<p>Improvements in genomic technology, both in the increased speed and reduced cost of sequencing, have expanded the appreciation of the abundance of human genetic variation. However the sheer amount of variation, as well as the varying type and genomic content of variation, poses a challenge in understanding the clinical consequence of a single mutation. This work uses several methodologies to interpret the observed variation in the human genome, and presents novel strategies for the prediction of allele pathogenicity.</p><p>Using the zebrafish model system as an in vivo assay of allele function, we identified a novel driver of Bardet-Biedl Syndrome (BBS) in CEP76. A combination of targeted sequencing of 785 cilia-associated genes in a cohort of BBS patients and subsequent in vivo functional assays recapitulating the human phenotype gave strong evidence for the role of CEP76 mutations in the pathology of an affected family. This portion of the work demonstrated the necessity of functional testing in validating disease-associated mutations, and added to the catalogue of known BBS disease genes.</p><p>Further study into the role of copy-number variations (CNVs) in a cohort of BBS patients showed the significant contribution of CNVs to disease pathology. Using high-density array comparative genomic hybridization (aCGH) we were able to identify pathogenic CNVs as small as several hundred bp. Dissection of constituent gene and in vivo experiments investigating epistatic interactions between affected genes allowed for an appreciation of several paradigms by which CNVs can contribute to disease. This study revealed that the contribution of CNVs to disease in BBS patients is much higher than previously expected, and demonstrated the necessity of consideration of CNV contribution in future (and retrospective) investigations of human genetic disease.</p><p>Finally, we used a combination of comparative genomics and in vivo complementation assays to identify second-site compensatory modification of pathogenic alleles. These pathogenic alleles, which are found compensated in other species (termed compensated pathogenic deviations [CPDs]), represent a significant fraction (from 3 – 10%) of human disease-associated alleles. In silico pathogenicity prediction algorithms, a valuable method of allele prioritization, often misrepresent these alleles as benign, leading to omission of possibly informative variants in studies of human genetic disease. We created a mathematical model that was able to predict CPDs and putative compensatory sites, and functionally showed in vivo that second-site mutation can mitigate the pathogenicity of disease alleles. Additionally, we made publically available an in silico module for the prediction of CPDs and modifier sites.</p><p>These studies have advanced the ability to interpret the pathogenicity of multiple types of human variation, as well as made available tools for others to do so as well.</p> / Dissertation

<p> <i>Chromobacterium violaceum</i> (CV) produces a violet color pigment known as Violacein. It has been reported that violacein has anticancer activity. This compound is produced by CV a gram-negative facultatively anaerobic bacterium found in soil and water environmental samples. The purpose of this study was to determine the effect of purified violacein on select cancer cell lines. Violacein used in this study was purified from CV strain (14N23), a strain isolated from environmental samples collected in the Tennessee Copper Basin. The previous reports used a crude extract preparation of violacein; thus, it was of interest to determine the effect of the pure compound on cancer cell growth was similar to that of the crude extracts. The compound purified following the method of Mehta, et al. was exposed to cancer cells and cell death assessed using the Alamar Blue procedure. It was found that violacein had no effect on A549, BT549, and PC3 cancer cell growth; however, there was a significant effect on Colo-320 cancer cells. It was concluded that further studies are required to assess the effect of violacein on enzymes and proteins involved in the cancer cell apoptotic pathways. Such studies will explain why cancer cell death was observed in certain cancer cells and not others.</p>

Le mélanome est le cancer de la peau le plus rare, mais celui qui cause le plus de mortalité. L’étapecritique de sa progression est l’angiogenèse, processus détourné par la tumeur pour s’oxygéner et senourrir. Les cellules tumorales peuvent alors former des métastases dans les ganglions lymphatiques,notamment. D’autre part, la tumeur inhibe les réponses immunitaires de l’hôte à son encontre. Tousces mécanismes font intervenir le monoxyde d’azote (NO ). Le but de ce projet a été d’approfondir lerôle du NO dans l’angiogenèse et le recrutement leucocytaire, mécanismes basés sur lareconnaissance cellulaire de l’endothélium.Nous avons montré que le NO est nécessaire à l’angiogenèse, mais que des doses plus fortes sontanti-angiogéniques. Cet effet inhibiteur peut s’expliquer par la diminution des interactions cellules-cellules,et l’inhibition de l’expression de PECAM-1/CD31, principalement.D’autre part, nous avons établi que l’adhésion des leucocytes est inhibée par le NO ce qui a été reliéà la modulation de l’expression des molécules capables de fixer des chimiokines : lesglycosaminoglycannes et les récepteurs de chimiokines, ainsi qu’à la sous-expression des moléculesd’adhésion CD34, ICAM-2/CD102 et VCAM-1/CD106.En conclusion, le NO est capable de réguler des mécanismes cellulaires majeurs de la progressiontumorale, par modulation de l’expression des molécules de surface de l’endothélium. Tout au long dece travail, nous avons observé que les différences de réponse sont dépendantes des doses de NO etdu type cellulaire, ce qui démontre le rôle pivot du NO dans la progression du cancer. / Melanoma is the rarest skin cancer, but one that causes the most of deaths. The critical step of itsprogression is angiogenesis, a physiological tumor-activated process which allows the delivery ofoxygen and nutrients. Tumor cells may then metastasize to the lymph nodes, in particular. Moreover,the tumor inhibits the host immune responses toward itself. All these mechanisms are regulated bynitric oxide (NO ). The aim of the project was to deepen the role of NO in angiogenesis andleukocyte recruitment, two mechanisms based on endothelial cell recognition.We have shown here that NO is necessary for angiogenesis, but that high concentrations are antiangiogenic.This inhibitory effect of NO can be attributed to the decrease of cell-cell interactions andthe inhibition of the PECAM-1/CD31 expression, mainly.Besides, we have demonstrated that leukocyte adhesion on endothelium is inhibited by NO . This canbe explained by the modulation of the expression of molecules able to bind to chemokines:glycosaminoglycans and chemokine receptors, as well as by the down-regulation of the adhesionmolecules CD34 and ICAM-2/CD102 VCAM-1/CD106.In conclusion, NO is able to regulate the main cellular mechanisms of tumor progression bymodulating the expression of surface molecules on endothelial cells. Throughout this work, we haveobserved that these modulations depend on NO concentrations and on the cell type, demonstratingthe pivotal role of NO in cancer progression.

<p> Angiogenesis, the formation of new blood vessels, requires the reorganization of microtubules (MT), which are polar cytoskeletal structures, consisting of a free&#129;-plus end and a minus&#129;-end that is free or anchored. CAMSAP2 and CAMSAP3 have been shown to bind and stabilize MT minus&#129;-ends; yet, this activity&#129;'s contribution to MT organization and directional migration is unknown. To investigate this contribution, we performed live&#129;-cell imaging of polarized HUVECs expressing CAMSAP2 or CAMSAP3. Our results show that CAMSAP2 and CAMSAP3 localized to the trailing edge of cells. Pharmacologic disassembly of MTs resulted in CAMSAP reorganization to the leading edge. MCAK expression is not sufficient for CAMSAP reorganization, but may recruit CAMSAP to the MT minus&#129;-end. MT growth dynamics analysis revealed that CAMSAP2 and CAMSAP3 promoted dynamic MT growth. These results suggest that CAMSAP2 and CAMSAP3 protect MTs against MCAK&#129;]mediated disassembly and also function to nucleate new, dynamic MTs at the leading edge.</p>

<p> Growth is one of the fundamental characteristics of biological systems. The study of eye growth regulation presents an interesting window that allows for the investigation of the role of the visual environment on internal processes. We now know that there is an intricate circuitry within the eye, independent of higher brain processes, that controls the growth of the eye but more needs to be elucidated about these local regulatory circuits. An improved understanding of this circuitry is critical to developing new therapies for abnormalities in eye growth regulation such as myopia, which is impacting more and more individuals around the world each day and in its more severe from, is linked to potentially blinding ocular complications. </p><p> The role of retinal glucagon, a neuropeptide, in the regulation of eye growth and refractive error has attracted the interest of researchers over the past 15 years yet there remain many unresolved questions. The research described in this dissertation aimed to elucidate the respective roles in eye growth regulation of specific subpopulations of retinal glucagonergic amacrine cells, which have been the subject of much speculation as the source of inhibitory growth signals, i.e. stop signals, yet not thoroughly investigated. </p><p> The approach taken to investigate this problem is to ablate glucagonergic amacrine cells in vivo using different neurotoxins, and to examine how this affects the sign-dependent circuitry of eye growth regulation. In addition, with the advent of advancements in high resolution imaging and electrophysiology, we were able to characterize the effects of these neurotoxins on the region-specific and time-sensitive changes in the structure and function of the living retina. </p><p> That the inhibitory response induced by imposed myopic defocus remains intact, in spite of total ablation of glucagon cells (Chapter 5) or elimination of the peripheral glucagon cells (Chapter 3) and other unintended adverse retina effects, compared to findings from previous studies involving QUIS (Chapter 2) of this thesis, is a novel finding. These results point to the same conclusion that glucagon cells themselves are not responsible for the decoding of the sign of optical defocus, but appear to have a role in fine-tuning of compensatory growth responses. The results of our experiments also suggest that the choroid may serve as an intermediate relay, and the altered anterior chamber development raise the further possibility that retina-derived growth modulatory factors also regulate the anterior segment, perhaps reaching this more remote site by diffusion forward through the vitreous chamber or via the uvea.</p>

<p> A living cell is a complex soft matter system far from equilibrium. While its components have definite mechanical properties such as stiffness and viscosity, cells consume energy to generate force and exhibit adaptation by modulating their mechanical properties through regulatory pathways. In this dissertation, we explore cell mechanics by stretching single fibroblast cells and simultaneously measuring their traction stresses. Upon stretch, there is a sudden, drastic increase in traction stresses, often followed by a relaxation over a time scale of about 1 minute. Upon release of stretch, traction stresses initially drop and often recover on a similar time scale of about 1 minute. We show that a minimal active linear viscoelastic model captures essential features of cell response to stretch. This model is most successful in describing the response of cells within the first 30 seconds of stretch. While perturbations of myosin and vinculin change quiescent traction stresses, they surprisingly have no significant impact on the stiffness or viscoelastic timescale of the cells. On longer time scales, cells may show an adaptive response to stretch that contradicts the minimal mechanical model. The probability of an adaptive response is significantly reduced by myosin de-activation and vinculin knockout. Therefore, we find that while vinculin and myosin are not important in determining passive mechanical properties of cells, such as stiffness and viscosity, they play a significant role in the adaptive mechanisms of cell response to stretch. To perform this work, we have built a novel micro stretching device compatible with live cell microscopy and developed a computational tool to analyze data from large traction stresses. Therefore, this dissertation's contribution is two-fold: (1) a novel experimental approach to explore the mechanics of living cells, and (2) a new model and framework for understanding the mechanical response of cells to stretch.</p>

<p> The membrane-bounded lysosome is a dynamic organelle responsible for macromolecule degradation, receptor down regulation, stress survival and pH balance. Intact lysosomal function is critical for proper cell function, as defects in lysosomal trafficking have been associated with neurodegenerative diseases such as Alzheimer&rsquo;s disease. <i>Saccharomyces cerevisiae </i>, budding yeast, is an ideal model organism for studying lysosomal protein trafficking because its vacuole is functionally analogous to the mammalian lysosome and both share conserved trafficking machinery components. </p><p> A previous yeast genomic deletion strain screen in our lab uncovered fourteen deletion strains with h&barbelow;ypersensitivity to <u>hy </u>gromycin B (<i>hhy</i> mutants), all of which exhibited defective vacuolar trafficking, morphology and/or function. Additionally, recent microscopic findings in our lab support that the late-endosome dependent pathway is the affected pathway and that <i>hhy</i>s have compromised T&barbelow;arget of R&barbelow;apamycin C&barbelow;omplex 1&barbelow; signaling. TORC1 regulates cell growth and cell proliferation and its hyperactivity is associated with cancer and metabolic diseases. As with lysosomes, the vacuole also serves as the platform for TORC1 signaling. </p><p> To further assess if the late-endosome dependent pathway is affected by hygromycin B treatment in <i>hhy</i> mutants, vacuolar delivery of two vacuolar markers was assessed. Alkaline phosphatase (ALP) delivery was assessed as a late-endosome independent pathway marker and carboxypeptidase Y (CPY) delivery was assessed as a late-endosome dependent pathway marker. To date, we have observed that hygromycin B treatment disrupts CPY trafficking via the late-endosome dependent pathway while ALP trafficking via the late-endosome independent pathway is unaffected by hygromycin B treatment. </p><p> Rapamycin is the direct inhibitor of TORC1. Rapamycin and its analogs (rapalogs) are currently administered in the treatment of TORC1 hyperactivity. However, therapeutic inhibition of TORC1 signaling by rapamycin is associated with severe toxicities. Since our recent results implicate compromised vacuolar trafficking of Tor1 kinase in <i>hhy</i> mutants, we tested vesicular trafficking inhibiting drugs in combination with rapamycin in order to explore a cumulative effect on TORC1 inhibition assessed by growth of wild type cells. Additionally, we explored the effects of combining two vesicular trafficking drugs on wild type cell growth. </p><p> We established a cumulative effect on wild type growth upon using low concentrations of rapamycin in combination with vesicular trafficking inhibitory drugs. Thus, rapamycin and vesicular trafficking inhibitory drugs have potential for drug combination therapy against TORC1 hyperactivity at lower drug concentrations. Drug combination treatment may be a new and effective way to regulate TORC1 function at sub-toxic levels of rapamycin.</p>

<p> HIV-infected cells can reach the central nervous system (CNS) and release HIV virions, which productively infect macrophages and microglia. Infected cells release both viral and host factors that can induce inflammation in bystander immune cells, to initiate neuropathogenesis beyond the initial site of the infected cell. While antiretroviral therapy can effectively control CNS viral loads, HIV-1 RNA and Tat protein can persist in cerebrospinal fluid of treated patients. Ongoing neurotoxic insults can result in HIV associated neurocognitive disorders (HAND). While the incidence of the severe form of HAND has declined with increasing treatment, milder forms of HAND have increased in prevalence. Delineating the viral determinants of cytotoxicity that can lead to HAND is critical to developing effective neuroprotective agents.</p><p> HIV-1 Tat protein contributes to HIV neuropathogenesis in multiple ways, including direct neurotoxicity and recruitment of HIV infected monocytes to CNS. Tat can promote transcriptional transactivation of cellular proinflammatory cytokines in the infected cells. Tat's ability to be secreted and subsequently taken up by bystander cells can lead to an induction of inflammatory cytokines in the uninfected cells as well. Tat's cellular uptake is mediated by an Arginine-rich region termed the cell penetration peptide (CPP). We now describe the functional consequences of a polymorphism in the Tat basic domain (residues 48-57) that diminishes extracellular Tat's uptake by cells. Earlier work, using lade B HIV-1 Tat CPP containing sixArginines, showed that each of the sixArginines are essential for maximal Tat CPP uptake. However, we observed significant variability in the conservation of R57 residue within group M clades &ndash; while R57 is well conserved (>75%) in many, other clades contain a non-Arginine residue at this position.</p><p> We hypothesized that the absence of R57 reduces Tat uptake. Cells incubated with fluorescently-labeled peptides corresponding to residues 48-57 in Tat, with R57, S57 or G57 residues revealed a 3-4 fold higher level of CPP-R57 uptake compared to the others. In order to further study the biological implication of this variation, we chose to compare clade B (most studied clade) with clade C (globally pre-dominant clade). To test the effects of R57S substitution on full-length Tat protein, we established a transcellular Tat transactivation system, where HeLa cells are transfected with expression constructs for Tat-B (R57), Tat-C (S57) proteins or their R57S or S57R mutants respectively. Media are collected from the transfected cells, relative levels of Tat determined by ELISA and equal amounts of Tat applied to TZM-bl reporter cells. We demonstrated that R57S mutation in Tat-B diminished its transcellular reporter signal response, while the S57R mutation in Tat-C enhanced the transcellular response. Furthermore, we investigated the role of R57S substitution on the establishment of an inflammatory environment by exposing monocytic and microglial cells lines to Tat-containing media and quantifying the inflammatory cytokines gene induction. We found significantly higher levels (~2-3 fold) of expression of several cytokine genes, in cells exposed to the R57-containing Tat variants compared with the S57-variants.</p><p> Based on our results, we conclude that Tat R57 is an important viral determinant for efficient cellular uptake of Tat. It contributes to the intercellular, systemic spread of inflammation, and may contribute to the inflammatory phenotype in the CNS of HIV-infected individuals. Intriguingly, a small data set of Tat sequences from CNS samples in HIV-1 C patients indicated an enrichment for R57 variants, compared with the consensus peripheral sequences (77% vs 13%, respectively), suggesting that the rare CNS HIV disease that is observed in some clade C patients may be associated with a R57-containing Tat. Our studies have identified a novel signature of HIV-1 Tat that enhances the induction of inflammatory signal, and may play an important role in neurovirulence. </p>

Heart Failure is the state in which the heart is unable to pump enough blood to meet the needs of the body, resulting from events that progressively damage the myocardium and/or its ability to contract normally. Several compensatory mechanisms allow for short-term maintenance of adequate delivery of blood to the body. Progression from the initial events to heart failure is caused by the accumulation of pathologic changes and their effects, many of which result from chronic activation of these compensatory mechanisms. One of these mechanisms is the activation of the neurohormonal β-adrenergic system, to which the β₃-adrenergic receptor (β₃-AR) belongs. Research on the role of the β₃-AR in the heart has focused on the short-term protective role of the receptor, based on its ability to reduce the effects of over-stimulation of the heart's other adrenergic-system components. However, during the course of heart failure, its up-regulation and functional persistence may also contribute to the progression of the disease. There have been few large-animal studies, no human trials, and no long-term studies of β₃-AR agonists for use in the treatment of heart failure. Ultimately, individualized treatment strategies that, over time, modulate the relative levels of agonistic and antagonistic effects across each of the three β₃-AR subtypes, may be most effective for management of heart failure. However, further research and trials with β₃-AR agonists are realistic and necessary places to start.

<p> In order to adapt to changing conditions and life stages, cells must remodel the composition of the proteome by specifically removing obsolete or damaged proteins. Failure to remove proteins at the appropriate time can cause a variety of cellular dysfunctions that can lead to cell death and conditions such as cancer, Huntington's, Parkinson's and Alzheimer's disease. Protein quality control is especially important during aging and stress, as the accumulating load of damaged, misfolded, and aggregated proteins can overwhelm the cell's ability to maintain the proteome. The ubiquitin-proteasome system is responsible for the specific degradation of many proteins within eukaryotic cells. The 26S proteasome degrades proteins that have been &ldquo;tagged&rdquo; with a chain of ubiquitin molecules. Ubiquitin is a highly-conserved 8.5 kDa protein that is conjugated post-translationally to a target protein after passing through a complex series of enzymes. Using the nematode <i> C. elegans</i> as a model, we sought to 1) Characterize the roles of specific ubiquitin-conjugation enzymes (UBCs) in the response to aggregation of a polyglutamine-containing protein in body wall muscle cells and 2) Examine the subcellular localization of proteasomes in these cells during normal aging and stress. Q82::GFP, a fusion protein expressed from a transgene encoding a polyglutamine tract fused to Green Fluorescent Protein (GFP), was found to aggregate rapidly (~58 minutes) in a manner that is not directly dependent on ubiquitin but dependent on the concentration of Q82::GFP protein, which was altered in response to RNA interference of several E2 ubiquitin-conjugating enzymes. In a separate transgenic worm strain, a fluorescently-labeled proteasome subunit, RPT-1, was observed in live animals to localize to the nucleus and cytoplasm during the adult stage. Within the nucleus, RPT-1::GFP localizes diffusely and to foci, and was excluded from heterochromatin. Within the muscle contractile apparatus, RPT-1::GFP localizes to I-bands, regions of thin actin filaments, and is excluded from dense bodies. After a prolonged heat stress, RPT-1::GFP in adult worms relocalizes to dense bodies. After starvation, these foci are associated with high proteolytic activity. These studies demonstrate some of the spatial dynamics of the ubiquitin-proteasome system in response to development, aging, and stress.</p>