Barth Syndrome is a rare X-linked genetic disorder that can cause cardiac myopathy, muscle weakness, fatigue, immune dysfunction, and childhood mortality. Like many such diseases, Barth Syndrome is the product of a mutation encoding for a single protein, Tafazzin, a phospholipid-lysophospholipidtransacylase found primarily in cardiac and skeletal muscle, where it plays an integral role in modifying cardiolipin, a component of the mitochondria, or energy production organelles of the body. When Tafazzin, and consequently the modified form of cardiolipin are absent, the function of the mitochondrialelectron transport chain, the primary pathway by which the body converts food into useable energy, is reduced. We hope to rescue defective cardiolipin modification through Tafazzin protein therapy and learn more about tafazzin structure and function. To this end we used X-Ray Crystallography to obtain a three-dimensional electron density map corresponding to the diffraction pattern of the protein, based on the distance and angle between each atom. The determination of structural properties illuminated binding sites and chemical interactions in both functional and mutant Tafazzin. This was supplemented by the synthesis of enzymatically active and stable human and mouse Tafazzin using E. coli (BL21(DE3)pLysS). Overexpression of Tafazzin results in relatively pure inclusion bodies which we we isolated through protein metal affinity chromatography. We produced several protein coding variants of Tafazzin in order to study the enzymatic activity of different mutants. Understanding of structure-activity relationships in Tafazzin will direct future work in Barth Syndrome treatment by facilitating  in vivo studies of Tafazzin protein delivery.

Lead zirconate titanate (PZT) is a piezoelectric material commonly used in sensors and actuators. The goal of this project is to formulate a process of producing PZT nanoparticles to be imbedded in a polymer ink that could be used in a 3D printer. The particles must be small enough to make a homogenous colloidal ink that will not obstruct the printer nozzle. My project analyzes the synthesis procedure in detail and how it can affect the piezoelectric properties and morphology of the product. Hydrothermal synthesis was performed at 200 degrees Celsius to control particle size, agglomeration, and chemistry of the PZT particles. It was found that the previous method of synthesis produced particles on the order of 1 to 3 microns in size. In order to reduce particle size below 1 micron I have proposed to reduce time spent growing the particles at elevated temperatures.

As our understanding of the genetic basis for the pathophysiology of many diseases has broadened, gene therapy, which is the delivery of genetic material into cells in order to supplement or alter defective genes, is being explored as a potential treatment option. Despite its promise, no gene therapies have been approved for clinical use due to the lack of safe and effective vectors. There are many obstacles that must be overcome in order to successfully transfect a cell with exogenous DNA. First, the vector-DNA complex must navigate through the extracellular environment and be uptaken into the target cell. Then, the genetic material must achieve endosomal escape, be released from the complex, and then translocate into the nucleus where it can be transcribed and translated into functional or therapeutic proteins. In the Pun Lab, we previously synthesized a block-statistical copolymer comprised of different hydrophilic and hydrophobic segments aimed at providing a plethora of functionalities to the formed polyplexes. Due to this polymer’s unique architecture, it exhibits transfection efficiencies higher than branched polyethyleneimine, the gold standard for nonviral gene delivery, but still fails to reach the same level of transfection efficiency as seen with viral vectors. In this project, two additional modifications will be made to the copolymer and a library of well-defined polyplexes with varying formulations and structures will be synthesized and evaluated in vitro and in vivo. These modifications, guanidinylation and conjugation of a targeting peptide, aim to increase uptake into cells through electrostatic interactions and increase localization to the target cell type. From these studies, the ideal method to incorporate both modifications into one system to improve transfection efficiency and the structure-function effects on transfection efficiency will be evaluated.

Ammonia-oxidizing archaea (AOA) are essential to the nitrogen cycle in the environment.  By processing ammonia into nitrite, AOA impact plant growth and other environmental factors such as greenhouse gas emissions and groundwater chemistry- mainly leaching of mobile nitrite and nitrate.  To optimize agricultural practices and minimize ecological impacts, a better understanding of the role of these microorganisms in the environment is needed.  AOA have been identified as the dominant nitrifying group at long-term agricultural study sites in eastern Washington.  Enrichment cultures of AOA have been established from these sites.  However, the relationship of the enriched populations to other AOA remains unknown. The goal of my research is to scale-up existing AOA enrichment cultures for nucleic acids extraction and molecular analyses.  Isolated DNA will be PCR amplified for phylogenetic and functional markers, mainly the 16S rRNA and ammonia monooxygenase subunit A gene, respectively.  These PCR products will then be cloned, sequenced, and compared to other AOA in public databases to assess their relationships with other populations.  These data will greatly aid in the design and interpretation of further physiology and environmental genomic surveys of AOA in the environment.

Thaumarchaeota are widespread in marine and terrestrial environments. Many of them are thought to carry genes coding for a putative ammonia monooxygenase (AMO), suggesting a capacity for ammonia oxidation. However, strong evidence from isotopic studies and molecular surveys, as well as direct physiological characterization shows some populations of ammonia-oxidizing archaea (AOA) have a capacity to use alternative fixed carbon for biosynthesis, and suggests their mixotrophic lifestyle. In this study, we investigate the influence of organic substrates on growth and activity of the only two available isolated representatives of marine AOA, Nitrosopumilus maritimus strain SCM1 and strain HCA1. The certain intermediates of the TCA cycle are supplemented into AOA pure cultures, and the growth response of each strain to different organic substrates are monitored by measuring nitrite production and cell counts. The calculated specific growth rate, doubling time and specific cell yields are compared between organic carbon amended cultures and organic carbon-free controls. The SCM1 and HCA1 growth rate and cell yield were greater in cultures supplemented with alpha ketoglutaric acid (from 93.4 to 112.6 x 1012 cells mol-1 NH4+ for SCM1, and no growth to 80.8 x 1012 cells mol-1 NH4+ for HCA1), which suggest that marine AOA may prefer a mixotrophic lifestyle. This data expands our knowledge of thaumarchaeal metabolic capabilities and can provide information on the competition between AOA and other microbial groups to improve our understanding of microbial ecology.

Biological systems coordinate themselves through cues such as ion currents and membrane electric potentials. This communication utilizing membrane proteins such as membrane receptors, channels, and pumps allows biological systems to form hierarchies of control over their systems that have yet to be achieved in conventional electronic systems. Integrating such biological components with nanoelectronics could allow exciting new developments in the field of biomimetic energy conversion. However, this approach requires the seamless incorporation of biological structures into electronics. In order to use membrane proteins, they must be integrated with a lipid bilayer which is essential to create a cell membrane mimicking environment. Lipid bilayers have previously been integrated on solid substrates for electrophysiological measurements and the construction of organic-inorganic hybrid devices, but have yet to be formed on PdH_x. In this work, we build the foundation towards harnessing this biology-based method of control by forming a lipid bilayer composed of DPhPC/DPhPE/PI/Cholesterol on PdH_x contacts. The formation and deposition of electrochemically robust liposomes on a PdH_x surface was confirmed using dynamic light scattering (DLS) measurements, atomic force microscopy (AFM), and resistance measurements with a PdH_x proton field effect transistor (H⁺-FET). Vesicle formation was proven through DLS by determining size of the lipid structures in aqueous solutions. These vesicles were then deposited onto a PdH_x contact and formed solid-supported bilayers on PdH_x surfaces by the process of spontaneous vesicle adsorption and rupture as confirmed by AFM. Finally, the electrical stability of the bi-lipid membrane was characterized through electrical measurements with an H⁺-FET, confirming minimal H⁺ leakage across the bilayer. Future work includes embedding an ion pumping membrane protein, bacteriorhodopsin, into the bi-lipid membrane to enable electrochemical gating of the PdH_x H⁺-FET through optical or other environmental cues for biomimetic energy conversion.

Chitosan and alginate are widely used natural polymeric biomaterials that are biocompatible, biodegradable, and non-toxic. Chitosan is cationic as well as hydrophobic in nature whereas alginate is anionic and hydrophilic in nature. Ionic interaction between the amine and carboxyl group causes the formation of polyelectrolyte complex in the scaffold and thus enhancing the mechanical properties of the scaffold. Mechanical properties of the scaffolds are dependent on the fabrication process, cross-linking process, and sterilization process. Similarly, porosity plays a crucial role in determining the mechanical strength of the scaffold which relates to the lyophilization process and molecular weight of the polymer used. Chitosan-Alginate (CA) scaffolds are prepared by mixing equal wt% amounts of each polymer and due to chitosan's hydrophobic nature, acetic acid was added. The solution was mixed using a Thinky mixer at 2000 rpm for five minutes. After obtaining a homogeneous mixture, pH was tested and solution  casted into desired well plates. The solution was then refrigerated at 4 degree Celsius for 12 h at 20 degree Celsius overnight, and lyophilized for 24 hours. Scaffolds were then cross-linked with 70% CaCl2, and sterilized with Phosphate-buffered saline (PBS). Scanning electron microscope (SEM) is used in to image pore morphology of the scaffold. High mechanical strength and porous structure is presented in pore size ranges of 100-500 micrometers which is required for bone tissue engineering. Mechanical strength of the scaffold is obtained by performing compressive test. Samples are compressed at 4.00 mm/min using micro-tensile tester with 1N load cell. Dry samples were found to have a young's modulus (YM) of 1-2 MPa, whereas wet samples were found to have a YM of 1-2 KPa.  CA scaffolds were found to be mechanically strong with good porosity which was directly impacted by fabrication process. Mechanically strong CA scaffolds with proper porosity were achieved with the fabrication process used. Further work will allow an optimization of CA scaffolds for specific applications and cell lines.  

Planet earth has been experiencing droughts of devastating proportions. With these droughts becoming more frequent and widespread, many areas of the world are unequipped to deal with the economic and agricultural impacts. About 44 percent of the world’s population resides in and around coastal regions. Thus, resorting to the oceans for clean drinking water is an obvious solution. Zwitterions are environmentally friendly molecules that maintain a positive and negative charge simultaneously. Research has shown that these types of molecules possess non-fouling or fouling release capabilities in a variety of marine applications due to their hydrophilic properties. Therefore, using zwitterionic polymer coatings in an effort to eliminate the membrane fouling issue that plagues ocean water reverse osmosis (RO) desalination plants is promising. Currently, harmful chemicals are added to the water to reduce membrane fouling, but this approach merely suppresses the issue. RO membrane fouling has prevented ocean desalination from emerging as a viable source of fresh water due to high maintenance and operation costs. Most commercially available RO membranes are comprised of either cellulose acetate or aromatic polyamide, and both are susceptible to fouling. I will synthesize two zwitterionic polymers, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA), that will be grafted to the membrane surface. I will then expose the coated membranes to solutions of fluorescently tagged protein and compare the non-fouling performance to an uncoated membrane by measuring the fluorescent light intensity of the surface binding proteins. In addition, enzyme-linked immunosorbent assays (ELISA) will be used to quantify the surface protein binding. Utilizing the zwitterionic coatings to eliminate the fouling issues experienced by desalination plants will reduce the monetary and environmental costs. This action will further prevent the addition of harmful chemicals that are released into the environment.

Cerebellar pathology has been associated with cognitive and affective deficits and cerebellar dysfunction may play a role in schizophrenia and autism spectrum disorder. Understanding how cerebellar abnormalities contribute to characteristic features of psychiatric disorders may pave the way to creating better therapies. One cerebellar output region, the lateral dentate nucleus (LDN), is associated with cognitive and affective roles in behavior. This region has reciprocal connections with the limbic system, where dopaminergic neurotransmission has been linked with psychiatric disorders. Cells expressing dopamine receptor-1 (D1R) can be found in the LDN, but their function is unknown. To determine whether D1R-expressing neurons in the LDN modulate cognitive, affective, and/or social behavior we used a pharmacogenetic approach in mice. Specifically, we conditionally expressed a yellow-florescent-protein--tagged HM4Di, the inhibitory DREADD Receptor (designer receptors exclusively activated by designer drugs) or green florescent protein (GFP) in D1R-expressing cells of the LDN by injecting mice expressing Cre recombinase, an enzyme used for precise gene expression manipulation, under control of the endogenous D1R gene (Drd1a^Cre/+) with conditional viral vectors. Systemic injection of the DREADD-specific ligand, Clozapine-N-Oxide (CNO), reversibly inhibits HM4Di-expressing cells allowing us to test their function in behavior. In the experiments I performed, in the presence of CNO, Drd1a^Cre/+; DNC-HM4 mice showed improved motor performance, decreased prepulse inhibition of acoustic startle reflex (a reflexive jolt triggered by sound), increased anxiety-like behavior in an elevated plus maze, reduced cognitive performance in a spatial navigation memory task, and impaired differentiation of novel and familiar mice compared to controls. No significant differences were seen between groups in an instrumental conditioning experiment, a measure of motivation for food reward. D1R-expressing neurons in the LDN influence specific cognitive, affective, and social behaviors. Alterations in dopaminoceptive neurons within specific cerebellar nuclei may play important roles in the etiology of symptoms of mental illness.

Stochastic modeling has been broadly used in the financial market and actuarial industry containing certain derivative investment products. It utilizes past or present information to measure unknown future values. These models are based on Brownian motion, which is the continuous analog of random walk, and takes the randomness of future paths into account. An effective tool to analyze these models is stochastic calculus. I have read and studied some past scholars’ works, which had come up with quite many variations of stochastic models in different financial situations, such as markets involving at least one risky asset (e.g. stock) and a riskless asset (e.g. money market). However, I have noticed that more study needs to be done in incomplete markets where there exists arbitrage opportunities (i.e. riskless profit). Because in real life, when major events such as bankruptcy, financial crisis, and natural disaster happened, original pricing models based on the Black-Scholes model cannot make valuable predictions during a period of time due to the incompleteness of the market. I am trying to estimate an optimal portfolio under those circumstances by utilizing probability theories to modify the existing models. In this model, volatility of assets will be treated as random variable. By utilizing machine learning techniques, synthetic data generated by computer programs will be used to test the fit and efficiency of the model.

Genome-Wide Association Studies (GWAS) determine the places in the genome where differences exist in the genetic sequences of individuals who have a disease as compared to a control group. Combining publicly available data from GWAS increases the ability to identify locations in the genome that may be shared by Autoimmune Diseases and thus share increased risk. It is well-known that many disease traits are polygenic and that it may actually be the smaller, combined contribution of many genes that influences risk and is therefore a better indicator of disease probability. Previously, I elicited the number of genes in common among autoimmune diseases, finding 32 genes associated with a cluster of five diseases and two clusters of two diseases each associated with 22 genes. Aware of the need for a rigorous analysis to determine whether these patterns are both novel and of statistical significance, I am developing multi-locus analytical methods to elicit the additive effects of each genetic location that may underlie the pathway toward disease development. To overcome single locus limitations, I am performing gene enrichment analysis on the genes that show these patterns to discover if single locations associated with these diseases are also associated with biochemical pathways and thus are of biological relevance. Finally, I am examining pair-wise analyses across the autoimmune diseases in question by using Biostatistics software packages such as Generalized Multifactor Dimensionality Reductions (GMDR). Continued identification and further characterization of genetic variants that may be causative for shared biochemical pathways and subsequent triggering of autoimmune diseases can be used to not only improve the health of those currently suffering by developing therapies that may be effective across multiple diseases, but also to develop targeted treatments designed to help prevent their development in those individuals who are genetically predisposed.

Tissue engineering is a field of study where porous scaffolds made of materials such as polymers and/or ceramics are used to promote tissue regeneration. This is important for treatment of tissue defects resulting from trauma or disease where repair does not occur via normal, physiological processes. In this work we fabricated 3D, porous scaffolds made of naturally-derived polymers, chitosan and alginate, using a phase separation and lyophilization technique. This chitosan-alginate (CA) scaffold is promising for tissue engineering because it is made of sustainable materials and is biocompatible, biodegradable, and cost-effective. We characterized the porosity of the scaffolds using scanning electron microscopy and observed interconnected pores on the order of 100 microns in diameter. As a first step in determining the potential of this CA scaffold for muscle tissue engineering, we cultured C2C12 mouse myoblasts and evaluated cell viability and proliferation using the Alamar Blue assay. The scaffolds support cell attachment and viability and we expect to observe cell proliferation.

The goal of my research is to  find an efficient drug delivery system to kill brain cancer cells by considering that some hydrogels change their viscosity dramatically at different temperatures (called thermosensitive). The whole project is regarded as “rocket”system, with a drug-loaded device that delivers content into the right place as a rocket and the drug  inside as an astronaut. The innovative idea of the project is that the drug-loaded system has the capability to be liquid-like at low temperature while becoming gel at body temperature, making it easy to produce and inject into tumor tissue to concentrate the effect at the tumor site. To find a stable, biocompatible and biodegradable material to be drug-loaded, we tested the in vitro stability and drug-release during 1hr, 24hr and 96hr incubation of various polymer gels including PEG-chitosan, alginate, and methyl cellulose. We then studied the drug delivery system with brain cancer cells in vitro and monitored the migration of the cancer cells towards the gel.

Ischemic preconditioning (IPC) is the neuroprotective phenomenon in which a brief ischemic episode induces genetic reprogramming of affected brain cells, causing physiological changes that minimize cell death from subsequent, sustained ischemic insult. We hope to characterize the cellular and molecular mechanisms of IPC. Our lab previously has shown that IPC is dependent on Toll-Like Receptor 4 (TLR4) and involves the production of interferon stimulated genes (ISGs) in microglia. Following ischemia, damaged neurons release endogenous TLR4 agonists called damage-associated molecular patterns (DAMPS), which induce interferon (IFN) release by microglia. The IFNs initiate a biochemical signaling cascade that activates transcription factors which bind to the interferon-stimulated response element (ISRE), promoting ISG transcription which alters the immunological function and phenotype of microglia, mediating neuroprotection. My project is to confirm the cell signaling mechanism by which hypoxic/hypoglycemic (H/H) conditions induce ISG expression in vitro. To accomplish this, I am using a reporter assay to detect ISRE activation with luciferase protein expression, looking specifically at the effects of H/H experimental stimulations on ISRE activity in microglia from the N9 cell line. I am first optimizing a transfection protocol using green fluorescent protein (GFP) as a reporter. I will use the optimized protocol for experiments, transfecting microglia with DNA encoding the luceriferase enzyme linked to the ISRE promoter, then incubating the cells under H/H conditions, and finally using a plate reader to quantify fluorescence. We anticipate that H/H stimulation will result in increased luminescence in wild-type microglia, and we would interpret these results as indicating that ISG expression in H/H-stimulated microglia is mechanistically due to transcriptional activation at ISRE sites. Identifying the genetic actors in IPC will reveal intracellular targets for pharmaceuticals that can mimic this neuroprotection. We can then administer these medications pre-emptively to patients at risk for stroke or emergently to acute stroke patients.

Cardiac cell death has been implicated in the pathogenesis of various forms of cardiomyopathy and end-stage heart failure. Here, a novel signaling molecule, TAK1 (TGFβ-activated kinase 1, gene name Map3k7), is identified as a nodal regulator of apoptotic and necroptotic myocyte death, myocardial remodeling, and heart failure propensity. I found that genetic or pharmacological inactivation of TAK1 in cardiac myocytes markedly induced programmed necrosis and apoptosis in response to TNFα (tumor necrosis factor alpha), an inflammatory cytokine. Conversely, overexpression of the constitutively active mutant TAK1ΔN (N-terminal truncation), or TAK1 plus its activator TAB1 protected cardiac myocytes from TNFα-induced cell death. Mechanistically, inactivation of TAK1 promoted formation of the necroptotic cell death complex consisting of RIP1 (Receptor-interacting protein 1), RIP3 (Receptor-interacting protein 3), caspase 8 and FADD (Fas-Associated protein with Death Domain). In addition, my data showed that genetic ablation of cytoplasmic cell death signaling molecules such as RIP1, RIP3, caspase 8, or FADD largely blocked TNFα-induced cell death in TAK1-deficient cells, whereas deletion of mitochondrial cell death proteins, Bax/Bak or cyclophilin D, showed no effects. Further, the transcription factor NFκB-mediated cell survival signaling was greatly impaired in TAK-1 deficient cardiac myocytes. Taken together, the data indicate that TAK1 functions as a critical “molecular switch” in TNFα-induced programmed necrosis in cardiomyocytes by interacting with the RIP1/3-caspase 8-FADD cell death pathway as well as the NFκB cell survival pathway. These results also reveal a new paradigm where TAK1 functions as a necessary survival factor in the heart by directly antagonizing necroptosis.

Stroke is the fourth leading cause of death and the leading cause of long-term disability in the United States. Microglia are the resident immune cells in the central nervous system and play critical roles in stroke pathophysiology. During ischemic stroke, cytokines known as interferons (IFNs) are released from CNS cells and bind to microglial cell surface receptors. This binding initiates a signaling cascade that leads to increased expression of Interferon-Stimulated Genes (ISGs). We hypothesize that increased expression of ISGs contributes to post-stroke neuroprotective pathways. There are three types of IFNs, and Type 1 includes IFNs α and β, which are well-known immunomodulatory factors. Type 2 IFNs include IFNγ, a prototypical pro-inflammatory activator. Type 3 IFNs (IFNλs) are the most recently discovered and least well characterized. IFNλs are effective in treating several viral illnesses and they are better tolerated (fewer side effects) than Type 1 IFNs. We have recently shown that Type 1 IFNs can induce increases in ISG expression in microglia. My project goal was to characterize the ability of IFNλs to induce ISG expression in microglia and compare their pharmacological potency to that of Type 1 IFNs. To do this, I stimulated primary microglia with varying dosages of IFNλ3 and for varying amounts of time. I extracted RNA and prepared protein lysates from the stimulated cells. ISG expression was quantified by qRT-PCR and immune (Western) blotting. I also collected conditioned medium and assayed for the release of specific ISG chemokines, such as CCL5 and CXCL10 using ELISA. I obtained dose response curves and time course data. Our results showed robust up-regulation of multiple ISG mRNA transcripts and increases in release of specific chemokines following stimulation of microglia with IFNλ3. These studies will help us continue to characterize important cellular and molecular targets for pharmacological intervention in stroke.