Published ETD Collection
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Browsing Published ETD Collection by Department "Biology and Biochemistry, Department of"
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Item A Genome-Wide Search for Tumor Suppressor MicroRNAs in Ovarian Cancer(2014-08) Hernandez Herrera, Anadulce 1982-; Gunaratne, Preethi H.; Flores, Elsa R.; Wang, Yuhong; Zhang, Xiaoliu Shaun; Widger, William R.Ovarian cancer is one of the most lethal cancers among women. The Cancer Genome Atlas (TCGA) is a collaborative effort, which seeks to characterize the complete set of molecular changes associated with cancer and provide a public resource that will allow the development of new therapies and better diagnostic tools for cancer. Much of the focus is on protein coding genes and our understanding of the contribution from non-coding RNAs is lagging behind. MicroRNAs are small non-coding RNAs that can bind and repress hundreds of gene targets to regulate gene networks. Therefore, defining and understanding the miRNA-regulated genes offer new insights that can be clinically applied for many of the disease. In order to identify new tumor suppressors for ovarian cancer and downstream targets that drive key aspects of this disease such as drug resistance and metastatic spread, 3 candidates were selected from the microRNA-mRNA bioinformatic analyses from the TCGA. A combination of molecular and functional studies confirmed that miR-29a that can regulate genes from the histone modifier and cell cycle pathways, inhibit proliferation and moderately increase cisplatin response in the p53-WT HEYA8; miR-509-3p which targets genes from the ECM/EMT networks, inhibits cell proliferation in p53-WT HEYA8 and p53-mut OVCAR8 and correlated with improved overall survival when analyzed by in situ hybridization in an independent cohort; miR-130b increases apoptosis by 3-fold in p53-mutant OVCAR8 and p53-wild-type HEYA8 and significantly induces TAp63 and BCL2L11 (BIM). Forced expression of TAp63 decreases cell viability by 60-80% and miR-130b-ABT-737 (BCL2L11-mimetics) combination increases apoptosis by 9-fold suggesting TAp63 and BIM are critical effectors of the tumor-suppressive mechanisms driven by miR-130b, and can be used to develop new therapeutic strategies that will target p53 WT and p53 mutant tumors.Item A Novel Function for Juvenile Hormone in Male Courtship Behavior of Drosophila melanogaster(2013-08) Wijesekera, Thilini P. 1976-; Dauwalder, Brigitte; Roman, Gregg; Williams, Cecilia M.; Mattox, WilliamJuvenile hormone is a significant insect hormone controlling development as well as reproduction, migration, and social behavior. Juvenile hormone-binding proteins of many insects are similar to Drosophila melanogaster ‘Takeout’, a protein preferentially expressed males that influences courtship behavior. This raises the possibility for a role for Juvenile hormone in male courtship behavior. This hypothesis was tested by creating flies with reduced Juvenile hormone levels and examining their mating behavior. To achieve a reduction in levels, a key enzyme of the Juvenile hormone synthesis pathway was targeted by RNA interference (RNAi). Juvenile Hormone Acid Methyl Transferase (JHAMT) catalyzes one of the last steps in the hormone’s synthesis in the Corpora allata, the organ of Juvenile hormone synthesis. A new Corpora allata-specific GAL4 driver line was created and used to direct JHAMT-RNAi to these cells. Courtship assays of the resulting males showed that RNAi against JHAMT results in a mutant courtship phenotype. Constitutive reduction as well as specific adult reduction reduced courtship. The same result was also obtained by conditional genetic ablation of the Corpora allata in adults. The courtship defects could be rescued by application of the Juvenile hormone analog Methoprene shortly before behavioral testing. Together, these results show that normal adult Juvenile hormone levels are physiologically required for normal male courtship behavior. It is unknown which protein(s) transport Juvenile hormone in Drosophila melanogaster. Since Takeout has many of the characteristics of JHBPs, we tested whether Takeout binds the hormone to act as a carrier for the hormone. No binding was observed between a Baculovirus produced Takeout protein and Juvenile hormone in an in vitro binding assay. The findings presented in this dissertation demonstrate a novel and important function for Juvenile hormone in the control of male courtship behavior in Drosophila melanogaster.Item A Novel Role of Fascin in Carcinoma Cells(2018-12) Ahrorov, Afzal 1986-; Khurana, Seema; McConnell, Bradley K.; Dauwalder, Brigitte; Chung, Sang-HyukColorectal cancer (CRC) is the second deadliest cancer in the US according to the 2014 National Cancer Institute data. Despite recent academic and medical advances, our understanding of the progression of CRC and, therefore, new therapeutic approaches to improve patient outcomes remains poor. Fascin, an actin-bundling protein, although absent from the adult epithelial cells of the colon, is often overexpressed in CRC. In this study we investigated the role of fascin in CRC. We also examined the cancer-related functions of fascin in non-transformed Madin-Darby Canine Kidney cells (MDCK) to validate fascin specific functions in CRC cells. CRC cells (HT-29/19A) and MDCK cells with or without fascin were analyzed for the formation of multilayered epithelium, a phenotype in which cells grow on top of other cells forming several layers of cells. We investigated cell proliferation and contact inhibition in low and high density cultures. Tumors were grown using CRC cells with or without fascin to evaluate the role of fascin in proliferation of cells in vivo. We measured the ability of cells to attach to a substrate. Cells were transduced with mCherry-tagged LifeAct lentivirus to observe the dynamics of stress fibers. Mechanistically, we investigated the role of actin-binding by fascin in stress fibers formation. The results show that fascin promotes multilayering of cells by evading contact inhibition in CRC and in non-transformed MDCK cells. Decreased integrin 1 protein and reduction in cell-matrix attachment causes this multilayering. Fascin disrupts stress fibers which in turn prevents the localization of vinculin and -actinin to focal adhesion sites. Mechanistically, by binding to LIMK1 (p-Lin-11/Isl-1/Mec-3 kinase 1), fascin prevents the phosphorylation of cofilin, which reduces polymerization of filamentous actin in stress fibers. We conclude that fascin promotes evasion of contact inhibition by dysregulation of stress fibers to promote multilayering of carcinoma cells.Item A Proteomic Signature of Dormancy in the Actinobacterium: Micrococcus luteus(2018-05) Mali, Sujina 1987-; Bark, Steven J.; Schwartz, Robert J.; Widger, William R.; Willson, Richard C.Dormancy is a protective state in which diverse pathogenic and non-pathogenic bacteria curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states including viable-but-non-culturable (VBNC) and persistence states that contribute to the antibiotic tolerance. Reactivation from latent infection are observed in many serious pathogens including Mycobacterium turberculosis, Staphylococcus, Streptococcus, and Borrelia bacteria. Despite the obvious threat presented by dormant bacteria, the protein mechanisms regulating these dormancy states are not well understood. We have studied VBNC dormancy in Micrococcus luteus NCTC 2665 by tandem mass spectrometry-based quantitative proteomics to uncover some of these mechanisms. M. luteus is a nonpathogenic actinobacterium exhibiting a uniquely well-defined and reproducible VBNC state induced by nutrient deprivation. Dormant M. luteus demonstrated a global loss of protein diversity accompanied by increased levels of eighteen proteins that are conserved across actinobacteria including M. tuberculosis. Four of these proteins have been previously associated with latent tuberculosis, but the other 14 proteins are novel protein targets for dormancy studies. We have developed rapid methods to quantitate dormancy-related proteins across growth phases by targeted proteomics. The proteins upregulated during dormancy implicate important roles for anaplerotic metabolism, redox and amino acid metabolism, ribosomal regulatory processes, and nucleoid associated proteins in dormancy. Our data show that M. luteus is a viable model system for dissecting the protein mechanisms underlying dormancy and we identified new protein targets for future studies on therapeutics active against dormant bacterial infections, which is a severe limitation of current antibiotics.Item A Requirement for the Lysine Methyl Transferase SMYD1 in Myoblast Differentiation(2015-12) Nagandla, Harika 1987-; Schwartz, Robert J.; Stewart, M. David; Chung, Sang-Hyuk; McConnell, Bradley K.; Wells, Dan E.The SMYD (SET and MYND domain) family of lysine methyltransferases harbors a unique structure in which the methyltransferase (SET) domain is intervened by a zinc finger protein-protein interaction (MYND) domain. SMYD family proteins methylate both histone and non-histone substrates and participate in diverse biological processes including transcriptional regulation, DNA repair, proliferation, and apoptosis. Smyd1 is unique among the five family members in that it is specifically expressed in striated muscles. Complete deletion of Smyd1 in mice caused embryonic lethality at E10.5 due to defects in heart development prior to proper onset of skeletal myogenesis program. Smyd1 is expressed in the skeletal muscle lineage throughout myogenesis and in mature myofibers, shuttling from nucleus to cytosol during myoblast differentiation. Because of this expression pattern, we hypothesized that Smyd1 plays multiple roles at different stages of myogenesis. The goal of our study was to decipher Smyd1’s role in mammalian skeletal muscle development. This was achieved by using Myf5-cre to knock Smyd1 out of earliest skeletal muscle precursor cells, thereby providing clues to its nuclear function. We found that Smyd1 is dispensable for initiation of skeletal myogenesis and Smyd1 conditional knock-out (CKO) embryos appear unaffected during the primary myogenic wave. However, by the second myogenic wave, both primary and secondary muscle fibers in Smyd1 CKO embryos had declined in number with no change in myoblast proliferation or apoptosis as compared to control embryos. A number of skeletal muscle-specific genes were found to be down-regulated at the mRNA level in Smyd1 CKOs by the secondary wave of myogenesis. Also, Smyd1 CKO embryos exhibited significantly higher percentage of Myog+ cells in the EDL during the second wave of myogenesis, indicating a block in downstream differentiation. Mutant embryos showed perinatal lethality and subcutaneous edema. Down-regulation of Smyd1 in C2C12 skeletal myoblast cell-line led to poorly differentiated, thinner myotubes, thereby recapitulating our observations in-vivo. Smyd1 appears important for proper down-stream differentiation of skeletal myoblasts.Item A Role for the Histone Chaperone Hira in Muscle Hypertrophy, Cellular Stress Responses, and Developmental Gene Expression(2016-12) Valenzuela, Nicolas 1988-; Schwartz, Robert J.; Stewart, M. David; Gunaratne, Preethi H.; McConnell, Bradley K.; Wells, Dan E.Chromatin modifications play a pivotal role in regulating gene expression. The deposition of histone variants by histone chaperones into the nucleosome plays a large role in influencing gene expression. Histone incorporation can be separated into two categories: replication-coupled and replication independent. The histone chaperone HIRA deposits the variant histone H3.3 into promoters and gene bodies of active genes in a replication-independent manner. HIRA is also responsible for H3.3 deposition into the “bivalent” promoters of regulatory genes in embryonic stem cells, required for transcription restart after DNA repair, and required for the formation of senescence associated heterochromatin foci. Hira null mutation in mice resulted in embryonic lethality by E11 which largely resulted from gastrulation defects including abnormalities in the heart. Cardio- and skeletal myocytes are post mitotic cells and thus the majority of chromatin remodeling should be performed in a replication-independent manner. Additionally, skeletal myocytes must alter gene expression in response to physiological signals. Because of this we hypothesized that HIRA is likely to play a large role in epigenetically regulating gene expression in myocytes. The objective of this study was to determine the consequence of HIRA ablation in cardio- and skeletal myocytes in vivo. We accomplished this by using Myf6-cre to delete Hira from myofibers, and αMHC-cre for cardiomyocytes, both of which remove Hira after these cells have terminally differentiated. Both HIRA CKO cardio- and skeletal myocytes exhibited hypertrophy, sarcolemmal damage, upregulation of fetal/developmental genes, and downregulation of genes associated with responses to cellular stresses and DNA damage. This resulted in focal replacement fibrosis and altered cardiac function in the heart, while mice lacking HIRA from myofibers exhibited decreased body weight, increased lean mass, increased grip strength and endurance, increased abundance of type I fibers, and centralized nuclei. Comparative analysis of gene expression sets suggest that loss of HIRA impaired transcriptional response to cellular stresses. The major discrepancies in these phenotypes can largely be attributed to each tissues mode of regeneration in which cardiomyocytes lack regenerative potential while skeletal myocytes can rapidly regenerate damage. Thus HIRA is an important factor in epigenetically maintaining myocyte homeostasis.Item A test of the Snowball model(2016-12) Kalirad, Ata 1989-; Azevedo, Ricardo B. R.; Bassler, Kevin E.; Cooper, Timothy F.; Ostrowski, Elizabeth; Zufall, Rebecca A.Genetic incompatibilities can emerge as a by-product of genetic divergence. According to Dobzhansky and Muller, alleles at different loci that have fixed in different genetic backgrounds may be incompatible when brought together in a hybrid. Orr showed that the number of Dobzhansky--Muller incompatibilities (DMIs) should accumulate faster than linearly---i.e., snowball---as two lineages diverge. Several studies have attempted to test the snowball model using data from natural populations. One limitation of these studies is that they have focused on predictions of the snowball model but not on its underlying assumptions. Here I use a computational model of RNA folding to test both predictions and assumptions of the snowball model. In this model, two populations are allowed to evolve in allopatry on a holey fitness landscape. I find that the number of DMIs involving pairs of loci (i.e., simple DMIs) does not snowball---rather, it increases approximately linearly with divergence. I show that the probability of emergence of a simple DMI is approximately constant, as assumed by the snowball model. However, simple DMIs can disappear after they have arisen, contrary to the assumptions of the snowball model. This occurs because simple DMIs become complex (i.e., involve alleles at three or more loci) as a result of later substitutions. I introduce a modified snowball model---the melting snowball model---where simple DMIs can become complex after they appear. The melting snowball model can account for the results of the RNA-folding model. I also find that complex DMIs are common and, unlike simple ones, do snowball. Reproductive isolation, however, does not snowball because DMIs do not act independently of each other. I also test the snowball model at the population level using an individual-based model. Using this model, I show that recombination rate, gene flow, and ancestral polymorphism can slow down the snowballing of incompatibilities between diverging populations. These factors result in selection for mutationally robust genotypes, and genotypes that are more resistant to mutations are also more resistant to introgressions, which reduces the number of DMIs.Item Aberrant Expression of Embryonic Mesendoderm Factor MESP1 Promotes Tumorigenesis(2019-08) Tandon, Neha 1986-; Liu, Yu; Frigo, Daniel E.; Dauwalder, Brigitte; Lin, Chin-Yo; Lin, Weei-ChinLung cancer is the leading cause of cancer-related deaths in the United States, with KRAS and EGFR known as driver oncogenes for the disease. Adding to these are “lineage-survival oncogenes” where tumor survival depends on the aberrant expression of master transcriptional regulators of cell-lineages. Here, by integrating various patient gene expression datasets, we identified that basic helix- loop-helix transcription factor MESP1, a master regulator of mesendoderm development, has a previously-unknown association with Non-Small Cell Lung Cancer (NSCLC). We found that MESP1 expression correlates with poor prognosis in NSCLC patients, and is critical for proliferation and survival of NSCLC-derived cell lines, thus, implicating MESP1 as a lung cancer oncogene. Ectopic MESP1 expression cooperates with the loss of tumor suppressor ARF to transform murine fibroblasts. Xenografts of MESP1-knockdown cells showed decreased tumor growth in vivo. Global transcriptome analysis revealed a MESP1 DNA-binding dependent gene signature associated with various hallmarks of cancer, suggesting that the transcription activity of MESP1 is most likely responsible for its oncogenic abilities. These observations demonstrate MESP1 as a previously-unknown lineage survival oncogene in NSCLC.Item Abiotic and Biotic Factors Shaping Plant-Microbe Interactions(2021-12) Locke, Hannah E; Crawford, Kerri M.; Pennings, Steven C.; Frankino, W. Anthony; Miller, Tom E. X.Symbioses between plants and soil microbes can shape plant traits and performance, population dynamics, community composition, and ecosystem function. However, the context-dependency of plant-microbe interactions remains an underexplored frontier of scientific understanding. Given that the biotic and abiotic contexts of ecological communities is rapidly shifting due to anthropogenic change, understanding the factors that shape plant-microbe interactions will allow us to make more accurate predictions of ecological interactions both presently and in the future. Here, we explore plant-microbe interactions across three different contexts—herbivory, ecological succession, and a water availability gradient. In each of these contexts we demonstrate of the importance of context dependency in shaping the outcomes of plant-microbe interactions. For example, in Chapter II, we found evidence that a multiple species mixture of mycorrhizal fungi in soils can facilitate aboveground herbivory, but only in the context of previous herbivory. The interaction between aboveground herbivory experiences and belowground microbial symbioses yielded entirely different directional outcomes for herbivore performance. In Chapter III, we explored whether soil microbes from three distinct ecological communities across a successional gradient differentially influenced plant defense strategies against herbivores. We found evidence that soil microbes from mid and late successional habitats increased plant capacity to reduce herbivore damage (resistance) but did not influence plant capacity to regrow after damage (tolerance). In Chapter IV, we pivot to assess an important abiotic context of plant-microbe interactions: water availability. We demonstrated through direct experimentation that the positive effect of mycorrhizae on plant performance increased as water availability increased. To our knowledge, is the first strong evidence that indicates the importance of mycorrhizae in facilitating the host plant’s ability to improve growth when provided ample water or even flooding conditions. In exploring the context-dependency of plant-microbe interactions, we gathered strong evidence that the biotic and abiotic environment shapes the outcomes of plant-microbes interactions ranging from negative (facilitation of aboveground herbivores) to positive (improved resistance against herbivores; increased biomass production in high water conditions). Taken together, this works emphasizes the need to incorporate variability in abiotic and biotic conditions into our understanding of ecological relationships between interacting species.Item Acute Cellular and Behavioral Outcomes of Focal Impact Traumatic Brain Injury in Xenopus laevis(2022-11-28) Spruiell Eldridge, Sydnee Lea; Sater, Amy K.; Dauwalder, Brigitte; Alward, Beau A.; Lekven, Arne C.Traumatic brain injury (TBI) is one of the leading causes of mortality and disability throughout the world. The Centers for Disease Control and Prevention estimate that up to 5.3 million people in the United States are living with a TBI-related disability, including cognitive, behavioral, and functional limitations. Although TBI represents a significant public health concern, we currently lack effective therapies to promote repair and recovery in the brain. TBI is a complex condition; characterized by an initial insult which sets in motion a secondary injury cascade including neuroinflammation, blood-brain barrier disruption, edema, and reactive astrogliosis. Reactive astrogliosis occurs on a finely graded continuum, encompassing both the initiation of essential recovery mechanisms and exacerbating inflammation. As it has been linked to chronic neurodegenerative diseases, TBI represents the initiation of a disease process, rather than an isolated event. While the initial insult causes irreversible damage, secondary injury begins minutes after and can last for years, presenting a promising opportunity for therapeutic intervention. We begin by investigating the response to focal impact injury in the optic tectum of Xenopus laevis tadpoles. This injury disrupts the blood-brain barrier, causing edema, and produces deficits in visually-driven behaviors. Injured brains show an early transcriptional activation of inflammatory cytokines, followed by astrocytes undergoing morphological alterations and upregulation of genes consistent with reactive astrogliosis. Since our results demonstrate that the response to focal impact injury in Xenopus tadpoles resembles that of mammalian models, we present a new, scalable vertebrate model for TBI. Next, we focus on characterizing the current literature surrounding sex differences in animal models of TBI and presenting preliminary findings in our model. Estrogen and its derivatives have recently been explored for their potentially neuroprotective capabilities. Previous works have suggested that estrogenic compounds are able to regulate reactive astrogliosis, control neuroinflammation, and reduce edema. Since treatment with estrogen comes with significant risk factors, many have suggested the use of selective estrogen receptor modulators (SERMs) like Tamoxifen. Our results support tamoxifen as an effective neuroprotectant when administered after a TBI and demonstrate a successful proof-of-concept pharmacological intervention in our X. laevis model of TBI.Item Alignment- and Alignment-refining Algorithms: Effects on Branch-length Estimation and Selection Pattern Analyses(2015-05) Zheng, Yichen 1991-; Graur, Dan; Cooper, Timothy F.; Zufall, Rebecca A.; Josić, Krešimir; Nakhleh, Luay K.This dissertation consists of a study of the effects of multiple-alignment method on phylogenetic analyses. First, I investigated the effects of multiple-sequence alignment quality on branch-length estimation, which can influence downstream bioinformatic analyses such as estimating rates of evolution and divergence times. To quantify the accuracy of branch-length estimates, I devised a scale-free measure of branch length proportionality between two phylogenetic trees that contain the same taxa and topology. This measure was named “normalized tree distance” (NTD). NTD is an ideal measure for detecting coevolutionary processes, in addition to measuring the accuracy of branch-length estimates. Using NTD as an error measure, I investigated the effects of multiple-sequence alignment quality on branch-length estimation. I simulated coding sequences and estimated the effects of multiple evolution parameters and choice of alignment- and alignment-filtering algorithms on the accuracy of branch-length estimation. I demonstrated that branch-length accuracy is indeed dependent on the method of alignment. Alignments with high-accuracy algorithms combined with methods for filtering out unreliable sites produce significantly better branch-length estimates. The optimal method combination depends on the evolutionary scenario. Thus, different alignment algorithms and different combinations of algorithms yield better branch-length estimates under different evolutionary conditions. A judicious choice of alignment- and alignment filtering algorithms is recommended for phylogenetic studies. Second, I studied the correlation between two types of purifying selection: against nonsynonymous mutations and against deletions using mammalian genomic protein-coding sequences. Intuitively, a codon that is intolerant of amino-acid altering substitutions is expected to be also intolerant of deletion. However, there has not been any comprehensive study on this purported correlation. In addition to the nine-species alignments of 8,595 genes, I simulated coding sequences along the same phylogenetic trees. The real data showed a much stronger correlation than the simulated sequences. I demonstrated that the correlation between amino-acid replacement and deletion rates exists and cannot be explained solely by alignment errors. Further investigations on nonsynonymous and synonymous mutations showed that this is most likely due to selection rather than mutation rates. Understanding selection on different types of mutations would help strengthen the link between population genetics and sequence evolution.Item Altered Glucose Metabolism in Cancer Metastasis and Drug Resistance(2013-08) Chen, Jinyu 1985-; Zhang, Weihua; Warner, Margaret; Khurana, Seema; Lev, Dina Chelouche; Gan, BoyiGlucose, one of the most important energy sources for living organisms, is first broken down through glycolysis then either undergoes oxidative phosphorylation in the mitochondrion or fermentation in the cytosol. Abnormal glucose metabolism was first discovered by Dr. Otto Warburg, i.e. cancer cells carry out irreversible fermentation of glucose in the presence of oxygen, which is also termed aerobic glycolysis. My studies focus on understanding altered glucose metabolism in cancer metastasis and drug resistance. We found that breast cancer brain metastasized cells developed enhanced oxidation of certain amino acids and gluconeogenic activity for survival and proliferation when glucose level is limited. We also found that drug-resistant colon cancer cells exhibited up-regulated aerobic glycolysis to meet the need of a higher amount of intracellular ATP to cope with chemotherapeutic stress. These results suggest alterations in glucose metabolism play critical roles in the development of cancer brain metastasis and drug resistance. The molecular mechanisms identified by this study may serve as potential therapeutic targets for cancer treatment.Item Amplification of Minor Pathogenic Stem Cell Variants in Crohn's Disease(2020-12) Neupane, Rahul Dev; McKeon, Frank D.; Xian, Wa; Dauwalder, Brigitte; Gunaratne, Preethi H.; Hou, Jason K.Crohn’s disease (CD) is a progressive inflammatory and fibrotic disorder of the intestinal tract thought to arise from defective interactions of immune cells, intestinal microbes, and intervening mucosal barriers. Here we perform novel clonogenic analyses of stem cells derived from the terminal ileum of pediatric and adult patients with CD. We show that CD stem cell libraries are dominated by two variants that display ingrained pro-inflammatory and pro-fibrotic signaling. Transplantation of these variants to immunodeficient mice triggers key features of CD including leukocyte infiltration and fibrosis. These variants, which exist at low levels in control and fetal terminal ileum, display an absolute commitment to gastric epithelial fates. Together, this work links CD to the amplification of minor stem cell variants whose conventional roles are in the response to ancient human pathogens and suggests mechanistic analogies to chronic obstructive pulmonary disease (COPD) and perhaps other chronic inflammatory conditions. If true, Crohn's disease can be mitigated by therapeutics that selectively target these variant stem cells allowing existing normal stem cells to compensate for the loss of those. We develop a high-throughput chemical screening platform to uncover small molecules that specifically target these variant stem cells. Furthermore, these small molecules, in a unique combination with promoters, protect and promote normal stem cells while eradicating variant stem cells. Lastly, the clonogenic mucosal stem cell variants tied to pathological features of the disease are selectively targeted by candidate small molecules in the xenograft model diminishing those features in parallel and leaving normal stem cells to repopulate and maintain homeostasis.Item An Account of Chemical and Mechanical Regulation of TRPC6 Channels in Podocytes(2013-05) Anderson, Marc 1981-; Dryer, Stuart E.; Gunaratne, Preethi H.; Sheikh-Hamad, David; Ziburkus, JokubasPodocytes play a dynamic role in regulating glomerular filtration. The focus here is on the regulatory mechanisms of podocyte expressed transient receptor potential 6 (TRPC6) channels, an ion channel implicated in certain forms of proteinuric kidney disease. TRPC6 channels are polymodal and can be activated by either chemical or mechanical stimuli. Chemical stimulation is mediated by surface-expressed receptors, and the roles of angiotensin II type 1 receptors (AT1R), insulin receptors, and N-methyl-D-aspartate receptors (NMDAR) on TRPC6 activity are studied here. In acutely isolated rat glomeruli, angiotensin causes an upregulation of TRPC6 activity, and this is mediated by the Gαq /PLC pathway. This angiotensin-evoked upregulation is partially dependent on the formation of reactive oxygen species (ROS). In mouse podocyte cell lines, insulin causes upregulation of TRPC6 through increasing ROS formation via NADPH oxidase 4 (NOX4). Similarly, NMDAR activation upregulates TRPC6, albeit through NOX2. The previously uncharacterized podocyte NMDAR has unusual properties with strong physiological implications. Specifically, podocyte NMDA receptors are essentially unresponsive to L-glutamate and L-aspartate and do not show glycine-mediated potentiation. These receptors respond to the agonists L-homocysteate and D-aspartate with large ionic currents that are potentiated by D-serine. Given their resistance to L-glutamate-induced activation, podocyte NMDA receptors likely do not act in a localized glomerular signaling system. However, their response to ligands that circulate in both the normal and pathological state suggests a role for podocyte NMDA receptors in normal glomerular function. Receptor-driven upregulation of TRPC6 comprises a class of potential targets for prevention and treatment of multiple acquired kidney diseases. Independent of receptor-mediated response, TRPC6 channels are mechanosentive and can be activated by membrane deformation in both podocyte cell lines and isolated glomeruli. This mechanosensitivity is repressed by podocin, a cholesterol-binding, membrane-associated partner of the TRPC6 channel. In addition, podocin mediates diacylglycerol activation of TRPC6, suggesting that podocin determines the favored mode of TRPC6 activation in podocytes. It is possible that disruption of the podocin-TRPC6 complex at the slit diaphragm contributes to Ca2+ overload and eventual foot process effacement. Drugs that selectively target and suppress TRPC6 mechanosensitivity could potentially serve as treatments for glomerular diseases.Item An Exploration of Fitness Landscapes: Theory and Experiments(2018-08) Karkare, Kedar 1992-; Azevedo, Ricardo B. R.; Cooper, Timothy F.; Zufall, Rebecca A.; Meisel, Richard P.; Burch, Christina; Bataillon, ThomasGenotypes, phenotypes, and fitness are the ultimate determinants of evolution. The relationship between these three components is collectively referred to as the fitness landscape. Evolutionary biologists have been working to understand the mechanisms and processes governing the fitness landscape since the early 20th century. However, it has proved difficult to unravel due to the tremendous combinatorics of genotypes, and the complex relationships between all components of the landscape. Here I study evolution on the fitness landscape through a combination of modeling and experiments. I identify a paradox within Fisher’s Geometric Model of Adaptation, and relax the model’s assumptions in an effort to solve this problem. I find that restricting the level of pleiotropy and restricting the number of maladapted traits simultaneously solves the paradox, and maintains fits to other experimental data. To complement this modeling, I spend the second two results chapters discussing experimental results. I focus on a case study of genetic divergence in the E. coli lac operon repressor (lacI), and aim to understand the underlying processes and mechanisms that cause divergence. Divergence at this site is due to the historical contingency of mutation fitness effect on epistatic interactions with other substitutions. I then examine the underlying mechanism of hange in lacI mutation fitness effects. I find that the cost of lac expression has decreased in evolved strains, due to an increase in translational capacity. The major benefit of lacI mutations is rendered obsolete, by other mutations that provide a similar growth benefit, and they do not fix.Item An Investigation into the Role of 27-Hydroxycholesterol and Estrogen Receptors in Adipose Tissue, Obesity, and Breast Cancer(2021-05) Asghari Khonakdari, Arvand; Umetani, Michihisa; Xu, Yong; Zhang, Yang; Dauwalder, Brigitte; Chung, Sang-HyukObesity is an emerging health crisis all over the world. With obesity comes several other health disorders such as type-2 diabetes, cardiovascular diseases, and cancers. Hence, understanding the underlying reasons for obesity is of paramount importance as it can guide us in developing new therapeutic approaches for preventing or decreasing the obesity rates. Breast cancer is the second cause of cancer-related deaths among women worldwide. Endocrine resistance in breast cancer which occurs after endocrine therapies, causes the tumors to relapse after years of dormancy. While estrogen receptors (ERs) mutations and malfunctions of other signaling pathways (e.g., MAPK signaling) are some of the underlying reasons for endocrine resistance in breast cancer, the underlying causes of 60% of endocrine resistance cases remain completely unknown. Estrogen and estrogen receptors play important roles in both obesity and breast cancer. 27-Hydroxycholesterol (27HC), the first identified endogenous selective estrogen receptor modulator, can modulate the activity of estrogen receptors in different tissues and thus can be one of the important factors in regulating the functions of ERs in the context of obesity and breast cancer. In this dissertation, I first showed that 27HC mostly works as an antagonist for ERs activity in different tissues. Next, I investigated the effects of 27HC on adipose tissues and obesity. My research showed that 27HC increases body weight gain in the presence of a high-fat, high- cholesterol diet in an ER-dependent manner. Moreover, 27HC increases body fat percentage regardless of the diet and affects adipose tissue gene expression and induces inflammation in the adipose tissue. I also showed that 27HC alters the morphology and function of brown adipose tissue. In regard to endocrine resistance in breast cancer, I showed that 27HC increases the growth rate of the endocrine-resistant breast cancer cells, and I also found a novel group of genes that can be the underlying reasons for the endocrine development and progression. All in all, the research presented in this dissertation confirms the importance of 27HC in obesity and breast cancer and opens new doors toward the development of potential therapeutics to decrease the obesity rates, as well as treatment of endocrine-resistant breast cancer.Item Antiproliferative and pro-apoptotic actions of Estrogen receptor β in prostate cancer(2013-05) Dey, Prasenjit 1978-; Gustafsson, Jan-Åke; Ström, Anders M.; Schwartz, Robert J.; Webb, Paul; Wang, YuhongHigh Gleason grade prostate cancers are aggressive. Currently, the major target for treatment is the androgen receptor. Recent literature points towards a tumor suppressive role of estrogen receptor β (ERβ), which has a potential to be exploited as a target for novel therapeutics used for treatment of prostate cancer. In Chapter 2 of the thesis, we showed that ERβ-selective agonists elicited an increase in apoptosis and this was accompanied by an increase in expression of the pro-apoptotic factor PUMA. Induction of PUMA was dependent on the presence of the transcription factor FOXO3 but was independent of p53. In the ventral prostates of ERβ-/- mice, expression of FOXO3a is lower than that in WT littermates demonstrating a relationship between ERβ and FOXO3a expression found in PC3 and LNCaP cells. Furthermore, in prostate cancers of Gleason grade 4 or higher there was a marked reduction of both ERβ and FOXO3a, while both genes were well expressed in BPH sections. In Chapter 3 of the thesis, we investigated whether the loss of ERβ (also called as ERβ1) and/or expression of its splice variant ERβ2 affected signaling pathways involved in proliferation and bone metastasis of prostate cancer. We found repressed expression of the bone metastasis regulator Runx2 and its target gene, Slug by ERβ1. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate cancer, was increased by ERβ2. In terms of cell cycle modification, of the two receptors, ERβ1, but not ERβ2, inhibited proliferation and expression of the proliferation markers Cyclin E, c-Myc, and p45Skp2. Xenograft studies using athymic nude mice confirmed the proliferative effect of ERβ2, as tumors in mice bearing PC3-ERβ2 cells were substantially larger than tumors in mice bearing PC3-control and PC3-ERβ1 cells.Item Apoptotic Properties of Platinum Antitumor Agents Phosphaplatins(2016-05) Dezvareh, Homa 1989-; Zhang, Xiaoliu Shaun; Chung, Sang-Hyuk; Merchant, Fatima Aziz; Bark, Steven J.Phosphaplatins are a group of non-DNA binding platinum compounds that exhibit excellent in vivo and in vitro efficacies against a variety of cisplatin- and carboplatin-sensitive and resistant ovarian cancers. Although, combinations of cis- or carbo-platin and paclitaxel therapies continue to be the choice of treatment for ovarian cancer, many patients develop resistance to the treatment. Phosphaplatins are pyrophosphate bound platinum-(II) and (IV)-platinum complexes that are bi-negatively charged at or near neutral pH. Both Platinum (II) and platinum (IV) complexes do not induce the overexpress of any DNA repair genes, consistent with the no-DNA binding property of phosphaplatins. In this report, we show the apoptotic properties of two representative compounds, (R, R-1,2-cyclohexanediamine)-(dihydrogen pyrophosphato)platinum (II) (RRD2) and (R,R-1,2-cyclohexanediamine)-trans-dihydroxo(dihydrogen pyrophosphato)platinum (IV) (RRD4) through a variety of experiments as described below. Phosphaplatins are found to activate and upregulate death receptors on cell surface such as FAS, DR5, and TNFR1. These three death receptors follow the common signaling path to trigger apoptosis by an extrinsic pathway via Death Inducing Signaling Complex (DISC) by recruiting FADD and procaspase-8 and activating caspase-8. A direct binding of FAS by platinum is implicated as the platinum compound was found to be co-localized in the lipid rafts. The PTEN-PI3K pathway involvement was confirmed by the down regulation of both PI3K and p-AKT. Phosphoplatins also upregulate tumor suppression genes P53 and PTEN. Apoptosis by p53 signaling follows intrinsic pathways involving BCL-proteins. In particular upregulation of BAX, PUMA, and downregulation of BCL2 were observed. To further confirm p53-signailng, we have identified a number of p53 target genes that include AEN, CYFIP2, TP53I3, TP53INP1, DPYSL4, LRDD, DRAM1, and a few others. Our data shows that phosphaplatins have the potential to not only treat resistant but also advanced metastatic ovarian cancers.Item Aptamers and Phage as Tools for the Development of Novel and Sensitive Point-of-Care Diagnostics(2014-08) Adhikari, Meena 1979-; Willson, Richard C.; Fox, George E.; Gao, Xiaolian; Williams, Cecilia M.; Bikram, MalavosklishImmunochromatographic lateral flow assays (LFAs) represent a well-established point-of-care diagnostic analytical method for the primary testing of diverse samples. The sensitivity of conventional LFAs that use the standard colored nanoparticles as reporters lags behind the more elaborate laboratory techniques, such as plaque counting, PCR or ELISA. To address this issue, we have introduced an innovative approach that utilizes functionalized M13 bacteriophage nanoparticles as reporters in the lateral flow diagnostic system. M13 phage offers a multitude of binding sites on its major coat protein pVIII for reporter enzymes, and for affinity agents specific for the target of interest. Furthermore we employed SAM-AviTag phage, derivatives of phage M13 in which the N-terminus of the tail protein pIII is replaced by the enzymatically-biotinylatable AviTag peptide (GLNDIFEAQKIEWHE). The lysine residue (K) in the AviTag is a substrate for biotinylation by the E. coli biotin ligase (birA) enzyme. Using streptavidin or Neutravidin, any biotinylated affinity agent can then easily be linked to these enzymatically-biotinylated phage particles. Viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). These particles were successfully integrated into a lateral-flow immunochromatographic assay detecting MS2 virus, a model for viral pathogens. The limit of detection of the assay was 10^4 MS2/mL, 1000-fold more sensitive than the conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments. Aptamers are short, library-selected nucleic acid molecules that can recognize and bind to pre-selected targets with high affinity and selectivity. They have been used as recognition elements in a variety of applications. We screened for DNA aptamers specific to the murine anti-lysozyme antibody, HyHEL-5. During the validation process, however, the originally selected aptamers did not show successful binding. As an alternative, literature DNA aptamers were employed to construct phage displaying aptamers and HRP, which were used as LFA reporters. We describe the first modification of bacteriophage particles with aptamers as bio-recognition elements, and demonstrate their use in ultrasensitive lateral flow assays detecting IgE and PBP2a (Penicillin binding protein) of Staphylococcus aureus.Item Are Parallel and Convergent Changes in Proteins Indicative of Adaptive Evolution?(2018-12) Yuan, Fei 1985-; Graur, Dan; Sater, Amy K.; Zufall, Rebecca A.; Meisel, Richard P.; Nakhleh, Luay K.The independent evolution of identical or similar phenotypical traits in different lineages is referred to as convergent evolution. At the phenotypic level, convergent changes are usually regarded as evidence for adaptation due to natural selection. It is, however, unclear whether such changes at the molecular level are also indicative of adaptive evolution. This dissertation attempts to tackle issues pertaining to parallel and convergent amino acid replacements and their evolutionary significance. First, I examined parallel and convergent replacements in all one-to-one orthologous proteins from nine mammals and twelve Drosophila species. I compared the numbers of inferred parallel and convergent amino acid replacements with the expectations derived from two evolutionary models: the JTT model and a new selection-free amino acid replacement model. When the selection-free model was used, no excessive parallel or convergent replacements were found and the observed numbers of such replacements could be explained without invoking positive selection. I also demonstrated that many parallel and convergent changes reported in the literature constitute false positives due to the discordance that sometimes exists between the gene tree and the species tree. Second, I investigated the effects of taxonomic sample size, tree balance, choice of multiple sequence alignment method, and choice of ancestral state reconstruction method on the accuracy of parallel and convergent amino acid replacement identification. Sample size has a profound impact, whereby identification accuracy increases significantly with sample size. I also found that the other three factors and some interactions (e.g., sample size by reconstruction method and tree balance by reconstruction method) influence identification accuracy, but to a much lesser extent. My study indicates that the best method combination to use is either T-COFFEE+CODEML or MUSCLE+CODEML. The Clustal Ω+FastML combination should be avoided. Third, to facilitate future studies of parallel and convergent replacements, I developed a Python package called ProtParCon that can be used to process molecular data and identify parallel and convergent amino acid replacements. As demonstrated in a case study of lysozyme c sequences, ProtParCon is capable of rapidly and efficiently analyzing parallel and convergent replacements in real biological datasets.