Pediatric rhabdomyosarcoma (RMS) is a rare and aggressive cancer that arises from skeletal muscle precursors in muscle and connective tissues. The molecular mechanisms underlying RMS progression, relapse and metastasis remain poorly characterized. CD82, a novel metastasis suppressor gene, has been shown to decrease tumor progression in a subset of human cancer types when expressed. Recently, two scientific publications investigated the role of CD82 in cellular differentiation and proliferation in muscle precursor cells. Uezumi et. al (2016) claimed that loss of CD82 results in premature differentiation and a depletion of muscle precursor cells. In contrast, Alexander et. Al (2016) discovered opposing results in which loss of CD82 in fetal muscle cells impairs the capacity of myogenic precursor cells to differentiate into mature muscle cells. In RMS, tumor cells keep on proliferating and have lost the capacity to differentiate into mature muscle cells. The role of CD82 in regulating cell proliferation and differentiation in RMS is unknown. In order to investigate the function of CD82 in differentiation of RMS, I used the CRISPR/Cas9 genome engineering system to target the gene in RMS cell lines. To characterize the knockout phenotype, I performed various cell-based assays to characterize the effects of CD82 gene knockout on the cellular phenotypes in tumor cell differentiation, proliferation, and self-renewal. Investigating the role of CD82 in regulating cellular differentiation and proliferation in RMS would provide important insight into the pathogenesis of cancer.

Predator-prey models provide invaluable insight into interspecies relationships and fluctuations in population sizes, but like all models, must rely on simplifying assumptions that reduce complex natural processes to functions with explanatory and predictive utility. Logistic equations for population growth predict future population size as a function of current population size and competition for limited resources; the related Lotka-Volterra equations use similar assumptions but with predator-prey interactions limiting population growth. In North America, the Canada Lynx-Snowshoe Hare cycle is an ideal demonstration of interconnected predator-prey population sizes. When vegetation is plentiful, hares have the resources to bear many litters of offspring and they provide a high level of nourishment to lynx. Lynx and hare populations increase in number until vegetation becomes scarce and predation by lynx reduces the size of the hare population. At this point, hares are too scarce to sustain lynx population growth and lynx numbers decrease while plants repopulate the region. Lotka-Volterra equations capture this cyclical relationship but assume genetic uniformity between individuals and no age structure in the population. Extensions of predator-prey models have incorporated age structure but not genetic variation. My model examines the dynamics of this system when both aging and genetic variation for fitness are incorporated into predator-prey dynamics. Using the statistical programming language R, I present a simulation of predator-prey dynamics with these two additional parameters—variation in relative fitness among individuals and age structure. Analysis of “the hare that never got caught” is an inquiry into the influence of age structure on fitness. More broadly speaking, this work attempts to model the effect of aging on the efficiency of natural selection and provides a basis for future research regarding the role of aging in evolutionary biology.

Cas9 is a versatile and effective genome engineering tool in characterizing biological effects of gene pathways due to its ability to activate and knock out genes with high efficiency and accuracy. When given truncated guide sequences 15 base pairs in length, Cas9 will bind to the specified site without cutting. By manipulating the binding sites of Cas9 in a gene, Cas9 can turn on or off gene transcription, thereby influencing gene activity. We constructed a Cas9-VPR system with the aforementioned features and have begun testing its ability to increase and decrease transcription of various cancer promoting genes. We quantified the level of each gene activity after introducing the Cas9-VPR system into the cells using quantitative Polymerase Chain Reaction (qPCR). Once we have optimized the system in modulating gene activity in cultured cells, we will move on to testing the functionality of Cas9-VPR in vivo using zebrafish as the model system. We plan to demonstrate Cas9-VPR’s efficacy by increasing expression of the Wnt pathway, which should result in the formation of a secondary embryonic axis in developing zebrafish embryo. Demonstrating Cas9-VPR’s ability to function in zebrafish will allow future research to examine the effects of manipulating multiple genes in order to understand the complex interaction of pathways driving a biological process.

Parkinson’s Disease (PD) is an age-related neurodegenerative disorder that leads to progressive decline in motor control. Tens of thousands of new cases are diagnosed each year. Many cases of PD are linked to chronic exposure to rotenone, a commonly used pesticide. As an inhibitor of complex I in the mitochondrial electron transport chain, rotenone causes deterioration of dopamine-producing neurons in the substantia nigra (part of the midbrain). Studies suggest that there is a genetic component to how likely one is to develop symptoms in response to rotenone exposure. To test this hypothesis, we used the Drosophila Genetic Reference Panel (DGRP), a set of 192 fully sequenced inbred lines of the fruit fly, Drosophila melanogaster. Exposure of Drosophila to rotenone has been shown to cause dopaminergic-specific degeneration, similar to the pathology observed in PD patients. When fed highly concentrated rotenone-infused food, we see significant variation in rates of survival among female flies from different lines of the DGRP, allowing us to capture natural variation in response to rotenone exposure. After most of the DGRP has been tested, we will carry out a genome-wide association study to identify specific genetic variants associated with rotenone resistance. In this way, we hope to uncover genetic factors that could point to specific mechanisms behind PD pathogenesis. With further related research, new genetic pathways could lead to new drug targets.

Zebrafish are a valuable model organism in scientific research, however there are limited genetic tools available for inserting/integrating engineered DNA into precise locations in the zebrafish genome. This project has developed a new method of precisely integrating engineered genes into specific zebrafish genomic locations. The heat shock-70 like (hsp70l) gene in the zebrafish genome is an ideal location for new gene (transgene) integration because the hsp70l promoter enables temporal regulation of the transgene through a heat shock-inducible mechanism. CRISPR/Cas9 genome editing technology was used to insert genetic markers, called attP sequences, flanking the green fluorescent protein (GFP) gene into the hsp70l gene locus in zebrafish. I built a construct containing attB sequences flanking the red fluorescent protein (RFP) transgene. These attP and attB markers are recognized by viral integrase proteins, bxb1 and fC31, which cut and recombine the attP and attB sequences causing directional recombination replacing GFP with RFP. I performed a proof-of-principle experiment by injecting zebrafish embryos with this new attB-RFP construct and the old attP-GFP construct with both the bxb1 and fC31 integrase enzymes to show functional integration in zebrafish. I optimized injection techniques to maximize the recombination frequency of the attB-RFP integration at the hsp70l gene in the zebrafish genome. This system allows for the rapid insertion of any transgene precisely into the endogenous hsp70l locus. I used this system to create heat-shock inducible Cas9 transgenic zebrafish as a tool for characterizing the function of novel genes essential for promoting or suppressing cancer growth in zebrafish tumor models. Inducing Cas9-mediated gene knockout is allowing us to determine the specific role a gene plays in cancer progression which helps us identify viable targets for the development of new cancer therapies.

Alzheimer’s Disease (AD), the most common form of dementia, is the sixth leading cause of death in the United States. With a prevalence of over five million cases, the number of AD patients is expected to increase as a new diagnosis of AD is made every sixty-six seconds. Early symptoms include short-term memory loss and gradually progress to devastating deficits in cognition, temperament, and behavior. AD is a neurodegenerative disease linked to the accumulation and aggregation of two proteins, Aβ_1-42 and hyperphosphorylated Tau, which form the characteristic amyloid plaques and neurofibrillary tangles found in the brains of AD patients. Previous studies support the notion that genetic variation underlies the wide array of phenotypes observed among AD patients, and several genes have been discovered to cause early onset AD. In sporadic (late onset) AD patients, it remains unknown what effects natural variation may have on the expression and severity of the AD phenotype. To explore the influence of genes on AD, our study utilizes a Drosophila model of AD that allows us to express both the Aβ_1-42 and Tau peptides in the fly eye. Expression of AD-related proteins in the eye leads to degradation of the hundreds of ommatidia that make up the fly eye. To quantify the extent of ommatidial degradation, we use a computer program, ImageJ, which measures the size and circularity of each ommatidium. To introduce natural variation, the AD fly is crossed with flies from the Drosophila Genetic Reference Panel (DGRP), consisting of 192 fully sequenced isogenic lines. By comparing the Aβ_1-42 and Tau-induced degradation in each background, we can identify naturally occurring modifiers of the AD phenotype. We have already identified promising hits, which could eventually help in the development of novel targeted therapies.

Rhabdomyosarcoma (RMS) is a soft tissue cancer that most likely develops from skeletal muscle stem cells. Like other cancers, RMS is a disease driven by critical mutation(s) in the patients’ genome, causing otherwise normal cells to divide uncontrollably without differentiating. Current treatments of RMS include chemotherapy, radiation therapy and surgical removal of the tumor. However, these treatments are limited in that they are toxic to both cancer and normal cells and are frequently ineffective in killing cancer cells because they do not directly target the genetic mutations causing cancer. One promising strategy to treat cancer is to develop therapies targeting the essential genetic mutations causing cancer. By utilizing the novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) gene editing tool that is more accurate and reliable than its predecessors, my project aims to target and knock-out the deleterious genes that promote growth and progression of RMS cancer cells. The two major subtypes of RMS are embryonal (ERMS) and alveolar (ARMS) with which they are defined largely by either activation of the RAS pathway (ERMS) or the presence of a fusion oncogene between PAX3 and FOXO1 (ARMS). To demonstrate the viability of the CRISPR-mediated gene therapy, I first targeted critical exons in the oncogenes of both RMS subtypes using inducible or viral CRISPR/Cas9 delivery systems. Targeting these essential genes resulted in significant regression of tumor xenografts. To further demonstrate that tumor-specific CRISPR/Cas9 gene therapy can also be achieved, precise targeting of only the mutation site(s) of the oncogene followed. This strategy disrupts the essential cancer oncogenes with minimal impact to normal cells that do not exhibit the unique cancer mutations. This novel approach to cancer therapy provides a promising alternative for traditional treatments while establishing a foundation for further development of CRISPR/Cas9 gene therapy for cancer treatment.

Traumatic brain injury (TBI) is an illness that affects people in all walks of life, from everyday citizens to soldiers in combat, and both young and old. Among all cases of TBI, there are some patients who recover quickly and some patients who take a long time to see any improvements, suggesting a range of what we call “TBI resistance”. To see if we could identify a genetic basis to this variation, we used the fruit fly, Drosophila melanogaster, as a model of TBI resistance. Specifically, we used a set of fly strains captured in Raleigh, North Carolina, and commonly known as the Drosophila Genome Reference Panel (DGRP). The DGRP is a sequenced set of inbred flies that researchers around the world experiment on, thus establishing a universal platform to identify genes associated with natural variation for a wide range of fly traits. For our study, we created a spring device to model TBI in flies. The device consists of vial of flies connected to a high resistant spring which propels it towards a hard pad causing damage. We compared the mortality rates between the different lines of flies. We hypothesized that we would see some lines display resistance and some lines display sensitivity. This turned out to be true, and we have already identified candidate genes associated with this variation. As a next step, to study TBI resistance on a cellular and molecular level, we will perform metabolomics with both resistant and sensitive lines to see if there is a significant difference between metabolite expression. The expression of certain metabolites in resistant flies, compared to sensitive flies, could hold the key to understanding how resistance is acquired against TBI and we will be one step closer towards understanding ways to protect people from the effects of TBI.

Exposure to ionizing radiation is the longest-established environmental cause of human breast cancer in both women and men. Evidence indicates that HER2-positive breast cancers are more likely to spread and are more resistant to anticancer-treatments. The goal of our study is to investigate genetic variations that are introduced during breast carcinogenesis. As a progressive stages of breast carcinogenesis cell model, normal human breast stem cells were sequentially transformed with SV40 large T antigen, x-ray radiation, and HER2 (ERBB2) overexpression to generate non-tumorigenic/immortalized cells, weakly tumorigenic cells, and highly tumorigenic cells, respectively. We sequenced 82 genes that are frequently mutated in breast cancer using next generation sequencing. We identified 26 radiation-specific mutations in exonic or intronic splicing regions that are not present in immortalized cells, but are present only in weakly tumorigenic cells. Among the 26 mutations, a single nucleotide deletion in RECQL gene, which is involved in DNA repair, is highly mutated (43%). Mutations induced by the overexpression of HER2 were identified by analyzing mutations present in highly tumorigenic cells but not in weakly tumorigenic cells. Two HER2 overexpression-specific mutations PRSS1 p.K225R and RET p.F683Y were identified with the mutation RET p.F683Y present at a high prevalence of 54%. The RECQL deletion induced by radiation and the two nonsynonymous mutations PRSS1 p.K225R and RET p.F683Y induced by HER2 overexpression have not been previously reported by others. Studies are underway to further characterize genetic variations that are induced by SV40-T Ag, radiation, and HER2 overexpression in breast carcinogenesis. The identified mutations that are specific to these oncogenic treatments aid in characterizing molecular subtypes of breast cancers and provide a foundation of identifying potential target mutations that can serve as biomarkers for the sequential stages of breast carcinogenesis.

Parkinson’s disease (PD) is a progressive, neurodegenerative disease with a global and national prevalence of four million people and one million people affected respectively. Though PD can be clinically characterized by dyskinesia, resting tremors and rigidity, it remains largely idiopathic biochemically. PD patients can be pathologically characterized post-mortem however, by the presence of Lewy bodies in the Substantia Nigra pars compacta (SNpc) of the ventral midbrain. While the major protein constituent of Lewy bodies is known to be α-Synuclein (α-Syn), the pathogenesis of α-Syn fibrillation in the SNpc remains elusive. One mitochondrial chaperone protein, mortalin, is thought to play a part in PD pathogenesis for its anti-apoptotic effects, role in stress responses, and through its regulation of transcription factor p53. We previously found mortalin to be down-regulated in the corpus striatum of manganese (Mn) miners, as well as primary murine glial cells. In the study at hand, we evaluated the connection between mortalin and p53 and the resultant effect on α-Syn oligomerization in astroglia with Mn-induced neurotoxicity. Using primary astrocyte cultures, western blot analysis, immunofluorescence and confocal microscopy, we evaluated this possible connection and its implications for α-Synucleinopathy. Within Mn-induced neurotoxic conditions, we saw decreases in total p53 of cortical and striatal mortalin knock-down astrocytes and increases in the density of cytosolic oligomeric α-Syn of these cells. Notably, we found that overexpressing mortalin under the same conditions decreased the density of oligomeric α-Syn dose-dependently. Taken together, these data suggest that the interaction of mortalin with p53 could have a role in α-Syn up-regulation, resulting in oligomerization within Mn-induced neurotoxicity. Further study is necessary to elucidate mortalin’s role in α-Synucleinopathy and Lewy Body pathology more precisely, but this work illuminates an important protein-protein interaction and provides implications for understanding PD pathogenesis and for developing therapies against α-Syn fibrillation in Parkinsonian diseases.
 

Aggregation of alpha-synuclein is known to be a critical step in the development of Parkinson’s disease and Dementia with Lewy bodies . Dementia with Lewy bodies and Parkinson’s disease are both chronic, neurodegenerative diseases that affect motor movements and cognitive development. The aggregation of alpha-synuclein induces neurotoxicity, which contributes to disruptive synaptic behavior which results in the neurodegenerative disease. Misfolded protein structures are a result of the accumulation of aggregated aSyn  that form fibrils. Recent studies have found that plant extracts from plants with longer lives have inhibitors that could decrease fibrillation of alpha-synuclein. Previous research from our lab included screening of extracts from different plants. We found that some of the plant extracts that contain molecules smaller than 3kDA had anti-aggregation activity. Acer Saccharum extracts had results that showed a similar inhibitory effect. In the present study, we used this extract to treat brain tissue of Dementia with Lewy bodies and a healthy control on tissue slides. Treatments included testing with Phosphate Buffered Saline as a control as well as using multiple concentrations of the Acer Saccharum extract. We applied Thioflavin T Fluorescence assay to quantify and analyze the relative amount of alpha-synuclein to the experimental conditions. Based on previous research, we expect the fluorescence intensity to be lower for tissue treated with the extract, especially for the higher concentration. Elucidating the inhibitory effect the plant extract has on fibrillation of aSyn can potentially lead to pharmacological developments for future therapies for Parkinson’s disease and Dementia with Lewy bodies Disease patients.

Extracellular vesicles (ECVs) are small vesicles secreted into the extracellular space to transfer proteins, lipids, and genetic material throughout the organism. Although ECV signaling is thought to be a conserved cell-signaling modality, seen by all cells from bacteria to human, very little is known how this signaling occurs in a living multicellular organism. I used the simple genetic worm model organism C. elegans to conduct genetic screens to understand the mechanisms by which ECVs are formed and secreted in a living animal. In addition, I generated worm lines with fluorescent ECV markers allowing myself and others to monitor ECV signaling in an intact organism. C. elegans uses the ECV machinery to secrete extracellular vesicles that are needed to form alae – two rows of three “racing stripes” that run down the lateral sides of the worm. This clear phenotype allows me to perform a reverse genetics screen using RNAi (RNA interference) to identify the key molecular components for proper signaling. In my first screen, I selected 20 genes that my mass-spectrometry experiments indicated were on ECVs. I found that several of them also prevented proper alae formation, indicating they are key for proper ECV signaling. I then screened all 21 members of the C. elegans tetraspanin family, an ECV-associated transmembrane protein family that is implicated in loading, secretion and cell targeting of the vesicles. Then I generated transgenic lines with artificially introduced DNA that produce fluorescent versions of the genes that are key for ECV secretion. This research is significant because it will uncover new genes necessary for ECV secretion in C. elegans and will find markers that will monitor ECV trafficking in vivo. This results of this will open up the study of ECV signaling in a powerful model system, allowing us to identify physiologically relevant signaling pathways that utilize this communication modality.

With dramatic increases in life expectancy over the past century, millions of people are living long enough to develop age-related neurodegenerative diseases often associated with cognitive decline and dementia. These neurodegenerative diseases are all proteinopathies, or diseases associated with misfolded proteins. Abeta and Tau protein aggregation is a key hallmark of Alzheimer’s disease (AD) while aggregated alpha-synuclein is a strong indicator of Parkinson’s disease (PD). This categorization is not definitive by any means because many patients with dementia exhibit multiple neuropathologies with combined Abeta, Tau, and alpha-synuclein aggregation. However, there has been almost no research studying the effects of the combined expression of multiple neurodegeneration-associated protein species. Therefore, we have developed the nematode C. elegans as a powerful genetic model for quantifying the functional consequences and mechanisms of action of interacting neurodegenerative-related proteins in vivo. To do this we have generated new transgenic worm lines with neuronal expression of different AD-related toxic proteins (both singularly and in combination) and developed new ways for assessing neuropathologies in these animals. We have generated worm lines with neuronal expression of the individual proteins as well as combined expression and seen that we can discriminate between the physiological effects of neuronal Abeta, Tau, and alpha-synuclein through our touch response and pharynx pumping assays we developed. Our escape response assay measures the number of reversals a worm completes upon stimulation by bending a piece of surgical thread over its pharynx. Our pharynx pumping assay measures the electrical activity as the worm feeds and allows us to extract precise measurements for pump frequency, amplitude, and duration. This proposed research is significant because it develops new neurodegenerative worm lines and uncovers new phenotypes that can be used to determine the physiological impact of individual and combined neurodegenerative toxic peptides.

Accurate quantification of DNA replication errors is difficult to measure since mutations are exceedingly rare. Recent attempts have relied on Next Generation Sequencing however, they are limited by its high error rate, with 5 x 10^-5 as their lowest detected mutation rate. Duplex Sequencing is an error correction method that reduces sequencing error through barcoding strands with unique identifiers and comparing the complementary strands, to achieve error rates as low as 10^-7. Our assay starts with a cloned single-stranded m13mp18 phage DNA with a base accuracy of 10^-7 as a template for replication. After annealing a forward primer we have our test polymerase extend the complementary strand and then we excise and isolate an 850 base pair region to undergo Duplex Sequencing. Through the improved baseline error rate of Duplex Sequencing, we hope to assay high fidelity polymerases, such as human polymerase delta. Accurately measuring the in vitro mutation rate and mutational profile of human polymerases is important in establishing the role of DNA polymerase generated misincorporations in somatic mutagenesis and human cancers. Furthermore, we have the potential to define a mutator signature that indicates the misincorporation of nucleotides in cancer by a specific DNA polymerase.

Repeated dopamine release through sustained amphetamine usage promotes long-term plasticity changes within the striatum that contributes to drug-related behaviors. Although addiction has been linked to dopamine release in the striatum, there exists other neurotransmitters whose function is less known. In the past, we have shown that after repeated amphetamine exposure, acetylcholine is also involved in mediating long-lasting plasticity changes in the dorsal part of the striatum which is linked to habit formation. However, it remains unclear whether this mechanism also plays a significant role in the ventral striatum, a crucial structure in reinforcement and reward processing. To address this question, we used mice in which acetylcholine production in the ventral striatum, specifically in the nucleus accumbens (NAc Core), can be abolished by genetic inactivation of choline acetyltransferase (ChAT) using an adeno-associated virus encoding Cre recombinase (NAc Core ChAT-KO mice). These mice were tested for a drug-related behavior called locomotor sensitization, a phenomenon where locomotor activity is progressively increased through repeated amphetamine injections. The procedure consisted of 2 days of saline injections followed by 5 days of amphetamine treatment and a 10-day break to induce withdrawal before an amphetamine challenge. Each day, locomotor activity (ambulations) was measured for 90 minutes by recording successive infrared beam-breaks in activity chambers. We observed a significant main effect of time and a significant time x genotype interaction effect where NAc Core ChAT-KO mice were less active than their sham controls on the fifth day of amphetamine treatment. We found no significant differences in locomotor activity during the withdrawal treatment between sham and NAc Core ChAT-KO mice. These results indicate reduced acetylcholine levels attenuated the extent of locomotor sensitization, suggesting that acetylcholine in the ventral striatum is involved in mediating long-term behavior adaptations following amphetamine treatment.

Aging is a process which causes the deterioration of cellular function in organisms. In order to understand the mechanisms of aging and to begin to identify ways to extend healthy life expectancy, research in the Kaeberlein lab uses Saccharomyces cerevisiae, a species of yeast. The technique that our lab uses to quantitate lifespan in yeast is known as the replicative lifespan (RLS) assay. When a yeast cell divides, the original “mother” cell and the newly produced “daughter” cell can be differentiated based on size. Daughter cells are microdissected away from mother cells, the cell division is recorded, and this cycle repeats until the mother is no longer able to produce daughters. We have tested the effects on lifespan resulting from the manipulation of thousands of genes and numerous environmental conditions. This data can be analyzed to understand the process of aging in yeast cells. By studying the mechanisms of aging in unicellular yeast cells, we can transfer these findings to identify conserved factors in the aging process that are also present in multicellular organisms. The study of the basic biology of aging in yeast can yield insights into the aging process that may contribute to our understanding of aging in humans and help facilitate the development of therapies aimed at decreasing age-related diseases.

Cytosolic pH is sustained by the movement of protons out of the cytosol. The vacuolar ATPase (V-ATPase) is a conserved protein complex that localizes to the membrane of organelles and transfers protons from the cytosol into the organelle lumen. The V-ATPase couples the energy of ATP hydrolysis to transport protons. Proper pH homeostasis is important for many organelles such as endosomes, lysosomes, and secretory vesicles. In Saccharomyces cerevisiae, the Pma1 protein localizes to the plasma membrane and pumps protons out of the cell, helping to maintain cellular pH homeostasis. In Saccharomyces cerevisiae, we have found that loss of activity of the V-ATPase results in a growth arrest at low temperature. We generated 66 yeast V-ATPase mutant strains that are able to suppress the low temperature induced growth arrest. In complementation tests of these mutants, our results suggest that all of the mutants belong in the same complementation group. We have performed whole genome sequencing on one strain and analysis of the whole genome sequencing data has identified a single putative mutation. We are validating that the identified mutation is causative for the phenotypic suppression, and performing targeted sequencing of the candidate gene in the other suppressor strains.

Cancer stem cells (CSCs) are small populations found within tumors and appear to be more resistant to anti-cancer treatments. Although evidence supports that CSCs play a major role in initiation and in the clinical outcome of cancers, the roles of breast CSCs in metastasis and invasiveness are not fully understood. Moreover, the association of the invasiveness of breast CSCs with genomic variations is unclear. The overall goal of this study is to investigate the migration abilities and genomic expression changes in breast CSCs. Human breast CSCs and immortalized/non-tumorigenic cells were derived from the same parental breast normal stem cells. To recapitulate tumor microenvironment, the cells were cultured on nanotopography-defined extracellular matrix (ECM)-mimetic platforms. We have employed a live cell imaging microscopy to measure migration at the single cell level as an indicator of invasiveness in breast tumorigenic and immortalized cells. Our preliminary results indicate that breast CSCs migrate significantly faster than immortalized cells on ECM mimics. Interestingly, we only find this difference under ECM-mimetic culture conditions, but not under flat control surfaces lacking ECM structures. This suggests that the invasive abilities of breast CSCs are lost when they are cultured in vitro on flat control surfaces without ECM structures. We will perform RNA sequencing to identify differences between the transcriptomes of breast CSCs and immortalized cells, and between cells cultured on ECM-mimics and substrates without ECM-mimics. The current study will characterize transcriptome profiling in breast CSCs and identify the genes that are differentially expressed between breast CSCs and immortalized cells. This will have implications in classifying molecular subtypes of breast cancers and in identifying potential therapeutic targets to regulate specific gene expressions for the inhibition of breast CSC invasiveness.

DNA replication is the process by which genetic information is duplicated and passed from cell to cell. The process of DNA replication occurs with the assistance of various enzymes, including DNA polymerases, which synthesize new DNA molecules using template DNA and deoxyribonucleotides (dNTPs). Faithful DNA replication is vital for population survival and protected by exonuclease domains on the major DNA polymerases, which proofread replication errors. A secondary error-correction pathway known as mismatch repair (MMR) also scans the genome for replication errors. Defects in proofreading and MMR lead to "mutator phenotypes" marked by elevated mutation rates and increased cellular mutation burden.  In microbial populations, mutators spontaneously emerge and can increase adaptation to new environments by fueling genetic diversity. However, at extreme mutation rates, the accumulation of harmful mutations can also drive extinction of mutators.  Some mutator cells survive this "error-induced extinction" (EEX) by acquiring antimutator mutations that suppress the mutator phenotype. We have identified Saccharomyces cerevisiae strains that suppress elevated mutation rates through a screen for mutants that survive EEX. Using these eex mutant strains, we are currently mapping the genetic changes responsible for the antimutator phenotypes. Changes in mutation rates in haploid spores derived from these eex suppressor strains are being tracked to identify antimutator segregants, which are then being subjected to whole genome sequencing. As mutator phenotypes are important drivers in cancer, our analyses may reveal novel pathways that influence cancer evolution.

Many in vitro and in vivo approaches (e.g. overexpression or depletion) have established that proteins’ expression levels can affect the cellular response to stresses and stimulus. Here, I describe an approach to examine the lowest protein amount necessary for a physiologic cellular response. My studies are focusing on Fanconi Anemia (FA), a heritable human genetic disease characterized by cellular and developmental abnormalities, including congenital abnormality, bone marrow failure, and an increased risk of malignancy. The FA pathway responds to inhibition of DNA replication due to DNA damage, particularly interstrand cross-links such as mitomycin-C (MMC). Mutations in the FANCA gene, however, can disrupt this pathway and cause cellular defects. The specific goal of this research is to investigate how much functional FANCA (wildtype FANCA) is required to suppress these cellular defects. To modulate FANCA protein amount, we are currently developing vectors with variable strength promoters that allow for graded expression (from 0-100%) of wildtype FANCA gene. These vectors will be integrated in a unique chromosomal location in corresponding FANCA-deficient isogenic cell line to generate a cellular library. The integrated transgene consists of two regions: a variable strength promoter and a common EGFP linked via a T2A self-cleaving sequence to the FANCA coding sequence. FANCA protein expression will, hence, be quantified using Western blot and using the co-translationally expressed fluorescent protein (EGFP). Lastly, the cellular library will be subjected to differing doses of MMC and promoter sequencing will be used to quantify promoter-specific survival (aka FANCA protein expression-specific survival) as a function of MMC dose. The findings from this study will enable FA-specific drug/therapeutic research by establishing the quantitative requirements for FANCA protein function in human cells.

Duplex Sequencing (DS) is a Next-Generation Sequencing technology employing double strand molecular tagging to reduce technical errors.The high accuracy of DS enables the resolution of low frequency mutations in nuclear and mitochondrial DNA (mtDNA). We are interested in using DS to analyze mtDNA mutations in ulcerative colitis (UC), an inflammatory bowel disease that predisposes to colorectal cancer. Tumor progression in UC is related to mitochondrial dysfunction. By sequencing mtDNA mutations in colonic biopsies from UC patients, we aim to elucidate the role of these mutations in tumorigenesis. A critical limitation, however, is the high number of low frequency mtDNA mutations present in these biopsies and the lack of appropriate computational methods to process large amounts of information generated by DS. The goal of this study was to develop computational methods to enable the extraction of mtDNA mutation information from DS data. DS identifies the reads corresponding to each strand of DNA by grouping them by their unique tags, making single-strand consensus sequences (SSCSs), which are then paired to form duplex consensus sequences (DCS). SSCS reads remove technical errors, but contain errors caused by DNA damage. The first computational method that we developed compared sequencing information from SSCS and DCS reads to assess the level and type of DNA damage in each sample. Other computational methods consisted of a program to classify mutations according to their sequence context, and a script that calculates mutation types and frequencies inside and outside the D-loop region as well as in all mtDNA genes and allows comparison of these parameters in large batches of samples. These tools will enable the processing of mtDNA DS information in a fast and high-throughput way, leading to assessment of the range of different types of mtDNA mutations in the progression of ulcerative colitis and colorectal cancer.

Glioblastoma multiforme (GBM) is the most common malignant adult primary brain tumor, accounting for 70% of malignant brain tumor diagnosis. However, regardless of therapy – surgery, radiation, and anticancer drug therapy – virtually all GBM patients eventually succumb to the progressive disease within 12 – 15 months of diagnosis. This is indicative of the proliferation and differentiation of neural stem cells into various cell types in the tumor which is thought to be a main contributing factor of malignant tumor resistance to standard treatments. This project aims to address this therapeutic challenge by identifying and testing potential new therapeutic regiments that leverage the underlying genetics of glioblastoma to improve treated response. We propose that certain genetic modifications (i.e. modifications in IDH1 or MGMT) can be made in glioma stem cells such that when a gene loses or gains a function, it will increase sensitivity of cells to a combination of therapies that is greater in magnitude than a single drug by itself. Towards this, we have generated whole exome sequencing data that will guide us in building a genetically-defined glioblastoma cell line and allow us to identify potential new therapeutic combinations predicted to have high specificity for GBM cells. Implementation of these predicted synthetic lethal combinations in a co-culture system will determine if the predictions are tumor, genotype, and treatment-specific. Results of this project will help us better understand therapeutic vulnerability and its determinants, and test specific combination therapeutic regiments that could be applied to improve the treated outcome of patients with this deadly disease.

The presence of protein biomarkers in specific human tissues can be indicative of diseases such as Parkinson’s Disease (PD). One such protein, α-synuclein (α-syn) commonly exists in brain and blood and can be transmitted between different cell types in secreted membrane vesicles called exosomes. The blood levels of α-syn are very high, and α-syn originally from red blood cells (RBCs) could be transported in exosomes.  In order to study α-syn in RBC exosomes, we have collected α-syn-containing exosomes from RBCs by culturing fresh blood samples. However, RBCs degrade quickly, meaning that in the past, RBC exosomes could only be collected from fresh samples on the same day as collection. So the purpose of this work is to save time and resources from culturing exosomes by determining if they can be pulled down from previously frozen blood plasma. RBC derived exosomes could be targeted by the RBC membrane specific cell surface protein CD235a and using antibody coated superparamagnetic beads to pull down the desired exosome from samples. We hypothesize that the technique of immunoprecipitation is able to make a pure preparation of RBC exosomes which can be use for studying exosome α-syn from RBCs.

Myocardial infarctions (MI) are ischemic events which cause heart tissue to die and scar tissue (fibrosis) forms to maintain integrity of the walls of the organ. Fibrosis is crucial in the initial healing process but continued formation can compromise the elasticity of the tissue and lead to heart failure. Fibrosis is formed by myofibroblasts. These cells differentiate from fibroblasts after MI. Currently, the differentiation process is not well understood. The RNA binding protein muscleblind like (MBNL1) is known to be critical in this process, however. This project aims to use fluorescent imaging to track the localization of mRNA bound by MBNL1, particularly serum response factor (SRF) which has also been shown to play a role in myofibroblast differentiation. We aim to use regularly interspaced short palindromic repeats (CRISPR) and nuclease-null, RNA-targeting CRISPR associated protein 9 (RCas9) to specifically bind to the desired sequence of SRF mRNA. Enhanced green fluorescent protein (EGFP) is bound to the RCas9 in order to be able to visualize the RNA. Two target sequences on SRF mRNA were identified and protospacer adjacent motif presenting oligonucleotides (PAMmer) and single-guide RNA (sgRNA) were designed to bind to these sequences. PAMmer and sgRNA allow Cas9 to bind to the target sequence. This system was transfected into mouse embryonic fibroblast (NIH 3T3) cells and differentiation induced using transforming growth factor beta. During differentiation, live cell imaging was used to visualize RNA dynamics and localization. Results are expected to show colocalization with MBNL1 in the cytoplasm. This data will allow for the correlation of RNA trafficking and cell differentiation. It will also allow for a better understanding of myofibroblast differentiation and fibrosis formation after a myocardial infarction.

The biological basis behind aging in eukaryotic species is defined by a wide range of factors, from diet to environmental conditions and metabolic predisposition. The genetic factors associated with aging are of particular interest because they are highly conserved among all eukaryotes. Determining longevity genes that are broadly conserved among eukaryotic species is a critical step in understanding aging, and is one of the primary goals of this project. The Kaeberlein lab has analyzed the replicative lifespan (RLS) of more than 4700 single-gene deletion mutants of Saccharomyces cerevisiae, a eukaryotic model organism. RLS experiments measure the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Mother and daughter cells are visibly differentiated under a light microscope and physically separated using a fiber-optic needle. The number of daughter cells dissected are quantitated and recorded. The total number of cell divisions are then summed for statistical analysis. The result of the investigation yielded 238 gene deletion mutants that exhibited a statistically significant lifespan extension. Longevity genes were found to be clustered within specific parts of an organism's biology. Ribosomal genes and genes related to metabolic responses to nutrients were particularly significant. One particularly significant gene is the LOS1 gene, which functions as a tRNA exporter from the nucleus. Deletion of the LOS1 gene resulted in a dramatic enhancement of yeast RLS, opening the possibilities of lifespan research regarding the subcellular localization of tRNA. Also significant was the discovery that a majority of the longevity genes were concentrated among highly evolutionarily conserved parts of the genome. Ultimately, the success of the yeast RLS project in identifying novel longevity genes and establishing a database of conserved longevity genes in eukaryotes points to the efficacy of such methodology.

The process of aging brings about susceptibility to disease and death, but its mechanisms are still poorly understood. Dietary restriction is a potent approach to slow down the aging process, which has shown efficacy in multiple species, including the roundworm, Caenorhabditis elegans, a popular model organism for aging studies. Specifically, pre-reproductive adult C. elegans subjected to starvation enter a state of adult reproductive diapause (ARD), which preserves their lifespan and reproductive potential. Intriguingly, diapaused animals show signs of age-related deterioration upon prolonged starvation, but exhibit dramatic morphological improvements and have normal lifespans following return to feeding. However, the mechanisms regulating this lifespan preservation and apparent rejuvenation are not known. In this project, we aim to identify the genes responsible for the recovery during diapause exit. For that purpose, we used an RNA interference (RNAi)-based screen to inactivate individual genes during exit from diapause and analyzed the resulting phenotypes. Our current results reveal many regulators of metabolism and cellular homeostasis as crucial mediators of post-diapause recovery. Further studies based on these findings will discover the critical steps required for post-diapause tissue rejuvenation and preservation of longevity.

Small cell lung tumor (SCLC) is a type of lung tumor that accounts for 10-15% of new lung cancer cases. It is an aggressive cancer that is often not detected before metastasis occurs. Even with chemotherapy and radiotherapy, SCLC has a very high mortality rate and patients tend to develop chemoresistance quickly. Unlike other types of lung cancers, SCLC has no available gene-targeted therapies. This project examines the development of chemoresistance in SCLC genetic mouse models . This approach deletes tumor suppressor genes, such as Rb1 and Tp53, and/or activates oncogenes like lmyc, using a Cre-expressing virus in mouse lung tissue, which leads to the development of SCLC. My role included verifying tumor burden following magnetic resonance imaging (MRI) and quantification of tumor volume in the lungs using MRI data. Once MRI confirmed tumor burden, mice are treated with chemotherapy and tumor volume measured over three weeks of drug treatment. The tumor response to chemotherapy will be compared in different genetic mouse models. At the end of treatment, lung tumors are collected and I use immunohistochemistry to measure the response of the tumors collected from the genetic mouse models. I examine markers of cell proliferation and cell death to assess drug effectiveness. These results could be relevant clinically if certain genetic mutations are found to be associated with chemoresistance.

Mutations are imbedded in every individual’s DNA. A key challenge in human genetics is to determine the functional phenotype of specific mutations. The goal of my research in the Monnat Lab is to develop an efficient, quantitative, cell-based assay to study the phenotype of mutations in a gene in parallel. Our target disease is Fanconi Anemia (FA), a recessive human genetic disease associated with cancer, bone marrow failure, and sensitivity to crosslinking agents such as mitomycin-C. Among of the 21 “FANC” genes that could result FA, we focused on FANCA gene as it is mutated in 2/3 of FA patients. A set of nearly 500 FANCA variants was then used to build a plasmid library encoding each protein variant. In order to achieve this, each variant, linked to a unique DNA molecular 'barcode', will be inserted at a unique chromosomal location in FANCA-mutant human cells in order to determine their cellular phenotype. We will assess cell survival as a function of mitomycin-C dose and the expressed transgene. Preliminary results have already been generated to confirm the feasibility of this approach. My effort thus far has been focused on generating a library of ~500 single nucleotide variants of FANCA. In addition to capturing each variant, I developed an optimized workflow to generate barcoded plasmids with only desired variant without additional mutations in the FANCA. In the coming quarter, I will work with other members to develop a corresponding cell library in which each of these FANCA variants is expressed in a common, FANCA-deficient background for functional phenotyping. Results of my research will help identify new and existing deleterious as well as innocuous FANCA variants, and thus will enable a more accurate molecular diagnosis of FA.

Organisms face a range of stressors over the course of their lifespans. As with most traits, we see significant variation in response to stress even between individuals within species. In our attempt to explain differences in stress responses between individuals, the Promislow lab studied the metabolome, the total profile of all small molecules in an organism, and found variation in stress response is correlated with variation in contents of the metabolome. Indeed, in a study of peroxide resistance in the fruit fly, Drosophila melanogaster, we found resistant flies tended to have increased galactose levels, compared to sensitive flies. This observation led to the novel hypothesis that flies fed supplemental galactose will have increased peroxide resistance compared to control flies. For this experiment, we chose flies among the most sensitive and resistant lines examined previously because the most sensitive flies, with low galactose levels, may show increased effects of supplemental galactose on lifespan. In this experiment, flies feed on food with varied galactose concentrations, including a negative control without galactose. We then place the flies into media with peroxide or water, the control. We record the number of flies that die in each condition multiple times a day, and the death rate provides a quantitative measure of peroxide resistance. By comparing the death rate of flies within a line kept in varying concentrations of galactose exposure, we can test the effect of galactose on resistance. If our hypothesis is correct, the flies receiving supplemental galactose should die at a slower rate than those without additional galactose. However, it is possible flies receiving supplemental galactose will die at faster rates, suggesting galactose makes flies more sensitive. Determining whether galactose makes flies more resistant or sensitive to peroxide may prove to be important in counteracting effects of oxidative stress in other organisms.

There are many age-related cardiac diseases that relate to impairment of muscle function, including heart failure and arrhythmia. Previous studies show that increased mitochondrial function results in the enhancement of cardiac and skeletal muscle function as mice age. In these studies, the SS-31 tetrapeptide was used, an agent that serves as an antioxidant by binding to cardiolipin in the inner mitochondrial membrane, enhancing electron transport and increasing ATP generation, and protecting against damage from Reactive Oxygen Species (ROS) by reducing their generation. However prior literature has suggested that ultrastructural morphology does not change in relation to age. The goal of this study is to quantitatively assess interfibrillar mitochondrial damage caused by these ROS', and to see if there is structural improvement after treatment with the SS-31 peptide. Eight 24 month-old mice were split into two treatment groups, four treated with SS-31 for 8 weeks and four untreated. Four 8 month-old untreated mice were used as controls for youthful, “ideal” mitochondrial structure. The mice were euthanized, and cardiac tissue was harvested, sectioned, and examined by transmission electron microscopy (TEM). Quantitative analysis using NIH ImageJ software allowed the comparison of the SS-31 treated and untreated old hearts to young hearts. This revealed stark differences when comparing the young and old ultrastructure. Untreated old mice exhibited higher amounts of vacuolation, greater lack of mitochondrial axial organization, and lower density of mitochondrial cristae than the younger untreated mice. Older mice treated with SS-31 appear to exhibit improvement in ultrastructural definition compared to untreated older mice, but additional quantitation is necessary to validate this observation. In further testing, we are examining the persistence of the SS-31 tetrapeptide's positive effects after its administration period ends, and how ultrastructure changes during this refractory period.

Oxidative stress arises from an imbalance between reactive oxygen species and an organism’s ability to avoid or repair the damage caused by these reactive molecules. The fruit fly, Drosophila melanogaster, demonstrates varying degrees of resistance to oxidative stress. To understand the genetic basis of this variation, our lab analyzed a collection of inbred, fully sequenced Drosophila lines, the Drosophila Genetic Reference Panel (DGRP), and tested the associations between resistance and each of more than two million single-nucleotide polymorphism (SNP) markers throughout the genome. We found a particular SNP at the second chromosome located within the gene u-shaped (ush), had the strongest statistical relationship with oxidaive stress resistance. We then hypothesized that this gene contributes to the degree of resistance in the flies. For my research, I am evaluating this hypothesis by comparing the peroxide resistance of two sets of genotypes to confirm if the SNP at 2L:531863 bp contributes to the trait. For this comparison, I used two groups of lines, one with the ‘resistant’ and the other with the ‘sensitive’ version of the SNP, and distributed mated females from each line into vials with or without peroxide added into the food. I counted the number of dead flies at regular intervals and used these data to calculate the mean lifespan for each line, where lifespan is a measure of peroxide resistance. If both groups show similar lifespans, it suggests that this particular SNP does not affect peroxide resistance and I may test other SNPs at nearby loci with a similar procedure. However, if the two groups show significant differences in resistance, this result would support the connection between the SNP and peroxide resistance. I would then go on to manipulate the expression of ush using RNA interference to test whether changing mRNA levels impacts peroxide resistance.

Aging is a phenomenon that brings about the onset of disease and eventually death. One of the hallmarks of aging is the decline in proteome maintenance that can lead to the accumulation of misfolded proteins. In extreme cases, protein misfolding can induce such devastating neurodegenerative diseases such as Huntington’s disease and Alzheimer’s for which very limited treatment is currently available. It has been observed previously that dietary restriction is a method that increases lifespan and delays the loss of function caused by age-associated pathologies. Specifically, complete food removal leads to robust increase in longevity in the roundworm Caenhorbabditis elegans, a popular model organism for aging research. Remarkably, when pre-reproductive adult worms are subjected to starvation, they enter a condition known as adult reproductive diapause (ARD). Upon reintroduction to food after prolonged starvation, ARD worms experience a pronounced morphological recovery and have normal lifespan. To understand how diapaused animals preserve this rejuvenation potential and normal lifespan we aim to analyze how proteome quality is maintained during the diapause. We hypothesize that proteome quality is either well maintained during the diapause or is restored back to youthful state upon exit from the diapause. To discriminate between these possibilities, we use C. elegans models of proteotoxicity wherein toxic protein species produced in muscle cells induce paralysis with age. By analyzing the accumulation of insoluble protein aggregates and motility during and after the diapause we will gain a better understanding of the physiological aspects of the ARD state. These insights will be useful in developing novel approaches to counter age-associated pathologies.

It is known that dietary restriction is a positive intervention in aging. Its efficiency was demonstrated in multiple species suggesting that dietary restriction acts through conserved mechanisms that are not well understood. Specifically, in Caenorhabditis elegans (C. elegans), dietary restriction can extend lifespan well beyond that of their fed counterparts. Remarkably, it has been found that upon starvation young adult C. elegans enter a state of adult reproductive diapause (ARD) that allows a full lifespan preservation. We aim to characterize the mechanism by which lifespan can be preserved and extended in the diapaused animals. To this end we will investigate whether ARD animals experience typical age-related changes during prolonged starvation and whether they are reset upon diapause exit. Through fluorescent microscopy, we intend to distinctively characterize phenotypic differences of the cytoskeleton and nuclear morphology between ARD individuals and their fed counterparts. We expect these age-related markers either remain intact throughout the diapause state, or repaired upon diapause exit. This will further allow a better understanding of aging as wells the mechanisms of lifespan and healthspan preservation.

Aging can be defined as the time-related deterioration of physiological functions that are essential to the fertility and survival of a living organism. In the Kaeberlein Lab, we study how cells change with age and how these changes contribute to age-associated diseases in order to help people live healthier lives. The eukaryote Saccharomyces cerevisiae (yeast) is used as a model for cellular aging. Due to its conservation of cellular functions through evolution, modeling aging using yeast can provide insights into how mammalian cells age. Through replicative lifespan (RLS) analysis, we are able to determine the effects of interventions on aging by measuring the number of divisions that a single yeast cell goes through before it stops dividing. We are testing various nutraceuticals in order to identify novel pharmacological interventions that increase lifespan. In a screen of twenty nutraceuticals, we found that Pterocarpus marsupium (PME) extract significantly extended lifespan. P. marsupium is a deciduous tree native to the Indian regions. It is well-known to Ayurvedic medicine thanks to its purported curative and lenitive properties. The flowers are said to be effective in lowering fever; its heartwood may possess purifying and detoxifying effects; its wood is used to treat chest and body pain as well as indigestion. Additional studies will test whether the RLS-extending effects of PME can be attributed to pterostilbene, a compound found in PME that is structurally related to resveratrol. We tested concentrations of pterostilbene ranging from 1-50 µM to identify its effect on replicative lifespan. These studies will allow for further development of PME and pterostilbene for use as an intervention to promote healthy longevity.

The Food and Drug Administration (FDA) approved drug rapamycin lengthens lifespan and rescues rodents from age-related diseases. Previous studies have shown that a 3-months regimen of rapamycin for old mice of the C57BL/6 strain changes the composition of gut bacteria that includes increased bacterial species such as lactobacillus, bacteroides and segmented filamentous bacteria (SFB). In mouse models with a mitochondrial disease, there was a decreased amount of bacteria and an extremely short lifespan. Injection of rapamycin increased the amount of bacteria and prolonged lifespan, showing an association between the quantity of bacteria and lifespan. We are interested in the effect that this experiment would have on a different strain of mice, Balb/c, which had not been previously tested for the rapamycin effect on lifespan nor the change in the composition of gut bacteria. First, we conducted a 90-day injection study on Balb/c mice, in which the mice were injected with different frequencies of rapamycin via an intraperitoneal (IP) injection. After collecting fecal samples at three different time stages – at the start of the 90-day injection period, immediately after the 90-day period, and 3 months after the 90-day period, we ran a qPCR (quantitative polymerase chain reaction) for each of the fecal samples collected, which quantifies the relative amount of microorganisms in the sample. We also monitored the lifespan of those rapamycin-treated Balb/c mice. This allowed us to see if the lifespan extension and gut microbiome change observed in the original model is reproducible in a different mouse strain. We hope that our findings help to support a future translational research strategy that applies the effect of rapamycin on specific types of bacteria to produce an intervention on gut bacteria to slow aging in pet animals and humans.

Alzheimer’s disease (AD) and cancer are two of the most devastating public health challenges in modern society costing 361 billion dollars and 685,240 lives each year. As these two classes of pathologies progress they both generate protein stress inside cells. Tumor cells are often characterized by greatly increased protein production while most neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease are classified as a “proteinopathies”; characterized by the accumulation of the misfolded proteins. Some recent reports suggest that the Parkinson’s disease related protein α- synuclein influences cancer growth in mice and cell culture models. The goal of our project is to uncover the cellular pathways that are shared between neurodegeneration and oncogenesis. We will study this using the C. elegans genetic model system. I have generated a transgenic line that ubiquitously expresses a human form of the AD-related protein, Amyloid-βeta, and observed that it develops similar tumors to cancer-model worm lines. We are also generating transgenic worm lines that ubiquitously express the other canonical neurodegenerative proteins α-synuclein and Tau to see if they also form tumors. These transgenic lines will allow me to knock down different cellular pathways using RNA interference (RNAi) which silences or inhibits the gene expression using double stranded RNA. First we will look chaperone functioning because this class of proteins is necessary for the cells to deal with protein stress. Research in our lab has already shown that RNAi of some chaperones decreases the occurrence of tumors in a worm cancer line. Next, we will generate worm lines with fluorescent reporters of the tumor altering genes and reporters of cancer-related cell-signaling pathways. Thus, we will begin to uncover the ways in which AD-related protein stress impacts cell processes that promote cancer. This could lead to new gene targets for future therapies.

Aging remains as the biggest risk factor for developing the most prevalent diseases found in developing countries: cancer, cardiovascular disease, and neurodegeneration. Reduced insulin/IGF signaling (IIS) extends lifespan in diverse organisms, but the underlying molecular mechanisms that regulate this remain unclear. Recently, non-coding RNAs, i.e. those RNAs not translated into proteins, have become a hot topic, being identified as regulators of gene expression during aging. Circular RNAs (circRNAs) are one such form of non-coding RNAs. Although they are identified in a diverse number of species, the function of most circRNAs is unknown. CircRNAs are distinct from their linear counterparts because they are devoid of the terminal structures (e.g. 5’cap or a poly A tail) that frequently determine the fate of RNA transcripts thereby escaping normal RNA turnover. It has been established that circRNAs are abundant and differentially expressed; they can serve as either transcriptional regulators, and/or microRNA sponges. Furthermore, some circRNAs are produced at the expense of linear RNA, thereby affecting gene expression and thus tissue or organismal function. CircRNAs are also evolutionary conserved, which begs the question why they are so important. Recently, the Partridge lab conducted gene expression profiling of long-lived IIS mutants and identified differentially expressed circRNAs, which may influence IIS-mediated longevity and thus aging. The Partridge lab have generated in vivo Drosophila models, mutating individual circRNAs through genomic engineering (CRISPR/Cas9) and the Gal4/UAS system, to study their function. Using these novel circRNA mutants, I will contribute to discovering the in vivo function of circRNAs in aging by performing quantitative experiments. I will analyze circRNAs mutant lifespan and phenotypes associated to longevity e.g. development, fecundity, stress resistance, neurodegeneration. Genetic epistasis experiments will determine the role of the given circRNAs in insulin signaling. Finally, I will use molecular biology techniques to unravel the mechanism underlying any observed phenotype.

Human pluripotent stem cells (hPSCs) are defined by their ability to self-renew, proliferate, and differentiate into the three germ layers. Controversy remains as to the role of canonical Wnt/β-catenin signaling (β-catenin dependent) in hPSC maintenance and mesoderm formation. Wnt/β-catenin signaling has shown to be important in the context of cellular homeostasis and embryonic development, and improper signaling can cause growth of cancerous tumors. By determining the role of this signaling pathway in hPSCs, we aim to improve our overall understanding of the molecular processes behind hPSC directed differentiations and early embryonic development. We assessed the role of Wnt/β-catenin signaling in hPSCs by generating a CRISPR/Cas9 mediated knockout (KO) of the signal transducer β-catenin. Expression levels of protein biomarkers of pluripotency (SSEA4 and OCT4) were evaluated and no difference was noted between wild type (WT) and β-catenin KO hPSCs. Additionally, the relative level of cell proliferation between WT and β-catenin KO cells was analyzed by quantification of EDU incorporation using flow cytometry, and no difference was noted between the two cell types. After confirming β-catenin KO hPSCs can self-renew and proliferate, we assessed the ability of β-catenin KO cells to differentiate to form mesoderm in vitro. In order to further demonstrate the level of pluripotency of the KO cells, we also attempted to differentiate the KO cells towards ectoderm. Analysis of the effectiveness of both the mesoderm and the ectoderm differentiations show that while KO cells cannot effectively form mesoderm, they are still able to form ectoderm. In this study, we demonstrate that hPSCs lacking β-catenin can still proliferate, fail to form mesoderm, but can still form ectoderm. Thus, β-catenin may not be required for maintaining the proliferative characteristics of hPSCs, but is required for formation of the mesoderm germ layer, and is therefore an important component of early embryonic development.

DNA polymerase (Pol) proofreading and mismatch repair (MMR) cooperatively guard against DNA replication errors and cancer. Defects in these activities produce “mutator” phenotypes characterized by elevated levels of base-substitutions and frameshifts. Haploid yeast with combined defects in these activities rapidly go extinct, with some strains failing to even form visible colonies - a process termed error-induced extinction (EEX). Haploid eex mutants escape extinction by acquiring antimutator mutations that suppress the mutator phenotype; however, sixty percent of eex mutants escape extinction through spontaneous polyploidization, which insulates them from mutation accumulation by doubling their number of chromosomes. In evolution experiments with pol2-4 msh2Δ strains, which display a synthetic-sick phenotype due to Pol ε proofreading and MMR defects, polyploids routinely beat out antimutator mutants, which maintain haploidy. Whether strong diploid mutators also escape EEX by polyploidization is unknown. To investigate whether polyploids arise during the evolution of mutator diploid strains, we propagated pol3-01/pol3-01 msh6Δ/msh6Δ diploids (Pol δ proofreading and base-base MMR defective) with a mutation rate an order of magnitude below the diploid error threshold. Mathematical modeling suggests that although these diploid mutators are not immediately inviable, they should be overtaken by an antimutator or tetraploid after 200 generations. We found a single tetraploid culture in 89 independent cultures propagated for 300 generations. We measured mutation rates of isolates from cultures that remained diploid and repeatedly found antimutator phenotypes. Thus, spontaneous antimutator alleles and polyploidization rescues haploid and diploid cells from EEX with markedly different efficiencies. Differences in the relative frequency of each escape mechanism may reflect the nature of the mutator alleles, the starting ploidy of the cells, or the magnitude of the initial mutation rate. Our findings in diploids suggest that mutator cancer cells near the edge of error-induced extinction may similarly be under selection for spontaneous antimutator mutations and polyploidization.

Prolonged activation of cardiac fibroblasts after acute wound healing post myocardial infarction results in excessive extracellular matrix deposition in the myocardium and formation of fibrotic scar at the site of injury. This adverse fibrotic remodeling disrupts physiological function in the heart and leads to increased risk for heart failure which is detrimental for patient outcomes. The reasons why activated fibroblasts, known as myofibroblasts, persist after wound stabilization are poorly understood due to the lack of a myofibroblast reporter system capable of direct longitudinal study. Current reporting modalities are invasive, resource and time intensive, wasteful, and indirect. The present project aims to design and develop an in vivo, repeatable, and real time reporter system for myofibroblast cell fate as a surrogate for cardiac fibrosis. To achieve this, we have designed a chimeric promoter to drive firefly luciferase vector expression specifically in myofibroblasts. We have built 9x tandem repeats of serum response factor (SRF) and nucleated factor of activated T-cells (NFAT) binding sequence since they are key mediators in myofibroblast activation. These repeats were ligated onto smooth muscle alpha actin (ACTA2) and collagen type 1 alpha 2 (COL1A2) minimal promoters, and these constructs were cloned into pGL3 luciferase reporter vectors. The specificity of the promoter was evaluated and optimized through in vitro luciferase assays with NIH 3T3 primary mouse fibroblasts.  With the successful creation of this reporter, wasteful use of animal models will be mitigated and direct mechanisms in the pathobiology of chronic cardiac fibrosis can be elucidated. Furthermore, efficacy of novel therapies can be evaluated in vivo.

Aging is characterized by time-dependent deterioration of cellular functions and increased risk for disease and death. The onset of age-related diseases including cancer, Alzheimer's, and other neurological disorders decrease the healthspan of humans. Healthspan is defined as the period of life without infirmity. My research focuses on understanding the basic biology of aging and determinants of healthspan within the emerging interdisciplinary field of geroscience. I use the model system Saccharomyces cerevisiae, a unicellular fungus, to study aging at the cellular level. To analyze cellular aging, I focus on replicative lifespan (RLS), which quantifies the number of daughter cells produced by individual mother cells before they reach replicative senescence and cease to divide. Our replicative lifespan team is composed of a group of undergraduate, graduate, and post-doctoral scientists at the University of Washington who use light microscopy and manual micromanipulation to quantify the RLS of hundreds of yeast mother cells each week. There are four important steps to analyze RLS in yeast: assay setup, incubation to outgrow cells, dissection of daughter cells from mother cells, and quantification of the number of daughter cells produced prior to replicative senescence. Using the replicative lifespan assay, I investigate genetic, environmental, and pharmacological treatments to determine their impact on cellular aging. Research produced by our RLS dissection team has led to a better understanding of evolutionarily-conserved factors that modulate aging, as well as potential treatments for certain human mitochondrial diseases.

Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch repair proteins lead to increased error rates during DNA replication that accelerate the evolution of cancer cells and contribute to chemotherapy resistance. Work in the yeast Saccharomyces cerevisae indicates that excessive DNA replication errors can lead to error-induced extinction (EEX), where every cell within the population dies due to a random lethal mutation. Thus, a possible direction for cancer therapy may be to modulate mutation rates of mutator cells – to either increase mutation rates to a lethal level or suppress mutation rates to their baseline levels in order to slow the rate of tumor evolution. Genetic pathways that may be targeted to modulate mutator phenotypes have yet to be fully elucidated. To identify such pathways, we isolated spontaneous haploid yeast eex mutants that escaped from error-induced extinction caused by combining a mismatch repair defect (msh2Δ) with the pol2-L439V allele, which models a human cancer mutation affecting DNA polymerase epsilon. We crossed the eex mutants to wild type yeast, generating diploid strains that were heterozygous for msh2Δ, pol2-L439V, and the eex mutation. Following sporulation, we measured the mutation rates of haploid MMR-proficient pol2-L439V progeny in order to assess the presence or absence of the antimutator mutation. For each mapping experiment, cells showing evidence of suppressed mutation rates were pooled separately from those exhibiting the normal pol2-L439V mutator phenotype. Unique mutations present only in the antimutator pool were identified by Illumina Next Generation Sequencing. Candidate eex mutations were engineered into fresh strains to assess the antimutator phenotype. Our compiled list of antimutator mutations and their functions in yeast may allow us to identify analogous mutations in the human genome and explore new methods of cancer therapy by which specific genes can be targeted to lower mutation rates to normal levels.