During an immune response, T cell activation is mirrored by a dramatic metabolic shift from oxidative phosphorylation to glycolysis. The upregulation of glycolysis allows the cell to generate the molecules needed to rapidly proliferate and to synthesize effector molecules. The resolution of the T cell response is characterized by equally fast death of most effector T cells. The remaining T cells shift back to oxidative phosphorylation, allowing the cell to survive as a memory T cell. The upregulation of glycolysis and proliferation during the effector phase is paralleled by an increased sensitivity to T cell receptor restimulation-induced cell death (RICD). Whereas cellular metabolism and cell death are important in the proper function and response of T cells, it is not clear how metabolism regulates susceptibility to cell death, nor whether T cell proliferation and contraction are directly connected. The work presented in this dissertation provides a mechanistic link between T cell proliferation and contraction by demonstrating the regulation of caspase-3 activity by the metabolic state of T cells. In effector T cells, the cytokine interleukin (IL)-2 mediates the upregulation of glycolysis, while IL-15 induces oxidative phosphorylation and a memory-like state. IL-2 is known to sensitize T cells to RICD, while IL-15 reduces RICD and increases survival. This results from the ability of IL-2 and glycolysis to increase caspase-3 activity, whereas IL-15 induces the opposite phenotype. Activation of caspase-3 during glycolysis is mediated through clustering in lipid rafts in the plasma membrane. IL-15 is shown to inactivate caspase-3 through the posttranslational modification of protein glutathionylation, which is mediated by ROS generation in the mitochondria as a by-product of oxidative phosphorylation. We further observe that glycolysis parallels the reduced activity of the electron transport chain and oxidative phosphorylation, further increasing caspase-3 activity. This is mediated by the decreased expression of electron transport chain complexes and an increase in expression of the negative regulator of complex I, methylation-controlled J protein (MCJ). IL-15 promotes reduced expression of MCJ by its methylation. Similar to IL-15-cultured T cells, MCJ-deficient T cells manifest reduced glycolysis, caspase-3 activity, and RICD. Collectively, these findings demonstrate an adaptation that links metabolism to both cell proliferation and cell death to safeguard that proliferating cells do not escape regulation that could result in autoimmune disease or lymphomas.

Francisella tularensis is the etiological agent of tularemia, a severe and potentially fatal disease in humans. It is extremely infectious by the aerosol route, being thought to cause disease in humans with an infectious dose as small as one to ten organisms, which led to its weaponization by several nations and classification as a category A select agent by the Centers for Disease Control and Prevention. An intracellular pathogen, relatively little is known about the mechanisms by which Francisella is capable of successfully modulating host cell processes to escape its phagosome and replicate within the cytosol and what genes beyond the Francisella pathogenicity island are required. Furthermore, in the context of aerosol exposure, it is unknown what cells F. tularensis initially interacts with and the overall contribution of those interactions to inhalational tularemia. I initiated this study by generating an in vitro model system to study interactions of F. tularensis with epithelial cell lines in tissue culture. Utilizing this system, I determined that F. tularensis LVS was capable of adherence to human epithelial cell lines of alveolar (A549), bronchial airway (HBE), and cervical carcinoma (HEp-2) origin. Furthermore, LVS was capable of invading these cell lines and growing productively within them. In order to detect genes important for virulence in this system, I generated a ~15,000 member transposon library in virulent strain Schu S4 that was could be screened in a high-throughput manner by transposon site hybridization. As uptake in the in vitro epithelial cell line system was relatively inefficient, I screened this library through human primary macrophages. Results of the screen implicated 207 genes as negatively selected in the human macrophage model. Of these, I generated mutants in genes residing in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238, to determine their virulence phenotypes. Mutants in these genes demonstrated significant vulnerability to complement-mediated lysis as compared with wild type Schu S4. Analysis of purified LPS and capsule from these mutants further showed that they had marked defects in O-antigen and capsular polysaccharide biosynthesis. Complementation of these mutants restored surface polysaccharide biosynthesis and further determined that FTT1236 and FTT1237 compose an operon, as a mutation in FTT1236 is polar onto FTT1237. Characterization of the intracellular defect of these mutants in the absence of active complement demonstrated that they were taken up more efficiently by primary human macrophages than wild type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic analysis of macrophages infected with mutant bacteria revealed that, as early as 16 hpi, these macrophages exhibited signs of cell death. In contrast, cells infected with Schu S4 exhibited a healthy, spread morphology as late as 32 h, despite significantly more extensive F. tularensis cytosolic replication. Quantitation of cell death by the release of lactate dehydrogenase, signifying membrane permeability, confirmed that mutants in FTT1236, FTT1237, and FTT1238 induced early cell death in infected macrophages as compared with wild type Schu S4. Together, this work contributes to our understanding of the factors, such as O-antigen and capsule, required for and genes involved in Francisella's lifecycle as an intracellular pathogen.

Includes a list of publications co-authored by the author during the preparation of this thesis. Thesis amendments in back leaf. Includes bibliographical references (leaves 123-168). The studies described concentrate on the cloning and characterisation of the two Drosophila caspases, DRONC and DECAY

This study assessed the consequences of a single binge exposure to ethanol on postnatal day (PN) 4 in the Purkinje cell population of the rat cerebellar vermis. The acute temporal pattern of apoptosis and the long-term permanent deficit in Purkinje cells across the entire vermis and on a lobular basis was investigated. It also investigated the lobular based Purkinje cell vulnerability to ethanol-induced apoptosis. On PN4 Sprague-Dawley rat pups from timed matings were randomly assigned to either an alcohol exposed (AE; 4.5g/kg, 10.2% (v/v) as 2 doses 2 hours apart) via intragastric intubation, or sham intubated control (IC) group. Immunolabelling methods were used to detect active capase-3 in cerebellar vermal Purkinje cells hourly from 1 to 12 hours and 14, 16, 20, and 24 hours, following the first intubation. Semi-quantitative methods were used to determine the ratio of active caspase-3, a marker of apoptosis, labelled Purkinje cells per total Purkinje cells within the entire cerebellar vermis and on a lobular basis. The onset of caspase-3 activation occurred 3-4 hours following the initial ethanol exposure and peaked 10 hours post-ethanol, the magnitude of increase in active caspase-3 varied across lobules. To determine the long-term consequences a second cohort of pups was treated as per above and the total number of Purkinje cells in the adult (PN60) cerebellar vermis, as well as in lobules I/II, VII and IX, was determined using the optical disector method combined with the Cavalieri technique. A significant deficit was found in the entire vermis (21%), as well as lobules I/II (48%), VII (25%) and IX (38%). A third cohort of suckle control (SC) pups was used to investigate the lobular based vulnerability to apoptosis in normal cerebellar vermal Purkinje cells on PN4. The levels of Bcl-2 and Bax mRNA expression were investigated using laser capture microdissection (LCM) to isolate the Purkinje cell lobular based populations combined with quantitative RTPCR (qPCR) technique. This method enabled the determination of comparative levels of mRNA expression attributable to the Purkinje cell population in identified lobules. Results indicated that Bcl-2 is expressed in cerebellar Purkinje cells and does not vary across the lobules. However, Bax expression could not be confirmed. This thesis demonstrates the effects of a moderate ethanol binge on cerebellar vermal Purkinje cells on PN4. Findings demonstrate that apoptosis is initiated rapidly and results in the permanent loss of Purkinje cells, the degree of which is lobular dependent and greater than seen in the vermis as a whole. Thus significant cell loss can occur in discrete regions which may have specific discrete impairments in cerebellar function. Importantly for the human population, this study indicates that a single moderate alcohol binge is sufficient to cause significant permanent neuronal loss in the developing fetus, the onset of which occurs rapidly, and may result in specific brain dysfunctions.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are critical for the protection of organisms against pathogens and cancer. The process by which these cells eliminate infected or transformed cells are through two basic mechanisms, receptor-mediated interactions, or delivery of contents from intracellular cytotoxic granules. Granules are comprised of perforin and a family of serine proteases, called granzymes. Upon entry into target cells, these proteins work together to initiate cellular death pathways. Previous and extensive biochemical studies had already established that granzyme B (GrB) was a powerful inducer of apoptosis, but sensitive assays to confirm its release from cytotoxic cells were lacking. We hypothesized that GrB release, measured by ELISPOT, directly assessed the lytic potential of antigen-specific cytotoxic cells. Indeed, data provided in this thesis established a strong correlation between GrB release and target cell lysis. Our results imply that GrB could be a promising tool to assess cell-mediated immunity during vaccine development. However, several other independent studies in grB-/- mice demonstrated that additional granzymes were capable of clearing viruses and tumorigenic cells. Granzyme H (GrH) is highly and constitutively expressed in human NK cells, and therefore, we hypothesized that it was also an effective cytotoxic molecule. Our experiments established that GrH-induced cell death by a mechanism distinct from those of GrB and Fas. We identified a GrH substrate, DFF45/ICAD, and showed that GrH induced mitochondrial damage through a Bid-independent mechanism. Furthermore, cell death was dependent on Bax and/or Bak, but independent of caspase activation. Hence, we have elucidated an alternative cytotoxic pathway that could be employed to eliminate target cells with immune evasion strategies targeted to GrB or Fas. Finally, control of serine proteases by endogenous inhibitors is important to numerous biological processes, including apoptosis. We hypothesized that as GrH displayed chymase activity, the serine protease inhibitor anti-chymotrypsin (ACT) would impair GrH function. Our data established that ACT effectively attenuated GrH cytotoxicity and prevented proteolysis of a GrH substrate. Collectively, this thesis describes a novel GrH inhibitor, provides a new tool to evaluate cell-mediated immunity, and provides evidence of an alternative mechanism of cytotoxicity.

Programmed cell death (PCD) is an essential feature during the development of the central nervous system in Drosophila as well as in mammals. During metamorphosis, a group of peptidergic neurons (vCrz) are eliminated from the larval central nervous system (CNS) via PCD within 6-7 h after puparium formation. To better understand this process, we first characterized the development of the vCrz neurons including their lineages and birth windows using the MARCM (Mosaic Analysis with a Repressible Cell Marker) assay. Further genetic and MARCM analyses showed that not only Myoglianin (Myo) and its type I receptor Baboon is required for neuron cell death, but also this death signal is extensively regulated by endocytic trafficking in Drosophila melanogaster. We found that clathrin-mediated membrane receptor internalization and subsequent endocytic events involved in Rab5-dependent early endosome and Rab11-dependent recycling endosome differentially participate in TGF-β [beta] signaling. Two early endosome-enriched proteins, SARA and Hrs, are found to act as a cytosolic retention factor of Smad2, indicating that endocytosis mediates TGF-β [beta] signaling through regulating the dissociation of Smad2 and its cytosolic retention factor.

FCH/CIP4 homology-Bin-Amphiphysin-Rvs (F-BAR) domain proteins are a subfamily of the Bin-Amphiphysin-Rvs (BAR) superfamily of proteins. They contain unique domains that bind and reshape the phospholipid bilayers of endosomal compartments during endocytosis. Using a functional assay for cell survival, we identified an F-BAR protein, FCH/CIP4 homology and double Src homology 3 domains 2 (FCHSD2), that confers drug resistance. Stable expression of shRNA against FCHSD2 in multiple cell types showed that loss of FCHSD2 sensitized cells to apoptosis by doxorubicin. Silencing of FCHSD2 also enhanced the ability of fibroblasts to grow colonies in culture. Mass spectrometry analysis of FCHSD2 protein complexes identified multiple interacting proteins that are involved in adhesion and endosome trafficking. We identified and confirmed a novel interaction between FCHSD2 and sorting nexin 18 (SNX18), a BAR domain protein that binds to endosomes. Our results suggest that FCHSD2 is involved in regulating cellular adhesion and cell death.

In the presence of a non-fermentable carbon source, nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae undergo a meiotic program called sporulation to form gametes called spores. While four spores are produced under standard laboratory sporulation conditions, spore number is known to be regulated by carbon availability: under carbon-depleted conditions, yeast cells package a portion of the four haploid meiotic nuclei into spores. Our lab has demonstrated that these unpackaged meiotic products undergo programmed nuclear destruction (PND) that is associated with apoptotic-like DNA fragmentation. Nevertheless, the mechanism that mediates PND remained to be elucidated. Here, I describe the execution of PND through an unusual form of autophagy that has not been documented previously in yeast. This form of autophagy is most similar to megaautophagy in plants and lysosomal membrane permeabilization in mammals. My results demonstrate further diversity in cell death programs in unicellular microbes that is potentially conserved across eukaryotes.

FCH/CIP4 homology-Bin-Amphiphysin-Rvs (F-BAR) domain proteins are a subfamily of the Bin-Amphiphysin-Rvs (BAR) superfamily of proteins. They contain unique domains that bind and reshape the phospholipid bilayers of endosomal compartments during endocytosis. Using a functional assay for cell survival, we identified an F-BAR protein, FCH/CIP4 homology and double Src homology 3 domains 2 (FCHSD2), that confers drug resistance. Stable expression of shRNA against FCHSD2 in multiple cell types showed that loss of FCHSD2 sensitized cells to apoptosis by doxorubicin. Silencing of FCHSD2 also enhanced the ability of fibroblasts to grow colonies in culture. Mass spectrometry analysis of FCHSD2 protein complexes identified multiple interacting proteins that are involved in adhesion and endosome trafficking. We identified and confirmed a novel interaction between FCHSD2 and sorting nexin 18 (SNX18), a BAR domain protein that binds to endosomes. Our results suggest that FCHSD2 is involved in regulating cellular adhesion and cell death.

In the presence of a non-fermentable carbon source, nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae undergo a meiotic program called sporulation to form gametes called spores. While four spores are produced under standard laboratory sporulation conditions, spore number is known to be regulated by carbon availability: under carbon-depleted conditions, yeast cells package a portion of the four haploid meiotic nuclei into spores. Our lab has demonstrated that these unpackaged meiotic products undergo programmed nuclear destruction (PND) that is associated with apoptotic-like DNA fragmentation. Nevertheless, the mechanism that mediates PND remained to be elucidated. Here, I describe the execution of PND through an unusual form of autophagy that has not been documented previously in yeast. This form of autophagy is most similar to megaautophagy in plants and lysosomal membrane permeabilization in mammals. My results demonstrate further diversity in cell death programs in unicellular microbes that is potentially conserved across eukaryotes.