancer; (2) it is found in almost all tumors; (3) it is expressed by pre-malignant high-grade prostatic intraepithelial neoplasia (HGPIN) lesions. Since AGR2 is specific to cancer cells and is secreted into urine, we find the clinical application of AGR2 to be viable and that a urine test used for prostate cancer diagnosis is possible. AGR2 is detected at picograms per millimeter level for cancer patients.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Soft tissue sarcoma is a cancer that develops in the muscle, fat, fibrous tissue, blood vessels, or other supporting tissue. RMS is categorized into two major subtypes: alveolar RMS (ARMS) and embryonal RMS (ERMS). Most ARMS express the PAX3-FOXO1 fusion oncoprotein generated by the 2;13 chromosomal translocation. Among RMS tumors, PAX3-FOXO1-positive ARMS commonly gives the worst prognosis. Sphingolipids are defined by a common structural feature (a long-chain (sphingoid) base). Sphingosine is the most common sphingoid base composed of an 18 carbon-chain. The overall goal of this study is to investigate roles of sphingosine on growth, apoptosis (programmed cell death), and MYCN oncogene expression in PAX3-FOXO1-positive ARMS cells (RH30 cell line). The specific aims of this study are to determine: 1) effects of sphingosine on apoptosis; 2) effects of sphingosine on MYCN expression; 3) effects of MYCN overexpression on growth inhibitory and pro-apoptotic activities of sphingosine. Our previous results have indicated that sphingosine caused time and dose-dependent inhibition of the growth and death of RH30 cells, suggesting sphingosine as a potential chemotherapeutic agent against ARMS. Sphingosine, at a concentration that can induce apoptosis, down-regulates the MYCN expression as early as two hours. In the present study, the data from flow cytometry demonstrates that sphingosine induces apoptosis identified by a well-known apoptosis marker, AnnexinV+/PI+. Experiments are in progress to determine whether the growth inhibitory effects of sphingosine are decreased in RH30 cells overexpressing MYCN. This will test a hypothesis that MYCN is a mechanism which mediates the growth inhibitory and pro-apoptotic effects of sphingosine on PAX3-FOXO1-positive ARMS cells. Thus, this experiment will determine if MYCN is a possible target for chemotherapeutic agents in ARMS.

Cancers contain large numbers of mutations. This reservoir of genetic variation can be used by cancer to produce new phenotypes in response to environmental barriers. In order to accumulate these mutations we have proposed that cancers express a mutator phenotype, i.e. an increased rate of mutation. Previous results in the lab demonstrate that sequential selection of cancer cell lines (colon cancer cells HCT116 and SW620) for novel phenotypes enriches for cell lineages with elevated mutation rates. After three rounds of sequential selection for resistance to non-mutagenic drugs (diphtheria toxin, puromycin, staurosporine), both cancer cell lines exhibit an increase in mutation rate. Specifically a 3-fold for HCT116 and 6-fold for SW620 was measured. I will extend these experiments using additional selection conditions (e.g., elevated temperature, hypoxia and nutrient deprivation) and will determine mutation rate of the resulting cell lines using the PIGA gene mutation assay. The random mutation load will be characterized using Duplex Sequencing. We believe the process of sequential selection favors mutator lineages as has been observed in bacteria. However, because an increased rate of mutation is potentially deleterious to a cell, we propose the existence of a mutation limit beyond which cells are no longer viable, i.e. the cells undergo apoptosis. I will screen mutagenic nucleosides for their potential to increase the mutation rate of cancer cells beyond this threshold. Targeting cancer’s error threshold may represent a novel and innovative therapeutic strategy for the treatment of human malignancies.

Angiogenesis is the development of new blood vessels from pre-existing vasculature and is a hallmark of Kaposi’s sarcoma (KS), a highly vascularized neoplasm prevalent among immunosuppressed individuals. The etiological agent of KS, Kaposi’s sarcoma-associated herpesvirus (KSHV), induces angiogenic phenotypes in endothelial cells during latent infection. Interestingly, KSHV infection leads to down-regulation of transforming growth factor-beta 2 (TGF-β2), an anti-angiogenic cytokine. In addition to six viral genes, KSHV also encodes 17 recently discovered microRNAs (miRNAs) during latent infection. These endogenously encoded RNAs of 19-23 nucleotides post-transcriptionally regulate gene expression through basepairing interactions with the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs) and either mark them for degradation or prevent ribosomal binding. These viral miRNAs appear to directly contribute to pathogenesis and angiogenesis by targeting host gene expression. We screened the miRNAs of KSHV to determine if any are involved in the down-regulation of TGF-β2 during KSHV infection. We found that two KSHV miRNAs, miR-K3 and miR-K8, are independently sufficient to downregulate TGF-β2. To determine if these miRNAs directly target the 3’UTR of TGF-β2 as opposed to altering genes that affect TGF-β2 expression, we used a TGF-β2 3’UTR luciferase reporter construct. We also used competitive inhibitors of miR-K3 and miR-K8 to determine if these miRNAs are necessary for the down-regulation of TGF-β2. By identifying specific targets of KSHV miRNAs such as TGF-β2, we can improve our understanding of how KSHV is able to maintain latent infection and target host gene expression to alter endothelial cells.

Genome-wide association studies indicate that the sequence polymorphism rs6983267 located in the 8q24 risk locus correlates with increased risk for colorectal, prostate, breast and ovarian cancers. This region is particularly interesting because it is located in a ‘gene desert’ in which no expressed genes are known; however, several reports suggest that it functions as an enhancer that regulates the expression of the Myc oncogene located more than 300 kilobases away from rs6983267. The enhancer region also contains a binding site for CTCF, a factor known for its contributions to long-distance chromosomal interactions and gene regulation. Here, we aim to elucidate the specific function of CTCF located close to the rs6983267 sequence variation in DLD1-colorectal cells. We test the hypothesis that the CTCF element at the 8q24 risk region is required for a physical interaction between the enhancer of the 8q24 risk locus and the Myc gene. This hypothesis predicts that deletion of the CTCF binding site will yield reduced Myc expression. In our study we introduced a series of genomic modifications with recombinant adeno-associated virus (rAAV) to generate site-specific deletions and to examine CTCF’s role in long-range chromosomal interactions at the 8q24 locus. Mutant cell lines were developed with modifications on either one or both 8q24 alleles in the colorectal cancer cell line DLD1. Cell growth characteristics, gene expression and chromosome conformation capture (3C) experiments will be performed to better understand the functional and physical interaction between the 8q24 risk locus and the Myc gene.

Around 650 health services professionals in the Washington State Department of Corrections provided care for over 24,000 inmates in the last year, and the number of people being housed in correctional facilities is on the rise. Discourse abounds regarding the necessity and adequacy of health care provided to this growing population in the US, yet the bulk of this discourse applies a biomedical lens. This exploratory study employs qualitative methods to examine the role of the health care provider in the prison environment in an effort to understand the factors within the correctional system that influence the provision of health care in prisons. Through participant observation, in-depth interviews, and cognitive mapping, this study aims to shed light on the interactions among the medical staff, correctional staff, and inmates. All research is conducted at a single Washington state prison. Interviews are conducted with around 10 health care professionals who volunteered to be interviewed; field notes are taken during the course of interviews and observations; and diagrams expressing inmates' perceptions of the process of receiving care are collected. For analysis, open coding is performed to identify significant themes in the data, and closed coding is performed to explore the themes more deeply and make connections among them. An understanding of how these major players in correctional health care perceive health care and perceive each other has the potential to help increase the effectiveness of care.

Epilepsy is a neurological disease characterized by recurring seizures and it affects millions. We hypothesize that modulation of p38 mitogen-activated protein kinase (p38 MAPK) will influence hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity, affecting the frequency of seizures in an animal model of epilepsy. p38 MAPK is a kinase activated by conditions of cellular stress. It strongly stimulates the HCN channels, which are voltage gated ion channels that are highly expressed in the cortex and hippocampus, brain regions where seizure onset occurs. A decrease in HCN channel expression or function has previously been observed in epileptic animals associated with a loss of p38 MAPK activity, which produced neuronal hyperexcitability. We hypothesized that increasing p38 MAPK activity might decrease neuronal activity and decrease seizure frequency. In this experiment, Epilepsy was induced in Sprague Dawley rats through treatment with pilocarpine, a convulsant drug. Recurrent, unprovoked seizures were seen after three weeks of treatment. An osmotic pump was placed in the rats, delivering drugs that modulate p38 MAPK. The drug SB203580 (SB), a specific inhibitor of p38 MAPK, was administered, as was anisomycin, a non-specific activator of p38 MAPK. Animal brain activity was analyzed with video electroencephalography through electrode implants in the skulls of the rats. Inhibition of p38 MAPK by SB increased seizure frequency by 179 % as predicted, however, anisomycin also increased seizures by 236%. We hypothesize that this may be due to non-specific actions on other signaling pathways like the Jun N-terminal kinase pathway (JNK). We’re investigating the role of JNK in modulating seizure frequency, and this may point us to some new therapies. The implications of this study may indicate a new approach to the control of epilepsy via anti-epileptic drugs targeting p38 MAPK.

There are a variety of air pollutants that can adversely impact health, including diesel exhaust from vehicles and other sources. In the Duwamish River valley of Seattle, WA, there are many sources of air pollution that emit diesel exhaust. Based on concerns of local residents, the overall objective of the Diesel Exhaust Exposure in the Duwamish Study, or DEEDS, was to measure exhaust levels throughout two Duwamish River valley neighborhoods: South Park and Georgetown. I assisted in nearly all spects of this pilot study which included the deployment of nitrogen oxides (NOx) air samplers to nine sites throughout the two neighborhoods to perform passive sampling over a ten day period in the summer. During NOx sampling, fine and ultrafine particle measurements were also collected for ten minutes at each of the nine sites using direct reading instruments and a mobile setup. These pilot study results collected from this preliminary assessment were used to optimize location selection for measurement of NOx, NO2, 1-nitropyrene, light absorbing carbon, and fine particulate matter during the DEEDS sampling campaigns taking place over the past year.

Circadian rhythms like the sleep-wake cycle are governed by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN). This clock is synchronized by the light-dark (LD) cycle and the cyclic-guanosine monophoshate (cGMP) pathway is known to mediate its phase advances. Phase-advancing light pulses increase the levels of cGMP in the SCN and inhibition of phosphodiesterase-5 (PDE5), the enzyme that degrades cGMP, by artificial drugs such as sildenafil, enhance light-induced phase advances. These inhibitors act by inducing PDE5 phosphorylation and converting it to phosphorylated-PDE5 (phPDE5). This evidence suggests that the inactivation of PDE5 through its phosphorylation could be a mechanism through which light induces phase advances, and that sildenafil utilizes the same mechanism to enhance phase advances upon light stimulation. We are testing this hypothesis by measuring PDE5 and phPDE5 in the SCN of mice treated with a phase-advancing light pulse. Mice are administered either a light or a dark pulse at the time that light causes phase advances. Animals are sacrificed at different times after the presentation of the pulses, their brains are dissected and alternate sections are processed for immunohistochemistry against either PDE5 or phPDE5. Our hypothesis predicts an increase in phPDE5-expressing cells in light-treated animals. Future experiments will determine if sildenafil- induced enhancement of phase advances are mediated by further increasing phPDE5. Our findings may lead to the development of pharmacological treatments to treat circadian rhythm disorders such as insomnia, fatigue, and jet lag, as well as to manipulate circadian rhythms to achieve periods of extended wakefulness and alertness.

Approximately 800,000 Americans suffer a stroke each year, making this neurological disease the leading cause of adult disability in the US. An ischemic stroke occurs when blood flow to the brain is interrupted, resulting in inadequate oxygen delivery to brain cells. Currently, only one intervention has proven to improve outcome after stroke and must be given shortly after stroke onset. Effective interventions given at delayed time points after stroke are needed. During stroke, breakdown of the blood-brain barrier allows for interactions between once-segregated central nervous system antigens and the systemic immune system. My project investigates the effects of a cellular immune response directed towards myelin basic protein (MBP) and the interactions between CD8⁺ on myelin and stroke outcome in experimental cerebral ischemia. In this model, Lewis rats are subjected to middle cerebral arterial occlusion (2 hours) and injected with either lipopolysaccharhide, shown to elicit an immune response to MBP, or saline (control). I performed a battery of behavioral tests, including a standard neurological score for rodents and evaluation of rotarod performance at set time points before and after stroke. Using double-label immunocytochemistry, the brain sections were labeled for CD8⁺ and MBP. Quantitative analysis of myelin between the infarcted and non-infarcted hemispheres was performed using Metamorph; the numbers of CD8⁺ lymphocytes were counted in a standard fashion. Preliminary data show prominent demyelination in the infarcted regions. Additionally, we observed a correlation between myelin loss and performance on the rotarod immediately post-stroke, though further results are pending. The goals for this project are to (1) determine if animals receiving MBP specific lymphocytes adoptively at stroke onset suffer more myelin loss than those receiving lymphocytes unreacted to MBP and (2) determine if the number of CD8⁺ lymphocytes within the infarct correlate with the extent of myelin loss, and degree of the MBP response among the adoptively transferred lymphocytes.

Nicotinamide adenine dinucleotide (NAD) is used in many biological processes such as metabolism and cellular signaling. Recently NAD is found to have an important role in many signaling pathways. As NADs role in cellular pathways is being discovered, it is important to be able to detect and quantify the amount of NAD in vivo and in vitro by nuclear magnetic resonance (NMR) analysis. NAD is found in cells at low concentrations and its peak intensity in a phosphorus spectrum is much smaller than that of other phosphorus containing molecules like ATP. By decoupling and Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) experiments we hope to isolate and increase the signal of the characteristic phosphorus nuclei found in NAD molecules. Isolating and increasing the signal to noise of the NAD peak will prove that we have the right peak and allow us to quantify the amount of NAD in vivo. This will provide information about health risks of a subject.

Mosquitoes are the principle disease vector of malaria, yellow fever, and dengue fever, which are responsible for more than a million deaths worldwide annually. This makes controlling transmission a global health priority, and underscores the importance of understanding their behavior. Although it is known that olfaction is the main sensory modality mosquitoes use to select prey, many behavioral patterns, such as changing prey preferences, suggest that associative learning has a role in mosquito behavior. Previous research has established mosquito’s affinity for human scent and characterized their olfactory receptors, but little is known about the neural substrates and molecular underpinnings that modulate olfactory learning. To gain insight into the importance of olfactory learning, I have been introducing controlled perturbations into the larval mosquito nervous system and assaying their behavior using a classical conditioning model. This is achieved through genetic manipulation and injection of drugs with known targets. Initially, I have focused on the role of dopaminergic neurons, because they are known regulators of associative learning in other species. To this end, I have developed microinjection techniques that allow for delivery of silencing RNAs (siRNAs) to knockdown dopamine receptor expression. Using this approach, I have found that Dopamine receptor deficient animals, as well as those injected with Dopamine antagonists, are unable to learn like their untreated counterparts. Knockdown animals suffer a much higher mortality rate in survival experiments that test their ability to avoid predation, underscoring the importance of these receptors in vivo. Currently, we are using cell biological approaches to visualize the relevant neurons. Together these data show that larval mosquitoes are able to learn within days of hatching, and that dopamine plays a crucial role in facilitating aversive learning. In addition to clarifying the mechanistic basis for olfactory learning, these findings may ultimately enhance the development of vector control strategies.

SRC-Family Kinases (SFKs) are a family of non-receptor tyrosine kinases that play key roles in intracellular signaling transduction.  They are multi-domain proteins containing two regulatory domains, SH2 and SH3, and a catalytic domain.  The two regulatory domains are vital in controlling kinase activity and interactions with other proteins in cell signaling events. Current therapeutic development strategies target the ATP binding site of the kinase domain to inhibit enzymatic activity. Recently, it has been shown that the binding of ligands to the active sites of these kinases can stabilize conformational changes in the SH2/SH3 domains modulating the accessibility of these domains necessary for protein-protein interactions, potentially providing novel kinase regulation through both enzymatic activity and protein-protein interaction. Disregulation of SFK kinases plays an important role in tumor metastasis. A recent unbiased screening of kinase mutations in cancer cells indicated a considerable amount of inactivating mutations, which indicates that there are important non-catalytic functions of kinases which play significant roles in cancer biology.  The long term goal of our study is to better understand these non-catalytic functions in SFK signaling pathways so as to help develop better approaches to treating cancers. Using an array of ATP competitive inhibitors, we have biochemically profiled some of these conformational changes in SFKs which modulate the accessibility of these regulatory domains in particular conformations to help elucidate the role these non-catalytic activities have in intracellular signaling.  So far, it has been shown that alpha C-helix-out inhibitors favor the SH2/SH3 intra-molecularly engaged, inactive conformation, while smaller Type1 inhibitors favor the extended, active conformations of these SFKs.  Currently, the plan is to further profile these conformations, as well as to determine how specific ATP binding site conformations affect SFK function in intracellular signaling. Towards this end, a panel of alpha C-helix-out and Type 1 inhibitors are being synthesized and tested.

Currently there are three major classes of known viruses: exclusively RNA viruses, which do not require a DNA intermediate in their replication and propagation; DNA viruses, which use DNA dependent processes to perform their genome replication; so-called retroviruses, which go through both an RNA and a DNA phase with the help of a reverse transcriptase enzyme. Until very recently there was no evidence that there was genetic exchange between these distinct virus classes. However, a single stranded DNA (ssDNA) virus genome, recently found using metagenomics in Boiling Springs Lake in Lassen Volcanic National Park, USA (BSL) contains elements homologous to both ssRNA and ssDNA viruses. The genome of this RNA-DNA hybrid virus (BSL-RDHV) contains a gene similar to that of ssDNA circoviruses that infect animals and a capsid protein (CP) gene similar to that of the ssRNA tombusviruses that infect plants. It is currently unclear how the RNA virus capsid gene integrated into the DNA virus genome and what, if any, evolutionary impacts are associated with this recombination. My current work focuses on the characterization of the BSL-RDHV CP using recombinantly expressed protein. The CP gene will be amplified from the BSL viral metagenome using Polymerase Chain Reaction and expressed in bacterial cells. The long term goal is to determine the structure of this protein using X-ray crystallography and use the protein for in vitro assembly reactions. Successful completion of this work will be an important step towards understanding virus evolution and possibly the emergence of novel virus disease.

Obesity is a growing epidemic. Consequently, the percentage of Americans living with Type 2 Diabetes Mellitus (T2DM) has increased; 25.8 million Americans suffer from diabetes, and 90-95% of adult diabetes is T2DM. Bariatric surgeries--gastrointestinal operations designed to encourage weight loss by rerouting and reconnecting various parts of the stomach and small intestine--have been developed in response to increased obesity. A ramification of these surgeries is the elimination of T2DM; 84% of patients with T2DM experienced complete remission of diabetes after bariatric surgery. At RYGB Mechanisms (RYGBM) Animal Lab, we seek to understand the mechanisms of weight loss and T2DM remission in bariatric surgery through the development of an animal model. The animal models selected for this research are Ossabaw pigs. These pigs, raised on high fat diets, are obese and naturally develop insulin intolerance similar to T2DM in humans. There are two leading hypotheses for the causal relationship of bariatric surgery and the elimination of T2DM: the Lower Intestinal Hypothesis and the Upper Intestinal Hypothesis. To test these hypotheses, we perform five variations of bariatric surgery on the animal models: Gastrojejunostomy (GJ), GJ with Duodenal Exclusion, Roux-En-Y Gastric Bypass (RYGB), RYGB with Vagotomy, and Sham operation. We perform these variations of surgeries in attempts to identify which procedures will decrease T2DM while being the least invasive and most recoverable. Pre-operative and post-operative weights indicate if the surgery worked as a weight loss mechanism, while the pre-operative and post-operative changes in insulin sensitivity and gut peptide hormones--ghrelin, PYY, GLP1--indicate if the procedure succeeded as a T2DM loss mechanism. Since the start of the study, survival rates have improved, and RYGBM compilation of data has increased, thus enhancing conclusive findings, though data to support either hypothesis is still in progress.

Recent studies demonstrate that certain reproductive factors such as parity (defined as the number of completed pregnancies) are disparately associated with various pathologic subtypes of breast cancer. One factor, fetal microchimerism - the term for persistent child-origin cells in a woman’s circulation and tissues long after delivery - is generally deficient in women with unselected breast cancer and suggests a protective role. Here, we are testing the hypothesis that high-grade triple negative breast cancer (positive association with parity) will be distinctly associated with fetal microchimerism compared to low-grade luminal breast cancer (inversely associated with parity). Buffy coat specimens (n=450) were obtained from breast cancer patients and control participants aged 18-44 from the REACH project (Fred Hutchinson Cancer Research Center, PI Christopher Li). Using quantitative polymerase chain reaction, DNA extracts from these specimens were tested for the presence and concentration of Y chromosome sequence DYS14, a marker of male fetal microchimersim. In an interim analysis from the first 87 specimens, 33% (9/27) of women who developed luminal breast cancer tested positively for the presence of DYS14, as did 33% (10/30) of healthy controls. 47% (14/30) of women who developed triple negative breast cancer tested positively for the presence of DYS14. Preliminary results demonstrate a higher prevalence of fetal microchimerism in patients with triple negative breast cancer compared to control or luminal participants consistent with the original hypothesis. Additional samples will be tested. Fetal microchimerism testing may represent a novel non-invasive method to identify women with poor prognosis breast cancers.

We present the results of a linear regression of exam scores for students in general chemistry versus performance in two online learning systems: WebAssign, which presents problems in a traditional manner, and ALEKS (Assessment and LEarning in Knowledge Spaces), which is an adaptive, tutorial-based system. Comparisons are made for students taking the same general chemistry course, taught by the same instructor, in the same quarter in subsequent years, where WebAssign was used one year, and ALEKS the next. We find that in WebAssign, a 1 point difference in homework score corresponded, on average, to a 0.46 point difference in final exam score (95% C I: 0.36, 0.56). In ALEKS, a 1 point difference in homework score corresponded, on average, to a 1.06 point difference in final exam score (95% C I: 0.98, 1.15). Controlling for gender and SAT scores did not change the overall conclusion. For a student, ALEKS homework scores provide a better predictor of exam performance, allowing them to focus their efforts where they are needed. For an educator, earlier signs of knowledge gaps arise in the lower homework scores, which can be addressed well before an assessment. 

The purpose of this study was to describe and compare sleep quality and emotional behavioral functioning in children with and without Juvenile Idiopathic Arthritis (JIA). JIA is an inflammatory disease with unknown cause and one of the most common rheumatologic chronic conditions in children. Common disease symptoms include joint pain, inflammation, fatigue, and sleep disturbances. In a previous study, we found poor sleep quality (difficulty falling asleep, daytime sleepiness, sleep disordered breathing (SDB)) associated with JIA. Forty-six children (6-to-11 years) participated in the study, 23 were diagnosed with JIA and 23 were age-matched controls. Parents completed the Children’s Sleep Habits Questionnaire (CSHQ), a 45-item measure of their child’s sleep habits and quality over the previous week. The CSHQ yields a total sleep disturbance score (higher scores indicative of more disturbed sleep) and eight subscales: bedtime resistance, sleep-onset delay, sleep duration, sleep anxiety, night awakenings, parasomnias, SDB, and daytime sleepiness. Parents completed the Child Behavioral Checklist (CBCL), a 113-item retrospective report of their child’s behavior. CBCL yields scores for internalizing problem (i.e. somatic complaints) and externalizing problem (i.e. hyperactivity). Scores were summed and converted to age and gender adjusted T-scores (M = 50, SD = 10), higher scores suggesting greater behavior problems. Descriptive analyses were calculated on demographics, clinical characteristics, sleep disturbances, and behavior. T-test and X² procedures were used to examine study group differences (JIA and controls). We compared the proportion of children above the CSHQ clinical cutoff score (>41) to those below (<41) for overall sleep disturbance. Bivariate correlations and linear regressions were used to examine relations among age, medication, sleep variables, and CBCL behavior problems. Behavioral problems may stem from poor sleep quality in children with JIA. Findings from this study may heighten clinician awareness to assess sleep in children with JIA.

Quantum dots are nanoparticles with several applications including cellular imaging and drug delivery. Because of quantum confinement, their ability to fluoresce under ultraviolet light is size dependent. However, quantum confinement not only plays a role in emissive properties, but is also a factor in the absorbance spectrum of quantum dots. As a result, there is not a simple relationship between the concentration of quantum dots and optical density. It is the goal of this project to craft a new approach in accurately measuring the concentration of quantum dots in solution. We have determined a correction factor relating response units as measured by surface plasmon resonance (SPR) to values of refractive index. With this, we further use SPR in conjunction with analytical ultracentrifugation (AUC) to determine the concentration dependence of the refractive index, dn/dc, for TOPO-PMAT quantum dots. The measurement of dn/dc for the quantum dots allows us to use an interferometric determination of their total fringe increment observed by AUC synthetic boundary to measure the concentration of quantum dots in solution. We have estimated a dn/dc for 565 TOPO-PMAT CdSe quantum dots. In addition, we have used AUC to show that total fringe increments scale with concentration. Furthermore, we have estimated mass extinction coefficients for all wavelengths at which absorbance is observed for these quantum dost. This allows for UV-vis determination of concentration. Our results indicate that our strategy holds promise for providing a new method for measuring the concentration of quantum dots in solution.

 Spinal cord injury accounts for 29% of paralysis patients in the US, and the recovery of hand and arm function is the highest treatment priority in individuals with tetraplegia.  Induced pluripotent stem (iPS) cells can be directed toward specific cell fates, such as functioning neurons or astrocytes, making them an exciting new tool in regenerative medicine for those that suffer from spinal cord injury paralysis.  We investigated the effect of neurally-fated iPS cells on recovery of forelimb function after a cervical spinal cord contusion injury in a rat model.  Neurally-fated iPS cells were injected above and below the lesion in an attempt to replace cells lost to injury.  We transplanted cells four weeks after injury to mimic a chronic spinal cord injury model.  Forelimb function was quantified before and after the injury for 15 weeks using three forelimb behavior tasks.  Tasks included a forelimb reaching task (FRT), a cylinder-rearing task, and a cereal-eating task (IBB).  Results from these tasks will be analyzed to quantify any improvement in the experimental animals that received iPS cells versus the controls.  If treatment provides a significant difference in recovery rate, then the introduction of iPS cells in damaged spinal cords could provide a possible regenerative treatment for paralysis patients.

Gamma activity refers to high-frequency neural activity (30-100 Hz) typically measured by electroencephalography (EEG). Evoked gamma activity is time-locked to stimulus onset which often reflects processing of stimulus properties such as the presence of edges and local luminance contrast. However, induced gamma activity is not time-locked to stimulus onset and evidence suggests it may reflect activation of the perceptual representation of the object presented. Here, we investigated whether induced gamma was altered by in-depth rotations of objects to elucidate the role of such activity in object perception. If induced gamma reflects a high-level object representation then it should not differ with rotation. However, if induced gamma reflects a representation of object features then induced should be modulated by in-depth rotation. We presented pairs of stimuli: an initial adapting stimulus followed by a test stimulus. Stimulus pairs were images of the same or different cars with the test stimulus rotated 0°, 90°, or 180° from the initial adapting stimulus. EEG gamma activity was measured using a 32-channel active electrode Biosemi, Active Two system. We analyzed gamma activity using Morlet wavelet decomposition that allows the measurement of induced gamma activity. The results revealed that induced gamma activity was related to the amount of rotation, with the lowest induced gamma activity during trials with no rotation and highest for trials with 180° rotation. This pattern suggests that induced gamma activity is related to processing of object features below the representation of the whole object. Moreover, the results suggest the cortical generator for the gamma oscillations may be lateral occipital object-selective regions that have similar sensitivity to in-depth rotation across similar paradigms and stimuli using fMRI. The results suggest a link between EEG induced gamma activity and object-selective visual cortex, and provide important information on the temporal dynamics of object-processing brain regions.

Horseradish peroxidase (HRP) is part of a family of heme-containing metalloenzymes that reduce the O-O bond of peroxides while oxidizing a wide variety of substrates; in the case of HRP, hydrogen peroxide is reduced to water. In the presence of an oxidant and the absence of a substrate to oxidize, hydrogen peroxide coordinates to the iron cofactor of HRP, forming a ferric-hydroperoxide intermediate known as Compound 0, and subsequent O-O bond cleavage releases water and a very oxidizing ferryl-oxo porphyrin radical cation intermediate, known as Compound I. The goal of this project is to probe the potential reversibility of Compound I formation, a step traditionally thought to be irreversible. A key experiment will react HRP with unlabeled H₂O₂ in ¹⁸O-labeled water, and determine whether mixed-labeled H₂O₂ is formed. The presence of mixed-labeled H₂O₂ indicates that there is reversibility of the O-O bond cleavage that forms Compound I and its existence will be determined by addition of a water soluble phosphine that is too bulky to fit into the active site of HRP. In this case, the phosphine cannot be oxidized by Compound I, but can readily react with free hydrogen peroxide in solution to give the corresponding phosphine oxide. ³¹P-NMR will be used to determine whether the phosphine oxide is labeled because the NMR shifts of ¹⁶OPR3 and ¹⁸OPR3 are distinct. If this reaction proves to be reversible, this knowledge could then be used to describe other enzymes that catalyze oxidations with similar intermediates. This would not only be important to enzymatic systems, but possibly catalytic chemical processes.

The goals of this project are to produce transgenic maize and wheat tissue expressing a gfp marker in chloroplasts and to regenerate homoplasmic plants from the transformed tissue. There are several advantages of transgenic chloroplasts, including multiple copies and high expression of the transgene without gene silencing. In addition, plastid DNA (ptDNA) is maternally inherited so that the transgene will not be present in pollen, thus providing containment for the transgene. We used circular and linear vectors that contain ptDNA sequences corresponding to the ends of the plastid chromosome and the gfp transgene. We used particle bombardment to deliver the vector to plastids of maize and wheat. Transgene expression was assessed by fluorescence microscopy and imaging of GFP in plastids. In addition, both standard PCR and quantitative PCR were performed using DNA extracted from the tissue and primers for the gfp transgene. Although we achieved successful plastid transformation for both maize and wheat, the tissues were heteroplasmic, containing both wild type and transgenic ptDNA. We found that a linear vector gave higher transformation efficiency than a circular vector. The next step is to develop procedures for generation of homoplasmic maize and wheat plants.

Author: Amanda Tanadinata, Karol Bomsztyk, Daniel Mar. Background: Apart from DNA mutations, alterations in chromatin structure, also referred to as epigenetic changes, have been shown to be among principal causes of tumor genesis that leads to cancer. However, unlike mutations, the usage of these chromatin alterations in gene expression is reversible. This feature, in fact, is what has nurtured the idea of creating an epigenetic-based treatment for cancer. One such treatment uses the compound 5-aza-2’-deoxycytidine (5-aza-dC), a DNA methyltrasferase inhibitor. DNA methylation can be crucial for transcriptional repression and activation, by altering recruitment of enzymes and transcription factors to chromatin . The goal of this study was to profile a set of pathways through the analysis of 5-aza-dC -mediated changes in DNA methylation, mRNA expression , RNA Polymerase II, histone marks, and enzymes. Method: Colorectal cancer cell line (HCT116) cells treated with or without 5-aza-dC were probed with different antibodies to see chromatin density differences at specific sites of the genome by Matrix Chromatin Immunoprecipitation (Matrix ChIP). Methylated DNA Immunoprecipitation (MeDIP), was used to assay DNA methylation sites at selective gene sites. The results were then quantified with real time PCR. In this study, SPARC promoter, H19, and UBE2b were examined specifically. Results: Treatment with 5-aza-dC increased SPARC mRNA levels, and reduced DNA methylation This was associated with changes in RNA polymerase II levels ,histone acetylation and methylation, and chromatin modifying enzymes at the SPARC but not the control H19 and UBE2b genes. Conclusion: These results imply that 5-aza-dC induced de-methylation of selective genomic sites alters enzyme binding and chromatin structure which then causes changes in rates of transcription.

GPSM2 is required for orienting the mitotic spindle during asymmetric cell division during development. The hearing loss and brain malformations in Chudley-McCullough Syndrome (CMS) suggest that this autosomal recessive condition is due to mutations in GPSM2. The clinical phenotype of CMS includes partial agenesis of the corpus callosum, arachnoid cyst, cerebellar dysplasia, gray matter heterotopia, and frontal polymicrogyria. This combination of brain malformations is highly distinctive and not seen in any other genetic syndrome. Homozygosity mapping and sequencing of affected individuals revealed eight molecular variations in the G protein-signaling modulator 2 gene, GPSM2, that underlie CMS. To identify additional GPSM2 mutations, I utilized a molecular inversion probe (MIP) method enabling minimal DNA input, high output performance, and low-cost sequencing for less than $1 per gene per sample. Each MIP is a common 30 bp linker flanked by an extension arm and a ligation arm giving a total MIP length always equal to 70 bp. The unique arms of each MIP target a specific 112 bp genomic region for a gap-fill and circularization. Post-capture PCR amplification with primers corresponding to the common linker was used to adjoin sample-specific barcodes. Samples were then PCR amplified, pooled together, and ran on Illumina MiSeq. Currently, I am sequencing six confirmed CMS patients in addition to eight samples we obtained from patients presenting only with agenesis of the corpus collosum and hearing loss. These results are pending. By sequencing patients who present with classic CMS features, as well as those who present with limited CMS features, I hope to identify a genotype-phenotype correlation in Chudley-McCullough patients.

MicroRNAs are a class of post-transcriptional regulators that silence messenger RNAs. MicroRNAs are interesting molecules to study because they are stable at room temperature and are long lived unlike messenger RNA. These characteristics make them useful molecular biomarkers to monitor disease and exposure status. For example, urine microRNAs have been used clinically as potential diagnostic markers of bladder cancer. However, their utility in non-clinical settings has yet to be elucidated. The goal of this study is to compare total RNA isolated from fresh and archived urine field samples to investigate their quality and quantity over time. RNA will be isolated from both whole urine and urine sediments and will be analyzed using multiple techniques. To date we have optimized the methodology required to isolate total RNA from the whole urine sample and the sediment. In a preliminary study, we isolated total RNA from samples under various conditions (fresh vs. field; whole urine vs. sediment) and compared them using the Nanodrop and Bioanalyzer. Our results demonstrate that RNA can be isolated from both fractions under both conditions. The molecular profiling of the RNA suggests that the urine samples contain only small RNAs because they lack ribosomal RNA peaks. Interestingly, the profiles look similar for all urine samples examined. In light of this, we performed an additional microarray experiment to characterize and identify the small RNAs present in the whole urine and sediment samples. These results are currently in progress. Using the information obtained from the preliminary microarray study, we will select the 3 most abundant microRNAs present in the urine for further analysis using RT PCR techniques to determine whether archival duration has an effect on their stability. The results of this experiment will provide valuable insight on the utility of archived field samples for the future development of urine biomarkers.

Gaze-following allows infants to identify social and cultural information. Ongoing research examines what experiences help infants learn about the gaze of others. This research finds that particular experiences help infants learn about visual perception and affects their gaze-following. 18-month-olds watched an adult’s goal-directed actions while wearing a trick blindfold that looked opaque but was see-through. When infants observed the blindfolded adult successfully perform visually guided behaviors (grabbing a toy), they later gaze-followed as though the adult could see through the blindfold. However, when they watched the blindfolded adult’s unsuccessful behaviors (missing a toy), they treated her as though the blindfold blocked her view. These results show that infants are able to use their observation of others’ motor behaviors to learn about their perceptions. The present research examines whether infants’ gaze patterns during the observational experience affect infant learning about perception. Infants’ looking will be coded during the observational experience to identify the number of gaze shifts from the adult’s action pattern (grabbing or missing) to the adult’s face, called action-to-face looking (A-F looking). We hypothesize that infants who frequently perform A-F looking will be more likely to learn whether or not the blindfolded adult can see through the occluder. We will analyze gaze-following scores as a function of high versus low frequency A-F lookers (median split) and observational experience group. We expect that a 2 (high vs. low A-F looks) x 2 (grab vs. miss) ANOVA will reveal a significant interaction. We expect infants who more frequently shift their gaze during observation will be more likely to learn about perceptual status and thus gaze-follow more frequently. Infants’ gaze-shift patterns have implications for how infants’ attention and looking patterns support their nonverbal learning and development.

 Personalized medicine aims to improve patient outcome by tailoring cancer treatment to individuals based on the specific molecular makeup of tumors. This approach is showing particular promise in the treatment of gliomas, the most common type of primary brain cancer. The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) removes O⁶-alkylguanine adducts from DNA produced by the therapeutic methylating agent temozolomide (TMZ). In glioblastoma multiforme (GBM) tumors, low levels or lack of MGMT activity is correlated with longer progression free survival (PFS) following TMZ treatment and other alkylating chemotherapy. However, in oligodendorgiomas and oligoastrocytomas the effect of MGMT activity on tumor resistance has not been established. Better clinical outcome is associated with 1p and 19q loss of heterozygosity (LOH), which occurs in an appreciable fraction of oligodendrogliomas. Other studies associate these deletions with CpG methylation of the MGMT promoter, an epigenetic indicator of low MGMT activity. However, there have been no studies linking MGMT activity to chromosomal deletion, and 1p/19q LOH is associated with better clinical outcome regardless of therapy, suggesting that other mechanisms may also be responsible for prolonged survival. To clarify the unknown role of MGMT in these tumors, we tested the hypothesis that 1p/19q LOH accompanies low MGMT activity by: [1] assaying MGMT activity by measuring the extent of transfer of tritium-labeled methyl groups at the O6 position of guanine to MGMT in substrate DNA extracts of deleted and non-deleted tumors; [2] assessing promoter CpG methylation status using methylation specific PCR; and [3] examining the correlation of MGMT activity to TMZ treatment response using a Cox regression analysis. This approach should help to clarify the relationship of MGMT activity and 1p/19q LOH.

Protein kinases are enzymes that take phosphate groups from adenosine triphosphate (ATP) molecules, the energy in your body, and add them to other specific molecules inside the cell. This transfer of the phosphate group can activate or deactivate the target molecule, thus regulating cellular activities such as growth, replication, and respiration. The kinases studied here are hemopoietic cell kinases (Hck kinases) under the class of Scr kinases. Hck kinases are present in bone marrow and are often associated with myeloid leukemias and myeloproliferative disorders. Studies have shown that in the active conformation, the Hck kinase detaches two of its domains, SH2 and SH3, resulting in a more extended conformation. Using a Waters Synapt G2 HDMS, ion mobility mass spectrometry (IM-MS) techniques were used to probe the size of inactive and active Hck kinases by measuring drift times of their gas-phase ions traveling through a neutral background gas under the presence of a weak electric field. Drift times were then converted into collision cross section (CCS) values, which depend on the size and shape of the ion of interest. Eight different small molecules (< 500 Da) were used to inhibit Hck kinases, and changes in conformation were investigated using IM-MS. Preliminary data shows no significant changes in global conformation or size, conflicting with the previous studies. To investigate more subtle changes in conformation, collision induced unfolding (CIU) methods are used. By selecting an ion of interest using a quadrupole mass filter, protein unfolding is monitored using ion mobility as a function of ion activation prior to ion mobility analysis. This process allows an investigation of the subtle conformational changes in the Hck kinase upon the binding of small inhibitor molecules.

Atherosclerotic cardiovascular disease is the leading cause of death in North America.  Hepatic lipase (HL) a liver enzyme that hydrolyzes lipoprotein triglyceride, plays a critical role in lipid metabolism and may influence the development of atherosclerosis.  Hepatic lipase may serve dual roles in the development of atherosclerosis: 1) HL may be pro-atherogenic by converting low-density lipoprotein (LDL) to small-dense LDL, causing build up of cholesterol in arteries. 2) HL may be anti-atherogenic by increasing the cholesterol-poor form of high-density lipoprotein (HDL) that removes cholesterol from the arteries, decreasing build up of cholesterol in arteries.  Previous studies have reported that elevated levels of human hepatic lipase (hHL) reduce the development of atherosclerosis.  However, those studies were done in transgenic mouse models with supra-physiologic levels of hHL and may not accurately reflect the role of physiologic levels of HL on atherosclerosis.  Therefore, we created a physiologic model of hHL expression to analyze the role of hHL in the development of atherosclerosis.  This “humanized” mouse model expresses hHL at physiologic levels in the liver.  We hypothesize that physiologic levels of HL will reduce the development of atherosclerosis compared to mice lacking HL.  To test this hypothesis we bred the transgenic hHL mice onto a low-density lipoprotein receptor knock out background (which is an established model for atherosclerosis).  Littermates with and without the hHL transgene were fed a high-fat, cholesterol enriched (“Western”) diet to accelerate atherosclerosis development, after which the aortas were harvested, dissected longitudinally, and pinned to expose the inner aortic walls for visualization.  Cholesterol buildup was stained and the ratios of atherosclerotic accumulation to total aortic surface area were quantitatively measured via Adobe Photoshop.  Results from this study will clarify the contribution of hHL to atherosclerosis development.  Ultimately, our results will guide the development of new treatments to prevent and treat atherosclerosis.

Glioblastomas (GBMs) are grade four astrocytomas, composed of heterogeneous cells with unique molecular features. Using brain tumor and tumor stem cell lines, we are developing ex vivo methods to use on-chip, single cell characterization and mass spectrometry to better understand brain tumor heterogeneity in order to personalize drug therapy and optimize treatment of GBMs. We first performed an on-chip, single cell culture, using a biocompatible poly dimethyl-siloxane (PDMS) microchip that contains 50-100 nm subnanoliter wells. A diluted solution of cultured cells was loaded onto the microchip while the chip was submerged in DMEM, and the cells settled into the wells by gravity. The PDMS microchip was then subjected to hybridization conditions, to simulate the technique of assaying cytokines. Cell viability was monitored using a live/dead fluorescence assay. To pursue the goal of analyzing protein arrays using single cell mass spectrometry, we loaded a PDMS microchip; first with bovine serum albumin digest, then with hemoglobin, as controls to obtain a MALDI (Matrix-Assisted Laser Desorption Ionization) spectrum. We found that a solution of approximately 7.14x103 cells/mL ensured that one cell was delivered per well. The neuroblastoma cells did not exhibit typical dendritic morphology within the microchip. They remained round due to the non-adherent properties of PDMS. When the loaded microchip remained submerged in DMEM and incubated, the cells grew to confluence and remained viable up to seven days. Under hybridization conditions, the cells remained viable up to five days. The MALDI controls were assessed, but did not produce a spectrum. The soft and thick PDMS surface may inhibit the MALDI laser’s accuracy to pinpoint proteins. Also, PDMS is not electrically conductive and impedes analyte ionization. Therefore, the project has been continuing to optimize the surface for analysis. This work is still ongoing with promising results.

The U.S. goal of producing 16 billion gallons of cellulosic ethanol by 2022 will be possible only by carefully making use of all available feedstock sources. Forest residues, including material remaining after logging or thinning, are an inexpensive and widely available feedstock, with estimated potentials between 30 to 120 million dry tons a year on the source. However, their heterogeneous nature and relatively low cellulose content renders them a challenge to current pretreatment and bioconversion methods. Forest residues typically consist of branches, needles/leaves, bark and wood in varying proportions and particle sizes, so some degree of homogenization prior to pretreatment is essential for consistent results. Our objective in this research was to develop methods to utilize this material for production of ethanol via SO₂ catalyzed steam explosion pretreatment, enzymatic hydrolysis and fermentation. To achieve this, we first homogenized biomass consisting of Douglas-fir and Hemlock residues. We pretreated this material at temperatures ranging from 195°C to 205°C, with residence times from 5 to 20 minutes to fractionate the cellulose and hemicellulose from lignin. We subsequently quantified the sugars remaining after pretreatment, in both the liquid and solid phases using gravimetric and HPLC analysis to determine which pretreatment resulted in the best sugar recovery from the original raw biomass. This data was then used to determine which pretreated solids were the best candidates for enzymatic hydrolysis, which we then conducted to convert all of the insoluble cellulose to soluble, fermentable glucose. From our data we compared sugar recovery, enzymatic hydrolysability and other factors to determine the best pretreatment conditions to use to increase the overall production of ethanol from heterogeneous forest residues.

Deficiencies of monoamine neurotransmitters including serotonin, norepinephrine, and/or dopamine have been associated with many CNS diseases including depression, schizophrenia, ADHD, autism, Parkinson’s disease, and Tourette syndrome. The intensity and duration of monoamine signaling is mainly determined by the clearance of the released transmitters through uptake1 and uptake2 transporters. The recently discovered uptake2 transporters including the plasma membrane monoamine transporter (PMAT) and organic cation tansporter3 (OCT3) have been less studied partly due to the lack of specific inhibitors. Recently, inhibition of PMAT and/or OCT3 has been proposed as a strategy for the development of antidepressant drugs with improved efficacy and faster onset of actions. The goal of this project is to characterize specific chemical inhibitors for PMAT and OCT3. Previously, 10-40 small molecule inhibitors for PMAT and OCT3 were identified from high throughput screening. We will choose two to three inhibitors for PMAT and OCT3 obtained from the initial screen and further analyze their potency, specificity and mechanism of inhibition towards PMAT and OCT3. We first will determine their inhibition potencies (IC50) towards PMAT and OCT3 using radiotracer uptake assays in cell lines stably expressing these transporters. Preliminary data showed that two compounds inhibited OCT3 with IC50 values of 1.97 ± 0.766 and 2.86 ± 0.532 µM. One compound inhibited PMAT with an IC50 value of 2.15 ± 0.537 µM. Further studies will be performed to determine the specificity and mechanism of inhibition for these inhibitors. The proposed studies may lead to novel, highly specific and potent PMAT and OCT3 inhibitors that could be further evaluated and developed in preclinical models for the treatment of depression and other monoamine-related diseases.

Vasodilator-stimulated phosphoprotein (VASP) is a focal adhesion protein that regulates actin filament polymerization, a process necessary for cell motility. Through cell locomotion, endothelial cells migrate to designated locations to form new capillaries (angiogenesis). Previous studies have shown that VASP regulates endothelial cell migration in human umbilical veins and during human placental development. Given VASP's role in endothelial cell movement, we hypothesized that VASP is required for angiogenesis. We are investigating VASP’s role in angiogenesis through both in vitro and in vivo assays in VASP null and wildtype mice. In addition, we measured blood flow recovery in VASP null and wildtype mice after hindlimb ischemia by laser Doppler perfusion imaging. The in vitro assays compare microvessel outgrowth from either tibialis anterior muscle or descending thoracic aorta in response to the vascular endothelial growth factor (VEGF). The in vivo assays compare capillary ingrowth into matrigel plugs containing (VEGF) and capillary angiogenesis stimulated by hindlimb ischemia generated by unilateral femoral artery ligation. Capillaries are identified through immunohistochemistry. Significant difference for the hindlimb ischemia blood flow recovery rate and capillary counts was evaluated through the T-test and the Analysis of Variance test, respectively. So far, we have identified no significant difference between VASP null and wildtype mice in blood flow recovery by laser Doppler perfusion imaging; however, this process is more dependent on arteriogenesis than capillary angiogenesis. Identification of potential regulators of angiogenesis would further our understanding of processes such as wound healing, growth and development, and recovery from ischemia, where capillary formation plays a key role.

Defects in joint cartilage are a common pathology found in the knee. Operative management aims at alleviating symptoms and decreasing the potential of progressing to osteoarthritis. One method of repair involves the insertion of a plug of analogous cartilage tissue taken from a cadaver, called an osteochondral allograft, providing function restoration of the affected joint surface. Traditionally "fresh" grafts were used within 24-48 hours and resulted in good clinical outcomes. Currently concerns with possible disease transmission have resulted in longer delays from graft harvest until surgical implementation. Prolonged graft storage however leads to death of chondrocytes, the cells that are responsible for producing cartilage. Trehalose is a linked form of two glucoses, and is present in bacteria, plants, and invertebrate animals. These organisms drastically increase cellular concentrations of trehalose during times of cellular stress such as severe dehydration, heating, exposure to cold, or oxidative stress. Following these observations, studies were conducted that have shown trehalose to be a useful cyroprotectant in the cold storage of cells and various tissues. The purpose of this study was to determine if supplementing standard storage medium with trehalose would preserve chondrocyte cell viability after prolonged graft storage. Osteochonral plugs were taken from rabbit joints and stored in two types of medium, one supplemented with trehalose and one without. At various time points after storage (0, 14, and 24 days) plugs were taken and analyzed for markings of apoptosis (self programmed cell death) and articular cartilage integrity. Trehalose medium preserved osteochondral grafts better then the control group after prolonged cell storage. Additional research is needed to determine success of trehalose stored grafts in the clinical setting, and in trehalose's role in the storage of other tissues.

 Double electron electron resonance (DEER) and electron nuclear double resonance (ENDOR) are two variants of electron paramagnetic resonance (EPR) spectroscopy that are useful in investigating the structures of chemical species with unpaired electrons. DEER is used to measure the distances between unpaired electrons, and ENDOR is used to measure the effect that nearby nuclear spins have on unpaired electrons. EPR experiments are conducted using microwave frequencies, with the X band (~10 GHz) and Q band (~34 GHz) being commonly used. Pulsed variants of DEER require the use of a resonator with a wide resonant bandwidth in order to accomodate two microwave pulses of different frequencies and to minimize measurement deadtime, while ENDOR requires the device to accomodate a wire coil through which radio frequency signals are sent to induce nuclear transitions, posing a technical challenge. Based on previous computational work, an tunable cylindrical resonator with a resonant frequency of ~34 GHz, has been built. The goal of this work is to evaluate the efficacy of this resonator design in pulsed EPR experiments. In addition, a radio frequency coil for use with ENDOR will be computationally designed and optimized using CST Microwave Studio, an electromagnetics modeling software package. 

Auxin is one of the most important plant hormones that regulates almost every developmental process during plant life cycle. Auxin response factors (ARFs), are DNA-binding transcription factors that regulate auxin-mediated transcription by activation or repression. In Arabidopsis, ARFs are encoded by a family of 28 genes, and each member is thought to play a central role in various auxin-mediated developmental processes. ARF2 has shown to be a transcriptional repressor. Nemhauser lab uses Arabidopsis seedling hypocotyl, the embryonic stem, as a model to study growth. Loss of ARF2 alters hypocotyl growth in Arabidopsis seedlings. arf2-9 is a single point mutation in DNA-binding domain that results in loss of ARF2 function. I have analyzed the hypocotyl growth in arf2-9 mutant in presence and absence of exogenously applied auxin. The results indicate that arf2-9 exhibits longer hypocotyl in comparison with wild type seedlings not only in control untreated condition but also in response to auxin application. This data suggests that ARF2 is a negative regulator of hypocotyl elongation. To investigate the mechanisms how ARF2 regulates hypocotyl growth and elongation, I have crossed arf2-9 mutant with a reporter line P_PKS4::GUS that marks actively growing hypocotyl region. I have used dCAP genotyping method for arf2-9 single point mutation and Basta resistance selection for P_PKS4::GUS transgene to isolate double homozygous arf2-9 X P_PKS4::GUS plants. My hypothesis is that long hypocotyl in arf2-9 is a consequence of indeterminate growth. Given that, my prediction is that GUS staining pattern of P_PKS4::GUS will be different in arf2-9 genetic background compared to the original line. This includes possibility of having GUS staining in different regions of hypocotyl or extended staining pattern over the time when in wild type background P_PKS4::GUS marker is no longer active. Those differences will help us understand the function of ARF2 better.

Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease with an unknown cause. Children experience joint inflammation, pain, fatigue and sleep disturbances. Sleep disturbances such as sleep disordered breathing and insufficient amounts of sleep may contribute to lower quality of life and poor health outcomes in JIA. In a recent study from our laboratory 40% of a sample of children with JIA showed signs of sleep disordered breathing. The aim of this study is to describe and compare sleep disturbances in JIA and age, sex matched control children, and its relation to quality of life (QoL). Forty-six children (6-to-11 years) participated in the study. Of the 46 children, 23 were diagnosed with JIA and 23 were age-matched typically developing children. Parents completed the Children’s Sleep Habits Questionnaires (CSHQ), a 45-item measure of their child’s sleep habits and quality over the previous week. Children underwent 1 night of polysomnography in the UW School of Nursing sleep laboratory, and completed the Pediatric Quality of Life (PedsQL) questionnaire, a 23-item report that assesses health-related QoL (day-to-day activities) and disease specific symptoms. The specific domains include physical, emotional, social, and school function during the month prior to completion of the questionnaire. The PedsQL 4.0 yields a total score for overall health-related QoL, physical and psychosocial function and 4 subscales: physical, emotional, social, and school function. Data will be analyzed using SPSS for Windows version 18.0 (SPSS Inc, Chicago, Ill). Data analyses will be blocked into conceptual categories and then analyzed for group differences. Findings from this study may provide new knowledge about sleep disturbances and QoL in JIA children. Missed days from school and lower QoL in JIA often are attributed to pain and fatigue, but untreated sleep disturbances and sleep disorders may be an important etiology that is unrecognized by both clinicians and parents.

Aging in the budding yeast Saccharomyces cerevisiae can be measured by its replicative life span, which is defined by the finite number of mitotic divisions of mother cells. The Kaeberlein lab measures the replicative life span of S. cerevisiae through the microdissection of dividing mother cells. This laborious process separates daughter cells from their mothers, which quantifies the replicative life span. We are assessing how various environmental factors affect the replicative life span of the cells. Our research project involves paraquat, a commonly used pesticide that produces reactive oxygen species within cells. This substance is normally considered to be toxic, and as expected, we have observed that the replicative lifespan of yeast is decreased in the presence of high concentrations of paraquat. However, we have also found that low concentrations of paraquat increased the replicative life span of the yeast. This observation is an example of hormesis- a phenomenon where beneficial effects are obtained at low doses of an agent that is otherwise toxic when given at higher doses. These findings are significant because a similar effect has been published with the nematode worm, Caenorhabditis elegans. C. elegans and S. cerevisiae are evolutionarily separated by approximately one billion years, suggesting that there is a conserved mechanism of life span extension influenced by paraquat. We are currently characterizing the effect of paraquat and other environmental conditions that will help us to find conserved pathways that modulate longevity.

There are currently an estimated 350 million people worldwide chronically infected with the Hepatitis B virus (HBV). These individuals, over the course of their life, are at an increased risk of developing liver cancer. While antiviral drugs can suppress HBV replication, latent HBV viruses, lying dormant but still potent in the hosts’ cells, so far have proven difficult to eradicate. Homing endonucleases (HE), enzymes that recognize long DNA sequences and induce DNA double strand breaks, could be a means to target and inactivate latent HBV. In particular, we aim to modify the recognition site of the WT I-GzeII homing endonuclease specific to the HBV genome and introduce DNA double-strand breaks. By exploiting the error prone nature of non-homologous end joining, the cellular process to repair double-strand breaks, these modified HEs will have a mutagenic effect on target sequences. Repeated HE activity will eventually cripple the replicative ability of latent viruses. To selectively modify the structure of homing endonucleases, a variant library of the I-GzeII gene is generated with randomized bases corresponding to the amino acid residues that interact with the DNA substrate. Variants that encoded for active HEs with the appropriate recognition site are selected for by In Vitro Compartmentalization (IVC). The selected variants will then be run through bacterial selection and in vitro cleavage assays to further select for structurally stable variants that function in vitro. The final step is to test the HE product on an in vitro HBV cell line. Through our research, we hope to prove that the target of I-GzeII recognition sites can be effectively altered and that latent viruses can be targeted and inactivated through mutagenesis by homing endonucleases. Hopefully, our research will be the first step to a more permanent cure for HBV infections and help to reduce incidences of liver cancer.

Pollination syndromes refer to suites of floral traits that influence biotic or abiotic vectors transporting pollen from one flower to another. These traits include flower color, position, shape, size, scent and reward. Scent is a major component of pollination, as the volatile organic compounds (VOCs) that compose “scent” often attract pollinators or deter predators. The genus Thalictrum in the Ranunculaceae (buttercup family) possesses both insect- and wind-pollinated species, enabling comparisons of the role of scent in floral diversification among recently diverged taxa with distinct pollination syndromes. To characterize floral scent, we identified and compared the bouquet of VOCs emitted by insect- and wind-pollination species, as well as by floral vs. vegetative tissue. Focusing on twelve species representative of the two pollination syndromes and spanning the Thalictrum phylogeny, we identified 8 primary compounds of interest. These compounds are known to be chemical communicators for various insects, including bees and syrphid flies, reported to visit Thalictrum. The overall purpose of this project is to determine the differences in scent emissions between species in an evolutionary context, and identify the key volatiles involved in pollination syndrome variation. This will further illuminate our understanding of the biochemical and, ultimately, the genetic basis of these starkly different pollination syndromes. In addition, the variation in distribution of VOCs across species will also be used to test implications of patterns of compound development and reuse during evolution.

In the United States prostate cancer (PCa) is one of the most commonly diagnosed cancers and estimated to affect 239,000 men in 2013. Although few prognostic biomarkers are currently used in the clinical setting, biomarkers that predict the risk of disease aggressiveness leading to prostate specific antigen (PSA) failure after prostatectomy may assist clinicians in selecting adjuvant therapies following prostatectomy. In studies utilizing co-registered pre-operative imaging data with post-operative pathology data, validated prognostic biomarkers may also be highly useful as “gold standards” against which to train imaging methods to assess disease aggressiveness pre-operatively. We previously identified 33 potentially prognostic biomarkers from the analysis of multiple gene expression datasets and current literature. Using tissue microarrays containing PCa tissue from 170 prostatectomy specimens from men with detailed PSA and follow up data, we quantified immunohistochemical (IHC) staining for these markers within selective tissue sub-regions (malignant epithelium) and identified 16 of the 33 biomarkers which were associated with shorter time to PSA failure. While our aim is to use validated biomarkers to pre-operatively train in vivo imaging methods, current imaging capabilities have lower resolution than digital histology and it is unclear whether biomarker expression in total tumor areas (encompassing malignant epithelium, benign epithelium, stroma, and other tissue structures) which are resolvable by imaging methods will also be prognostic. In this study, we have re-evaluated our list of 16 strong candidate biomarkers to determine if IHC expression within total tumor areas are associated with PCa aggressiveness (PSA failure following prostatectomy) using our TMA cohort.

Glioblastoma Multiforme, the most prevalent form of primary brain tumors, is characterized by its aggressive, invasive nature and short patient lifespan post-diagnosis - approximately 12 months. Through gene analysis of RNA sequencing data, we have found two miRNAs, miR-149 and miR-181, that target gene products whose increased expression has been shown to promote invasion and migration of U-87 human glioblastoma cells. miRNAs are short, non-coding RNAs that bind to messenger RNAs (mRNAs) - the body’s method of transferring genetic information, and target them to be degraded or prevent them from being translated into protein. As the miRNAs and the genes are inversely related, treatment of U-87 cells with the miRNAs would cause a decrease in migration, and blocking the miRNAs would cause an increase in migration. To quantify migration, we have employed a scratch test or wound healing assay, in which glioblastoma cells are grown on a dish and the middle of the plate is scratched, leaving space for the cells to migrate. The cells are treated with the miRNAs, and the percent of the gap filled after 24 hours is measured. We expect changes in miRNAs to affect the amount of migration in the gap compared to control - providing evidence that these miRNAs inhibit migration. miRNAs have been investigated as markers for disease, and the presence or absence of this miRNA in a patient’s glioblastoma may have implications as to the severity of their case. Additionally, characterizing these miRNAs adds to the database of knowledge concerning glioblastoma. The more that is known about this disease, the closer scientists are to finding a practical treatment.

Asymmetric cell division creates diverse cell-types that are critical for stem-cell maintenance and tissue patterning in multicellular organisms, including ourselves. Therefore, understanding the mechanism of asymmetric divisions can have a huge impact on regenerative medicine. In plants, asymmetric divisions are important for producing proper patterns of stomata, which are microscopic valves that regulate proper exchange of carbon dioxide and oxygen while simultaneously minimizing water loss. A precursor of stomata, called a meristemoid, divides asymmetrically and renews itself, while its daughter cell, called a stomatal-lineage ground cell (SLGC), shows rapid polar expansion and loses stomatal precursor fate. How can a meristemoid create two daughter cells with contrasting characters? Lines of evidence suggests that rapid expansion of SLGC associated with polar localization of novel proteins is essential for cell fate determination. In this study, I will test this hypothesis within Arabidopsis thaliana by expressing a known regulator of cell polarity, Rho-GTPase2 (ROP2), in stomatal cell lineages. I have generated transgenic plants expressing green fluorescent protein (GFP) tagged Rho-GTPases (ROPs) driven by the stomatal-lineage specific POLAR promoter, which is short for POLAR LOCALIZATION DURING ASYMMETRIC DIVISION AND REDISTRIBUTION (POLAR) in order to view potential ROP localizations using confocal microscopy within stomatal cells. I expressed three forms of ROP2: a wild type form, a constitutively active form (CA ROP2) that always binds GTP and causes isotropic cell expansion, and a dominant negative form (DN ROP2) which always binds GDP and in inhibits cell expansion. Visualization of these ROPs may provide evidence indicating their presence is a requirement for meristemoid differentiation. Because ROPs are present in both plants and animals, this study is an important player in understanding stem cell differentiations, which may resolve the modern challenges we face with respect to regenerative medicine, world hunger, and biomechanical operations.

In adults, acute myeloid leukemia (AML) generally remains difficult-to-treat, and most patients are currently expected to die either of their disease or treatment-related toxicities. The need for novel therapies is thus unquestioned. With increasing availability of specific small molecule inhibitors, one line of research has focused on targeting signaling pathways that are pivotal for the in vivo growth, survival, and propagation of AML cells as a means to improve therapeutic outcomes in this disease. Emerging evidence suggests that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, a key physiologic regulator of transcription, translation, cell cycle progression, differentiation, metabolism, and suppression of apoptosis, could provide a rational drug target in AML. Constitutive activation of AKT signaling is indeed observed in many patients with AML, and several groups have reported inferior relapse-free and overall survival in AML cases with AKT activation. Given these data, we recently conducted an early phase clinical trial testing MK-2206, a highly specific allosteric AKT inhibitor, in patients with relapsed AML. However, MK-2206 has insufficient anti-AML activity at tolerated doses. Therefore, the goal of my project in the preclinical studies that I am currently carrying out is to identify signaling pathways that are critically important for AML cell survival in the presence of MK-2206 with the goal of developing combination therapies that optimize chemosensitization that can be achieved with AKT inhibitors. This is being done by mixing the MK-2206 inhibitor with various conventional chemotherapic drugs and measuring their toxicity on different AML cell lines by use of flow cytometry.

Heart disease is often responsible for morbidity and mortality in the United States. Current treatments aim to slow the progression of the disease by modifying calcium handling in the heart, but do not restore function to healthy levels. Endogenous troponin C (TnC) is the calcium binding subunit of troponin, a complex that regulates contraction in muscle. The amino acid substitution of leucine for glutamine at position 48, L48Q, in cTnC has been shown to increase calcium binding affinity of the troponin complex. We hypothesize that the expression of engineered protein L48Q-cTnC will increase calcium sensitivity of a failing heart and restore function without disrupting calcium handling. The aim of this study is to determine the expression pattern of this engineered protein in vivo in healthy rat hearts after various dosages of adeno-associated viral vectors of serotype 6 (AAV6) containing L48Q-cTnC. In addition, we aim to determine the correlation between vector dosage and changes heart function. We will achieve these goals by administering our AAV6 virus to healthy rats and monitoring whole heart function over the next four weeks. Heart function will be assessed in vivo with non-invasive echocardiography ex vivo on a Neely whole heart apparatus four weeks post-injection. The Neely apparatus allows us to control the environment of the heart while measuring function, which is not possible in vivo. The expression of L48Q-cTnC will be determined by immunohistochemistry and Western blotting. From this data, we will be able to calculate the dosage necessary to achieve maximum expression and increased heart function in our healthy heart model. This initial study it vital to characterizing the expression and efficacy of a potential heart failure therapeutic and will directly impact the design of future studies in disease models.

The immune system has evolved to achieve a fine balance between recognizing and attacking foreign pathogens and preserving unresponsiveness to self-antigens. T cells that are unresponsive to self-antigens are called self-tolerant T cells and a hallmark of such tolerant T cells is their inability to attack cells expressing self-antigens in order to prevent autoimmunity. However, tolerance to these proteins impedes anti-tumor T cell immune responses because many cancer antigens that are targeted in immunotherapy are in fact self-antigens. Thus, a critical challenge in tumor immunology is to develop strategies that break T cell tolerance to tumor antigens without causing unacceptable autoimmune injury. It has been shown that tolerant T cells lack important proteins normally required for T cell effector functions, including two master transcription factors, Tbet and Eomes, which have been found to be critical for T cell function by repressing the expression of inhibitory molecules and mediating the expression of effector molecules. Therefore, the decreased expression of Tbet and Eomes in tolerant T cells might cause their functional unresponsiveness. Using a transgenic tumor mouse model, my research project investigates if enforced over-expression of Tbet and/or Eomes in tolerant tumor-specific T cells can re-program such tolerant T cells and restore effector functions, allowing re-programmed T cells to recognize and attack cancer cells. Tolerant T cells will be isolated from spleens of transgenic mice, retrovirally transduced in vitro to over-express Tbet and Eomes, and subsequently transferred into tumor-bearing mice. The main focus of my research will then be to analyze Tbet-, Eomes-, and/or control-transduced donor T cells for anti-tumor effector function in vivo to understand if and by what mechanism(s) T cells can be re-programmed via the over-expression of the master transcription factors and whether such re-programming could possibly become an effective strategy for the treatment of cancers in humans.

Multiculturalism has been a progressively addressed topic in psychology. While there has been a collection of empirical literatures on bicultural conflict relying on explicit measures, not much has been done to explore the topic of bicultural conflict with implicit measures. Although self-report measures, such as Bicultural Identity Integration Scale-2, are effective in measuring bicultural conflict, there are always limitations when subjects fail to report accurately. This research answers 1) whether it is possible to measure bicultural conflict implicitly, and if so, then 2) how explicit and implicit measures of bicultural conflict correlate. We took advantage of the large size of Chinese American and Chinese international student body at the University of Washington to test our theories. This research uses a balanced identity design that includes three Implicit Association Tests (IATs). The three pairs of concepts in our balanced identity design are “self” and “other,” “China” and “U.S.,” and “family” and “college life.” Our hypothesis was that a more unbalanced identity is associated with a higher level of bicultural conflict. The balanced-ness of an identity is calculated with mathematical algorithms from the three IAT scores. The level of bicultural conflict was scored based on several explicit measures—Bicultural Identity Integration Scale-2, Vancouver Index Acculturation, Multi-Item Identification, and a cultural value ranking survey. This research tests a new method to implicitly measure people’s bicultural conflict by correlating the balanced-ness of one’s identity and the level of one’s bicultural conflict. This research calls for future studies to examine the effect(s) of bicultural conflict by making correlations between people’s implicit bicultural conflict and other aspects of their lives. Since implicit measures are powerful in detecting things people are not aware about themselves, these studies are helpful in developing coping mechanisms to help foreigners and immigrants.

Pre-erythrocytic malaria vaccines may need to target numerous sporozoite and/or liver-stage proteins to be effective. If the protective antigens could be identified, multi-component vaccines could be produced. Using a high-throughput T cell screening, our lab recently identified a CD8⁺ response against the Plasmodium yoelii L3 ribosomal protein in sporozoite-immunized BALB/c mice. Unlike the CD8⁺ T cell response against the circumsporozoite protein (CSP) that increases with each parasite exposure in our system, the L3-specific response is not boosted by repeated exposures to attenuated P. yoelii sporozoites. Our lab has also shown that L3-specific cells have no cell intrinsic defects that counteract their re-expansion or function but rather that broad antisporozoite immune responses in secondary or later exposures eliminate expression of L3, thereby preventing any opportunity for activation of memory L3-specific CD8⁺ T cells. This T cell outcome following immunization may be emblematic of other T cells with liver-stage targets as well. In my project, we are studying the L3-specific T cell response in other murine parasite species (P. berghei) and other mouse strains (C57BL/6) by immunizing BALB/c and C57BL/6 mice with both species of Plasmodium and monitoring L3-specific CD8⁺ T cell responses via IFNγ ELISPOT to determine if this phenomenon is conserved in related models. Further, we are testing whether or not heterologous immunization of BALB/c mice with radio-attenuated P. berghei and P. yoelii followed by genetically-attenuated P. yoelii sporozoites bypasses the anti-CSP response and allows for boosting of late liver-stage antigens. If this occurs, such approaches could potentially yield more protective outcomes in human challenge studies and lead to more effective malaria vaccines in the future.

Despite recent sweeping advances in DNA sequencing technology, whole genome sequencing remains impractical as a routine medical service. Nevertheless, recent work suggests that rare genetic mutations may play large roles in modulating disease progression. More affordable targeted DNA sequencing is currently necessary to study variants of low frequency in the human population and attain sufficient statistical power to link these variants with specific disease phenotypes. Previous studies have demonstrated the effectiveness of a capture by circularization approach using molecular inversion probes (MIPs) whereby thousands of individual sites in the genome can be captured simultaneously for DNA sequencing. Yet, non-uniformity across large numbers of targeted sites persists as a major barrier to more widespread adoption. In this work, we show that statistical analysis of past MIP sequencing runs readily informs modifications to the process of probe design and selection. Additionally, logistic regression on carefully selected MIP features enhances in silico identification of ineffective probes that leave gaps in sequence coverage. Richer data sets from MIP sequencing runs representing a broader range of MIP characteristics and interactions may offer further guidance in perfecting the design of MIPs, achieving greater uniformity across targeted sites and ultimately lowering the cost of sequencing.