<p> The aim of this thesis is to evaluate the impact of somatostatin receptor subtype-4 (SSTR4) actions on microglia cell viability, towards the understanding and advance of pharmacological treatments for Alzheimer&rsquo;s disease (AD). As of March 2016, there were 5 million people living in the United States with AD. Current drugs for AD have highly variable effects from patient to patient and are palliative at best. This thesis project focuses on the study of the BV2 cell line and the compound NNC 26-9100 (NNC). BV2 cells are immortalized microglial cells that maintain most of their morphology. The data collected suggests that BV2 cells can phagocytize amyloid-? peptides (A?), respond to lipopolysaccharide (LPS), and have the somatostatin receptor subtype-4 (SSTR4). NNC is a selective agonist of the SSTR4 and we have found that it causes BV2 cells to increase in number. The effects of NNC were able to be reduced with a somatostatin receptor pan-antagonist, cyclosomatostatin, and the adenyl cyclase activator forskolin. NNC can alter BV2 numbers by binding to the SSTR4, creating an intracellular cascade that results in the inhibition of adenyl cyclase and an increase in cell count. Collectively, a potential therapeutic mechanism for AD is increasing the number of microglial cells to increase both amyloid beta (A?) phagocytosis and degradation of A? by neprilysin.</p>

<p>Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant <italic>Arabidopsis thaliana</italic>, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies. </p><p>Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.</p><p>Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (<italic>PBG</italic>), and one in which it cannot (<italic>NGB</italic>). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the <italic>NGB</italic> line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in <italic>NGB</italic> regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.</p><p>In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); <italic>hmr</italic> mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak <italic>hmr</italic> allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, <italic>SON OF HEMERA</italic> (<italic>SOH</italic>). The <italic>soh</italic> mutations rescue all of the phenotypes associated with the weak <italic>hmr</italic> allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null <italic>soh</italic> alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.</p><p>In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, <italic>SON OF HEMERA</italic>, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.</p> / Dissertation

<p> Throughout development, canonical Wnt signaling contributes to the formation and maintenance of a wide array of cells, tissues, and organs. Dys-regulated Wnt signaling during embryonic development is implicated in developmental defects known as neurochristopathies, including craniofacial and heart defects, as well as defects in neural development. Due to its roles in stem cell maintenance and self-renewal, tissue homeostasis, and regeneration, aberrant Wnt signaling in adult tissues can result in various forms of cancer, including colorectal cancer, breast cancer, lung cancer, and gastro-intestinal cancer, among others. Dys-regulated Wnt signaling is also implicated in other pathologies including bone disease, and metabolic diseases, such as Type II diabetes. Our lab has previously identified a novel crosstalk between canonical Wnt signaling and ephrin signaling. Ephrin signaling occurs through the interaction of ephrin ligands and Eph receptor tyrosine kinases, and is bidirectional. Due to the roles of ephrin signaling in tissue development and maintenance, aberrant ephrin signaling is implicated in many diseases including bone remodeling diseases, diabetes, and cancer. The molecular mechanism of the crosstalk between canonical Wnt signaling and ephrin-B signaling remains unknown. &beta;-catenin is a key intracellular effector of canonical Wnt signaling that transduces the signal to the nucleus, where &beta;-catenin interacts with the TCF/LEF transcription factors and activates transcription of target genes. Due to its central role in transducing the canonical Wnt signal to the nucleus, we predict that ephrin-B signaling antagonizes canonical Wnt signaling by affecting the stability and/or sub-cellular localization of &beta;-catenin, or the interaction between &beta;-catenin and TCF/LEF transcription factors. By employing mouse ephrin-B constructs in human cell lines, we show that the canonical Wnt - ephrin-B crosstalk is conserved between frogs and mammals. We also found that ephrin-B antagonism of canonical Wnt signaling is likely independent of ubiquitin proteasome system (UPS)-mediated degradation of &beta;-catenin. Furthermore, confocal immunofluorescence microscopy revealed that overexpression of ephrin-B in HEK293T cells treated with lithium chloride (LiCl) seems to promote membrane localization of &beta;-catenin, particularly at the apical Z sections. These results suggests that re-localization of &beta;-catenin to the cell membrane may contribute to the ephrin-B antagonism of canonical Wnt signaling.</p>

To exit a non-proliferative state and enter cell division, metazoan cells require external signals to facilitate activation and metabolic reprogramming. As cell growth is required before cell division, cells redirect their metabolism for de novo synthesis of cell building blocks, including phospholipids for cell membrane construction. How cells coordinate initial signaling events with metabolism is unknown. Lineage-specific factors transmit activating signals via cell surface receptor-ligand interactions. Among these are PI3K/AKT, MAPK/ERK, and JAK/STAT, all of which have been described to contribute to metabolic regulation. In particular, the signal transducer and activator of transcription (STAT) is a transcription factor with broad roles in cell cycle progression and glucose metabolism. Previous data from our laboratory found that one STAT family member, STAT3, was one of the primary signaling pathways activated when transitioning out of a resting state. Inhibition of STAT3 was found to suppress the initiation of cell growth and citrate levels, a main intermediate for fatty acid synthesis, suggesting a connection to cell metabolism. This thesis investigates the role of STAT3 in the regulation of metabolism in cells transitioning from a resting state to a cell growth state. The first chapter of this thesis provides relevant background information on the metabolic and signaling pathways involved in a resting and cell growth state. It also provides data that supports an important role for STAT3 during initial cell growth. The second chapter demonstrates the importance of STAT3 in multiple cell types using a small molecule inhibitor of STAT3, STAT3 knockdown, and knockout experiments. I also establish a potential link between STAT3 and the metabolic enzyme citrate synthase (CS) for the synthesis of citrate. In the third chapter I show that STAT3 transcriptionally regulates CS through two binding sites, CS1 and CS2. Finally, I determine that CS is essential for initial cell growth and that exogenous citrate can rescue the loss in cell growth and proliferation observed in the CS and STAT3 knockdown cells. Together, these findings describe a novel mechanism for initial cell growth whereby signaling and metabolic events are tightly linked to regulate the transition from a resting state to a state of initial cell growth. These results may uncover new strategies to block the initiation of proliferation in human pathological conditions including tumor recurrence and autoimmunity. / Graduate

<p>The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.</p> / Dissertation

Interleukin-8 (IL-8), a member of the C-X-C chemokine subfamily, is an important chemoattractant and cellular activator. This study was conducted to determine the role of IL-8 in the immunopathogenesis of HIV-I disease and tuberculosis. The first section involved determining the effect of infection with HIV-1, Mycobacterium tuberculosis and co-infection with both of these organisms on IL-8 j_ roduction in vivo. This was monitored by the determination of levels of serum or plasma EL-8 and peripheral cell-associated IL-8, assessing peripheral mononuclear (PBMC) and polymorphonuclear (PMN) cell capacity to produce IL-8 spontaneously or in response to various stimuli, and the detection of constitutive IL-8 mKNA expression in purified subsets of mononuclear cells. Results show that whereas there is evidence of detectable levels of cell-associated EL-8 (mKNA and protein) in peripheral cells of healthy individuals, this is largely lost in the disease states studied. Coupled with this was significantly increased circulating levels of EL-8 in serum and plasma found in HIV-1 infected individuals with or without concomitant pulmonary TB. On the other hand, the capacity of PBMC to produce IL-8 spontaneously ex vivo was enhanced in HIV-1 and TB patients and many of the HFV/TB group, but their corresponding capacities to respond to various stimuli was significantly diminished when compared to that of the normal donors. The release of IL-8 from PMN in the presence of an agonist was diminished mainly in individuals with pulmonary TB, which was further exacerbated by the presence of HIV-1 infection. HIV-1-infected individuals have an increased incidence of bacterial infections which could be related to defective functioning of PMN. The second section was aimed at detecting PMN abnormalities in HIV and I-HV/TB patients by monitoring EL-8-induced p-glucuronidase release and PMN chemotaxis in response to IL-8. IL-8-induced (I-glucuronidase release from PMN of normal individuals and TB patients occurred in a dose-dependent manner. In contrast, PMN from HTV-1 infected individuals, whether co-infected with M tuberculosis or not, showed a reciprocal response in that increasing IL-8 concentrations resulted in decreased enzyme release. This reciprocal slope of the IL-8 dose-response curve was altered for the majority of HIV-1 positive individuals tested irrespective of their CD4+ cell counts. In addition, PMN chemotaxis in response to IL-8 was also found to be significantly impaired in a group of HIV-1 infected patients coinfected w ithM tuberculosis when compared to healthy individuals. The third section of the study involved analysing the expression of the PMN cell surface markers, FcyRIII (CD 16), and the two human IL-8 receptors, designated IL -8RA and 1L-8RB. FcyRIII (CD 16) expression on the surface of PMN was significantly reduced in HIV-1 seropositive patients with pulmonary tuberculosis when compared to those individuals with either disease alone or healthy blood donors. A significant reduction in the percentage of PMN expressing IL-8RA and IL-8RB and in their respective fluorescence intensities was found in TB, HIV, and HTV/TB groups when compared to that obtained for the ND group. IL-8RA intensity of fluorescence was significantly decreased in the HTV/TB group when compared to the TB and HIV groups indicating a further down-regulation of IL-8RA expression owing to dual infection. On the other hand, IL-8RB fluorescence intensity was substantially reduced on PMN from patients with pulmonary TB and to a greater degree in those patients co-infected with HIV-1 and M. tuberculosis. Having found a reduction in the expression of both IL-8 receptors on PMN in all the infection groups, cellular events following the binding of IL-8 to IL-8 receptors on PMN isolated from dually infected patients, the group which showed the greatest reduction in IL-8 expression was analysed. Results indicated that the impairment of DL-8-dependent PMN functions such as degranulation and chemotaxis was associated with the reduced expression of IL-8 receptors on these cells. Increased circulating levels of IL-8 in HIV-1 infection and a diminished cellular capacity to produce IL-8 as shown in this study may have important implications for antimicrobial defences and normal immune processes. A dysregulated production of IL-8 in vivo is likely to play a role in the pathogenesis of HIV-1 disease, pulmonary tuberculosis, and dual infections with both organisms. In addition, cellular responses dependent on specific receptor engagement and the subsequent translation of signal transducing events that lead to phagocyte effector functions are clearly impaired in IL-8 receptor deficient phagocytes. Abnormal PMN functioning in HTV-1 infected individuals, as shown here by defective degranulation and chemotactic responses, have important implications in the pathogenesis of HIV-1 infection in terms of their ability to clear secondary microbial infections. Future attempts should be aimed at defining the mechanisms that bring about these changes in order to contribute to a greater understanding of the mechanisms that lead to an enhanced risk of superinfections in immunosuppressed individuals.

Pas de résumé / No abstract

Le pancréas est une glande amphicrine composée de cellules exocrines et de cellules endocrines. Parmi les cellules endocrines, organisées en îlot de Langerhans, les cellules ß sécrétrices d’insuline en réponse à des stimuli précis, sont essentielles pour l’homéostasie du glucose. Des perturbations tant au niveau qualitatif qu’au niveau quantitatif sont responsables de différentes pathologies telles les diabètes ou certaines formes de tumeurs endocrines. De récentes publications suggèrent que l’état de différenciation de la cellule ß pancréatique mature n’est pas immuable et montrent que le maintien d’un phénotype mature de la cellule est un processus dynamique. Différents modèles de souris mutantes (avec perte ou gain d’un facteur de transcription) montrent une perte de l’identité de la cellule ß. Cette plasticité altère la synthèse, le stockage et la sécrétion d’insuline. En plus de la perte d’identité, caractérisée par la diminution de l’expression de marqueurs de la cellule ß (MAFA, NKX6-1), les cellules ré-expriment des marqueurs de progéniteurs (NGN3, SOX9) : on parle de dédifférenciation. Cette dédifférenciation serait un mécanisme clé dans la diminution de la masse de cellules ß fonctionnelles au cours du diabète de type 2. Le but de ma thèse a été d’étudier le rôle du facteur de transcription SOX9 dans le contexte de la perte d’identité de la cellule ß humaine. SOX9 est exprimé dans les progéniteurs multipotents pancréatiques et joue plusieurs rôles cruciaux au cours du développement de l’organe. Bien qu’un rôle important de SOX9 fut attribué au cours de l’organogénèse du pancréas, il y a de plus en plus de données suggérant qu’il a des rôles additionnels dans le pancréas matures qui semble aussi importants que son rôle au cours du développement. C’est le cas notamment des cellules formant les canaux pancréatiques. D’un autre côté, pour les cellules endocrines, et plus particulièrement les cellules ß, SOX9, normalement absent de la cellule ß saine, est ré-exprimé dans ces cellules dans des conditions pathologiques (diabètes, tumeurs neuroendocrines du pancréas). Une expression ectopique de SOX9 dans les cellules ß induit un phénotype diabétique. Alors qu’il y a de plus en plus d’observation de l’expression de SOX9 dans la cellule ß, il y a très peu de connaissance sur les mécanismes moléculaires et les cibles de ce facteur de transcription dans les cellules ß humaines. Dans un premier temps, nous avons disséqué différents mécanismes impliqués dans l’induction de l’expression de SOX9. Pour cela, nous avons développé des conditions mimant des contextes pathologiques (diabètes, tumeurs neuroendocrines du pancréas VHL) en utilisant les cellules ß humaines EndoCßH1, récemment développées au sein du laboratoire. Dans un deuxième temps, nous avons développé des outils moléculaires afin d’identifier les cibles de SOX9 dans la cellule ß humaine (dominant positif, dominant négatif). Pour finir, nous avons analysé les cibles potentielles de SOX9 dans différentes conditions pathologiques. / No abstract

Femtocell is an effective approach to increase system capacity in cellular networks. Since traditional Femtocells use the same frequency band as the cellular network, cross-tier and co-tier interference exist in such Femtocell networks and have a major impact on deteriorating the system throughput. In order to tackle these challenges, interference mitigation has drawn attentions from both academia and industry. TV White Space (TVWS) is a newly opened portion of spectrum, which comes from the spare spectrum created by the transition from analogue TV to digital TV. It can be utilized by using cognitive radio technology according to the policies from telecommunications regulators. This thesis considers using locally available TVWS to reduce the interference in Femtocell networks. The objective of this research is to mitigate the downlink cross-tier and co-tier interference in different Femtocell deployment scenarios, and increase the throughput of the overall system. A Geo-location database model to obtain locally available TVWS information in UK is developed in this research. The database is designed using power control method to calculate available TVWS channels and maximum allowable transmit power based on digital TV transmitter information in UK and regulations on unlicensed use of TVWS. The proposed database model is firstly combined with a grid-based resource allocation scheme and investigated in a simplified Femtocell network to demonstrate the gains of using TVWS in Femtocell networks. Furthermore, two Femtocell deployment scenarios are studied in this research. In the suburban Femtocell deployment scenario, a novel system architecture that consists of the Geo-location database and a resource allocation scheme using TVWS is proposed to mitigate cross-tier interference between Macrocell and Femtocells. In the dense Femtocell deployment scenario, a power efficient resource allocation scheme is proposed to maximize the throughput of Femtocells while limiting the co-tier interference among Femtocells. The optimization problem in the power efficient scheme is solved by using sequential quadratic programming method. The simulation results show that the proposed schemes can effectively mitigate the interference in Femtocell networks in practical deployment scenarios.

Studies to date have failed to identify the most effective treatment regimes for patients with chronic genotype 3 HCV infection. There is controversy regarding the role of cirrhosis in modifying response and disagreement regarding the impact of ethnicity on treatment outcome. Given the importance of genotype 3 HCV in the global epidemic and the lack of high quality research into this genotype, the purpose of this work has been to address some of the deficiencies in our understanding of the optimal management of this strain of hepatitis C. Specifically we have examined the following hypotheses:- 1) Patients from the Indian sub-continent (South Asians) will respond differently to therapy with pegylated interferon and ribavirin when compared to Caucasians. 2) An analysis of viral and host factors underlying differences between treatment sensitive and treatment refractory cohorts will reveal new insights into the virology of Genotype 3 HCV infection. 3) Increasing the duration of therapy in ‘difficult to manage’ patients with Genotype 3 HCV will improve response rates. 4) Whether non-invasive methods of identifying liver fibrosis are valuable in identifying the stages of fibrosis in Genotype 3 HCV patients. Three different research methodologies were used to address these questions including a metaanalysis of factors associated with treatment failure in patients with genotype 3 HCV, virological and immunological studies on patients with genotype 3 HCV who had failed to respond to therapy and a clinical trial evaluating extended duration therapy in patients with Genotype 3 HCV infection and cirrhosis.