In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and to the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multi-subunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in other trafficking pathways. Contrary to these other pathways, our lab previously suggested that the exocyst subunit Sec6, a component of the exocytosis-specific tethering complex, inhibited Sec9:Sso1 SNARE complex assembly due to interactions in vitro with the SNARE protein Sec9 (Sivaram et al., 2005). My goal for this project was to test the hypothesis that Sec6 inhibited SNARE complex assembly in vivo. I therefore chose to generate Sec6:Sec9 loss-of-binding mutants, and study their effect both in vitro and in vivo. I identified a patch of residues on Sec9 that, when mutated, are sufficient to disrupt the novel Sec6-SNARE interaction. Additionally, I found that the previous inhibitory role for Sec6 in SNARE assembly was due to a data mis-interpretation; my re-interpretation of the data shows that Sec6 has a mild, if any, inhibitory effect on SNARE assembly. My results suggest a potential positive role for Sec6 in SNARE complex assembly, similar to the role observed for other tether-SNARE interactions.

Aging can be defined as the accumulation of changes affecting the maintenance of homeostatic processes over time, leading to functional decline and increased risk for disease and death. In its simplicity, aging is the systemwide deterioration of an organism. Genetic studies have identified many potential molecular mechanisms of aging including DNA damage, telomere shortening, mitochondrial dysfunction, increased oxidative stress, uncontrolled inflammation, and hormone dysregulation (reviewed in [1]). However, in reality, aging is likely to be a combination of some (or potentially all) of these mechanisms. Interestingly, aging and metabolism are tightly coordinated. Aging is a major contributor to metabolic decline and related diseases, including type 2 diabetes, metabolic syndrome, and cancer. One of the best characterized metabolic pathways implicated in aging is the insulin/IGF-1 signaling (IIS) pathway. Downstream signaling components of the IIS pathway receptor have been well studied and include an interconnected network of signaling events that regulate many physiological outputs. However, less is known about the role of upstream signaling components and how intracellular pathways and physiology are regulated accordingly. In Part I, I present my work towards understanding upstream IIS pathway components using a systems biology approach. The goal of this study is to gain insight into the redundancy and specificity of the insulin gene family responsible for initiating IIS pathway activity in Caenorhabditis elegans. The information gained will serve as a foundation for future studies dissecting the molecular mechanisms of this pathway in efforts to uncouple the downstream signaling and physiological outputs. The clear impact of metabolism on aging and disease stimulated questions regarding the potential of promoting health and longevity through diet and dietary mimetics. Recent findings indicate reduced food intake, meal timing and nutritional modulation of the gut microbiome can ameliorate signs of aging and age-associated diseases. Aging, therefore, is also the result of dynamic and complex interplay between genes of an organism and its environment. In Part II, I will discuss my efforts to gain insight into how diet influences aging. This preliminary study has demonstrated that diet can affect lifespan in the model organism, C. elegans. Additionally, we observe diet-specific effects on drug efficacy that, in turn, modulates C. elegans lifespan and reproduction. The implications of these experiments, while limited, illustrate a potentially greater role in diet- and drug-mediated effects on lifespan.

Protein-mediated sugar transport is nominally absent in normoxic (adequately oxygenated) pigeon erythrocytes. Following exposure to metabolic inhibitors (cyanide or carbonylcyanide-p-trifluoromethoxyphenylhydrazone), pigeon red cells transport sugars by a saturable, stereoselective pathway that is inhibited by cytochalasin B or forskolin. The sugar transport capacity of fully poisoned cells is consistent with a transporter density of approximately 30 carriers per erythrocyte. Immunoblot analyses and competition ELISA indicate that pigeon red cells contain approximately 200 copies of an integral plasma membrane protein immunologically related to the glucose transporter isoform GLUT1. GLUT1 is quantitatively restricted to the plasma membrane at all times. Pigeon red cells and brain lack proteins immunologically related to the sugar transporter isoforms GLUT3 and GLUT4. Specific immunodepletion of red cell GLUT1 content results in the subsequent loss of reconstitutable protein-mediated sugar transport. These findings demonstrate that avian erythrocyte sugar transport is mediated by a GLUT1-like sugar transport protein and that sugar transport stimulation by metabolic inhibitors results from derepression of cell surface sugar transport proteins. Lysis-resealing experiments suggest that derepression is a glutathione (OSH) dependent phenomenon. This mechanism of transport regulation contrasts with insulin stimulation of sugar transport in muscle and adipose tissue which is believed to result from recruitment of intracellular sugar transporters to the plasma membrane.

Mycobacterium tuberculosis, the causative agent of tuberculosis, can manipulate host cell death pathways as virulent strains inhibit apoptosis to protect its replication niche and induce necrosis as a mechanism of escape. In vitro studies revealed that similar to lytic viruses, M. tuberculosis has the ability to induce cytolysis in macrophages when it reaches an intracellular burden of ~25 bacilli. Base on this finding, we proposed the burst size hypothesis that states when M. tuberculosis invades a macrophage at a low multiplicity of infection it replicates to a burst size triggering necrosis to escape the cell and infect naïve nearby phagocytes, propagating the spread of infection. The first part of this study investigated if the in vitro observations of M. tuberculosis cytolysis were relevant to cell death of infected phagocytes during pulmonary tuberculosis in vivo. Mice infected with a low dose of M. tuberculosis revealed during TB disease, the major host cell shifted from one type of phagocyte to another. Enumeration of intracellular bacilli from infected lung cells revealed the predictions of the hypothesis were confirmed by the distribution of bacillary loads across the population of infected phagocytes. Heavily burdened cells appeared nonviable sharing distinctive features similar to infected macrophages from in vitro studies. Collectively, the data indicates that M. tuberculosis triggers necrosis in mononuclear cells when its number reaches the threshold burst size. The previous study showed during the period of logarithmic bacterial expansion, neutrophils were the primary host cell for M. tuberculosis coinciding with the timeframe of the highest rate of burst size necrosis. The second part of this study examined this link by infecting mice with one of four different M. tuberculosis strains ranging in virulence. Mice infected with the most virulent strain had the highest bacterial burden and elicited the greatest number of infected neutrophils with the most extensive lung inflammation and greater accounts of cell death. Treating these mice with a bacteriostatic agent decreased the bacterial load and infected neutrophils in a dose-dependent manner indicating necrosis induced by virulent M. tuberculosis recruited neutrophils to the lungs. Infected neutrophils can serve as a biomarker in tuberculosis as evidenced by poorly controlled infection and increased severity of lung immune pathology.

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration. Two causative genes have been identified so far, WFS1 and WFS2, both encoding endoplasmic reticulum (ER) localized transmembrane proteins. Since WFS1 is involved in the ER stress pathway, Wolfram syndrome is considered an ER disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome, the molecular mechanism linking ER to the death of β cells and neurons has not been elucidated. The endoplasmic reticulum (ER) is an organelle that forms a network of enclosed sacs and tubes that connect the nuclear membrane and other organelles including Golgi and mitochondria. ER plays critical functions in protein folding, protein transport, lipid metabolism, and calcium regulation. Dysregulation of ER function disrupts normal cell metabolism and activates an array of anti-survival pathways, eventually leading to disease state. Here we show that calpain is involved in both prototypes of Wolfram syndrome. Calpain 2 activity is negatively regulated by WFS2 protein, and hyper-activation of calpain 2 by WFS2-knockdown leads to cell death. Calpain hyper-activation is also present in WFS1 loss of function cells due to the high cytosolic calcium. Extensive calpain activation exists in the Wolfram syndrome mouse model as well as in patient cells. A compound screen targeting ER homeostasis reveals that dantrolene, a ryanodine receptor inhibitor, can prevent cell death in cell models of Wolfram syndrome. Our results demonstrate that the pathway leading to calpain activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites. The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD mice also exhibit worsened performance on the raised-beam test, a test for balance and coordination indicating that the PSA heterozygosity impairs the function of neurons with mutant huntingtin. In order to test whether increasing PSA expression ameliorates the HD phenotype in mice we created an adeno-associated virus (AAV) expressing the human form of PSA (AAV-hPSA). Unexpectedly, testing of AAV-hPSA in non-HD mice resulted in widespread toxicity at high doses. These findings suggest that overexpression of PSA is toxic to neurons in the conditions tested. In the second part of my dissertation work, I designed a model for following the silencing of huntingtin sequences in the brain. Firefly luciferase is a bioluminescent enzyme that is extensively used as a reporter molecule to follow biological processes in vivo using bioluminescent imaging (BLI). I created an AAV expressing the luciferase gene containing huntingtin sequences in the 3'-untranslated region (AAV-Luc-Htt). After co-injection of AAV-Luc-Htt with RNA-silencing molecules (RNAi) into the brain, we followed luciferase activity. Using this method, we tested cholesterol-conjugated siRNA, un-conjugated siRNA, and hairpin RNA targeting both luciferase and huntingtin sequences. Despite being able to detect silencing on isolated days, we were unable to detect sustained silencing, which had been reported in similar studies in tissues other than the brain. We observed an interesting finding that co-injection of cholesterol-conjugated siRNA with AAV-Luc-Htt increased luminescence, findings that were verified in cell culture to be independent of serotype, siRNA sequence, and cell type. That cc-siRNA affects the expression of AAV-Luc-Htt reveals an interesting interaction possibly resulting in increased delivery of AAV into cells or an increase in luciferase expression within the cell. My work presents a method to follow gene silencing of huntingtin targets in the brain, which needs further optimization in order to detect sustained silencing. Finally, in this dissertation I continue the study of bioluminescent imaging in the brain. We use mice that have been injected in the brain with AAV-Luciferase (AAV-Luc) to screen 34 luciferase substrate solutions to identify the greatest light-emitting substrate in the brain. We identify two substrates, CycLuc1 and iPr-amide as substrates with enhanced light-emitting properties compared with D-luciferin, the standard, commercially available substrate. CycLuc1 and iPr-amide were tested in transgenic mice expressing luciferase in dopaminergic neurons. These novel substrates produced luminescence unlike the standard substrate, D-luciferin which was undetectable. This demonstrates that CycLuc1 and iPr-amide improve the sensitivity of BLI in low expression models. We then used CycLuc1 to test silencing of luciferase in the brain using AAV-shRNA (AAV-shLuc). We were unable to detect silencing in treated mice, despite a 50% reduction of luciferase mRNA. The results from this experiment identify luciferase substrates that can be used to image transgenic mice expressing luciferase in dopaminergic neurons. My work contributes new data on the study of PSA as a modifier of Huntington’s disease in a knock-in mouse model of Huntington’s disease. My work also makes contributions to the field of bioluminescent imaging by identifying and testing luciferase substrates in the brain to detect low level of luciferase expression.

During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation. In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks). Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance. Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function. This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation. Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.

Mobile genetic elements represent a large portion of the genome in many species. Posing a danger to the integrity of genetic information, silencing and structural machinery has evolved to suppress the mobility of foreign and transposable elements within the genome. Condensin proteins – which regulate chromosome structure to promote chromosome segregation – have been demonstrated to function in repetitive gene regulation and transposon silencing in several species. In model system Caenorhabditis elegans, microarray analysis studies have implicated Condensin II subunit HCP-6 in the silencing of multiple loci, including DNA transposon MIRAGE. To address the hypothesis that HCP-6 has a direct function in transcriptional gene silencing of the MIRAGE transposon, we queried MIRAGE expression and chromatin profiles in wild-type and hcp-6 mutant animals. Our evidence confirms that HCP-6 does indeed function during silencing of MIRAGE. However, we found no significant indication that HCP-6 binds to MIRAGE, nor that HCP-6 mediates MIRAGE enrichment of H3K9me3, the repressive heterochromatin mark observed at regions undergoing transcriptional silencing. We suggest that the silencing of MIRAGE, a newly evolved transposon and the only tested mobile element considerably derepressed upon loss of HCP-6, is managed by HCP-6 indirectly.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. Complementation of a high-passage non-adherent B. burgdorferi strain reveals that the expression of DbpA, DbpB, or BBK32, is sufficient to confer efficient spirochete attachment to 293 epithelial cells. Epithelial cell attachment by DbpA and B was mediated by dermatan sulfate, while BBK32 recognized dermatan and heparan sulfate. The GAG binding properties of bacteria expressing DbpB or DbpA were distinguishable in that DbpB, but not DbpA, promoted spirochetal attachment to C6 glial cells. Furthermore, DbpA alleles from diverse Lyme disease spirochetes exhibit allelic variation with respect to binding decorin, dermatan sulfate, and epithelial cells. Targeted disruption of bbk32 resulted in decreased spirochete binding to fibronectin, GAGs, and mammalian cells. Thus, DbpA, DbpB, and BBK32 may play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains and targeted gene disruption provide a comprehensive genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.

The work in presented in this thesis identifies Drosophila Wnt8 as a novel zygotic target of the Dorsal/Twist/Snail network using a microarray analysis to identify differentially expressed genes in maternal dorsal mutant and gain-of-function Toll10b embryos as compared to wild type. In-situ hybridization with a Wnt8 antisense RNA probe revealed a fairly complex expression pattern in the early embryo. No maternal expression was observed and the first zygotic expression appeared at stage 4 at the poles. This was followed by patchy and relatively weak expression in the presumptive mesoderm with stronger expression in the mesectoderm and later the neuroectoderm. These expression required the Dorsal/Twist/Snail network with some input from Delta in the neuroectoderm. All embryonic Wnt8 expression ceased after late stage 10. Snail was found to repress Wnt8 in the presumptive mesoderm. The relevance of Wnt8 as a Snail target was tested by bypassing this repression in wild type embryos using a maternal Gal4 driver to drive UAS-Wnt8. This led to a loss of ventral furrow formation and a phenocopy of the snail mutant phenotype, thereby indicating that the repression of Wnt8 by Snail is important for gastrulation. Further investigation into the mechanism revealed a reduction in the expression of multiple target genes of Dorsal (including snail) in these Wnt8 overexpressing embryos. Ventral nuclear Dorsal protein was reduced as compared to wild type, suggesting that high levels of Wnt8 can antagonize Dorsal nuclear localization. Deficiency embryos lacking Wnt8 showed the opposite phenotype of expanded anterior and posterior snail RNA staining, as well as an expanded nuclear Dorsal signal in the posterior. This could be phenocopied using dsRNA against Wnt8, and was fully rescueable in the deficiency background using a Wnt8 genomic fragment. It has been reported that loss-of-function snail embryos lose the sharp lateral boundaries and high levels of snail expression more rapidly as compared to wild type. We hypothesize that this loss is due to derepressed Wnt8 antagonizing Dorsal and consequently its target, snail. In support of our hypothesis, double mutants of snail and the Wnt8 deficiency show a rescue of the snail pattern, though not a rescue of ventral furrow formation. Western blot analysis reveals a decrease in the levels of phosphorylation of Dorsal in Wnt8 overexpressing embryos as compared to wild type. Phosphorylation of Dorsal is required for its nuclear translocation. Hence, these data corroborate the observation of reduced nuclear Dorsal in embryos overexpressing Wnt8. Together, these data point to Wnt8 being an important target and a feedback inhibitor of the Dorsal/Twist/Snail pathway that achieves its effect by the inhibition of Dorsal.