<p> For many organisms, the immune system tends to deteriorate with age, leading to higher susceptibility to foreign pathogens. While several biological pathways are associated with immunity, the components of the Janus-kinase-Signal Transducers and Activators of Transcription (JAK-STAT) pathway on immunity at different age groups is unclear. This study explored the knock down effects of the <i>Drosophila</i> JAK-STAT pathway components and a candidate gene, <i>robo3,</i> in blood cells. Assessments of immune function were conducted through bacterial clearance assays and phagocytosis assay at one-week and five-weeks of age. This study suggests that some JAK-STAT pathway components important in other cell types seem to have less of a role in blood cells and immunity.</p><p>

<p> Obesity is known to lower the quality of life of organisms and much effort has gone towards reporting the interactions between obesity and the immune response with one example being the metabolic syndrome caused by obesity-related inflammation. Work using <i>Drosophila</i> has shown high-fat diets affect cardiac function, lifespan, and glucose homeostasis. To determine whether metabolic syndrome can be modeled in flies, <i>Drosophila melanogaster </i> were raised on a high fat diet. Several parameters of the stress and immune responses were assayed in the presence and absence of infection using Gram-negative bacterium, <i>Serratia marcescens</i>. This study found that a high fat diet increased expression of cytochrome oxidase C subunit COX4L. High fat larvae had a reduced bacterial load, higher expression of the antimicrobial peptide <i>Diptericin</i>, and improved survival rate following acute infection. This study supports using <i>Drosophil </i>a as a model to improve understanding of metabolic-immune interactions and reports antimicrobial benefits from a high fat diet.</p><p>

The intention of the work described in this thesis was to identify whether the Notch signalling pathway is utilized by antigen presenting cells in order to influence CD4+ adaptive immune responses. The notion that Notch proteins may be involved in polarising CD4+ T cells is relatively recent and most of the work that had been done in this area so far has concentrated on the consequences of Notch signalling within T cells. In contrast, the work that I have done has focussed on Notch ligand expression by antigen presenting cells and addresses the question whether Notch signalling is a redundant, necessary or irrelevant tool in the arsenal of antigen presentation.

<p>Foam cell formation from arterial intima macrophages and defective clearance of apoptotic foam cells drive the progression of the inflammatory disease atherosclerosis. The role of innate immune protein C1q in autoimmune disease and pathogen defense is well characterized, however its role in atherosclerosis remains largely uninvestigated. Prior studies have characterized the complement independent role of C1q in polarizing macrophages towards an anti-inflammatory phenotype during uptake of apoptotic cells and modified lipoproteins. To further understand the role of C1q in programming human monocyte-derived macrophages during foam cell formation, we used RNA-sequencing to elucidate pathways that are modulated by C1q during clearance of atherogenic lipoproteins. Expression of genes in JAK-STAT, PPAR, apoptotic, and TLR signaling pathways were modulated by C1q in this study. In addition, C1q suppressed STAT1 and PPAR transcriptional activity. This study identifies potential molecular mechanisms that support a beneficial role for C1q in early atherosclerosis. </p>

This dissertation was to study the sites of B cell development, the mechanisms of Ig diversification, the time when diversification occurs, the B cell repertoire during development and usage of Ig light chains in cattle. The expression of TdT, RAG-1 and RAG-2 was detected in spleen in young animals but not in old animals indicating that gene rearrangement of B cells takes place in spleen and is restricted to a short period of time during early development in cattle. To determine the mechanisms by which cattle create a diverse Ig repertoire, genomic rearrangement patterns and V$\lambda$ sequences of cDNA and germline genes were examined. Few rearrangements, potential donor sequences in germline pseudogenes and point mutations in the cDNAs were found. These data suggest that cattle primarily use gene conversion to create a diverse Ig repertoire and somatic hypermutation may be used to refine the Ig repertoire upon antigen challenge. To investigate when diversification occurs, V$\lambda$ IPP cDNA were compared between young and old animals. Many diverse nucleotides were found in young animals as that in old animals indicating that the diversification takes place in the very early stage of development. To determine the peripheral Ig repertoire during development, expression of peripheral light chains of an animal at different ages was examined. Diversity clustered in the CDRs was increased and different clones which were present in the periphery at a low level in neonatal animals were found to be expanded in older animals. These data indicate that the peripheral Ig repertoire is ligand-selected and clonally expanded by positive selection. To investigate the control of $\kappa$/$\lambda$ usage, expression and germline $\kappa$/$\lambda$, V$\kappa$ and C$\kappa$ repertoire were examined. The ratio of expressed $\kappa$/$\lambda$ was high in IPP and spleen, but it was low in the periphery. Additionally, similar numbers of $\kappa$/$\lambda$ genes were found in the germline and V$\lambda$ cDNA sequences were found to be as diverse as that of V$\lambda$. In contrast, C$\kappa$ possesses a low homology to other primarily $\kappa$ expressing mammals. These data suggest that a post-transcriptional control may govern the usage of $\kappa$/$\lambda$ in cattle.

Thymocytes undergo negative and positive selection during their development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis. In the first part of this dissertation, I examined the role of oxidative stress in thymocyte apoptosis. My initial observations show that thymocytes require molecular oxygen to undergo apoptosis, and treatment with N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. Various apoptosis-inducing stimuli increase intracellular hydrogen peroxide (H2O2) levels in thymocytes ex vivo, and treatment with NAC reduces the levels of intracellular H2O2 during apoptosis. The degree of reduction of H2O2 by NAC correlates well with the decrease of apoptosis, except in cells treated with γ-irradiation. These results indicate that the level of intracellular H2O 2 influences a cell's vulnerability to undergo apoptosis under many conditions, but not all. I also show that cell death-related mitochondrial events are attenuated by NAC treatment in protected cells. By using various inhibitors of the mitochondrial electron transport chain, I identified the production site for H2O2 under all apoptosic conditions tested as complex III of the mitochondria. The results show that when the inhibitors decrease the production of H2O2 at the mitochondria, the mitochondrial cell death events are also significantly reduced under all conditions. I also show that the production of H2O2 and the mitochondrial cell death events are controlled by proteosomal activities during thymocyte apoptosis. The second part of this dissertation focused on the role of hyperbaric oxygen (HBO) in enhancing apoptosis and/or suppressing cellular proliferation. This study provides evidence that HBO treatment increases intracellular H 2O2, which is partly responsible for enhancing apoptosis in HL-60 cells, a granulocytic cell line. Since HBO is effective in treating chronic wounds, these results suggest HBO may exert its beneficial effect by inducing apoptosis in neutrophils, known to mediate chronic inflammation. I also provide a piece of evidence that exposure to HBO can stop the proliferation of breast cancer cells at various stages of the disease. This could be due to abrogated antioxidative defense mechanisms, which are commonly found in rapidly dividing cells.

Apoptosis is a type of programmed cell death that is essential for the development and maintenance of tissue homeostasis in multicellular organisms. Because apoptosis is important in many biological situations, laboratories are working to delineate the signaling pathways responsible for this process. This thesis work attempts to identify new components of the signaling pathways by examining a role for mitochondria and proteasomes in T cell apoptosis. Interest in mitochondria was initiated when a library screen performed in the laboratory revealed that portions of the mitochondrial genome, called apt-1 and apt-3, were downregulated in thymocytes during negative selection. In this thesis work, two aspects of mitochondrial function were examined in the T cell hybridoma, DO11.10: the integrity of the mitochondrial genome as assessed by levels of apt-l and apt-3 and the integrity of the mitochondrial membrane as assessed by membrane potential. Both the steady-state levels of apt-l and apt-3 RNA and mitochondrial transmembrane potential declined rapidly in apoptotic cells. Recent studies from other laboratories indicate that these changes are the result of the active participation of mitochondria in apoptosis. Apoptosis relies heavily on proteolysis, and this dependence has encouraged investigators to analyze the significance of many proteases and proteolytic pathways. The second part of this thesis work focused on describing a role for the ubiquitin-proteasome pathway in apoptosis. Proteasome inhibitors were used to determine whether the proteasome is required in thymocyte apoptosis. Treatment of thymocytes with acetyl-leu-leu-methioninal (LLM), acetyl-leu-leu-norleucinal (LLnL), carbobenzoxyl-leu-leu-leucinal (MG132), or lactacystin inhibited death induced by dexamethasone, ionizing radiation, or phorbol 12-myristate 13-acetate (PMA). These results suggest that proteasome activity is necessary for the progression of many apoptotic pathways in thymocytes. Inhibitor studies also indicate that the proteasome functions upstream of many apoptotic events, including: PARP cleavage, caspase-3 activation, and mitochondrial transmembrane potential depolarization. Although a precise role for the proteasome in apoptosis cannot be assigned without an identification of its proteolytic targets, the work described in this thesis provides the first definitive link between proteasomes and apoptosis in mammalian systems.

Runx1 regulates the expression of several important target genes and plays critical roles in the process of hematopoiesis. Runx1 by itself is a poor regulator of transcription and instead nucleates transcription complexes through its C-terminus to transactivate or repress the expression of target genes. We generated a C-terminally deleted Runx1 construct (Runx1.d190), which lacks important co-factor sites, to further investigate the function of Runx1 in development. A potential role for Runx1 in regulating the expression of another potent transcriptional regulator, c-Myc, has been suggested by published studies, which show that Runx1 and c-Myc collaborate in oncogenesis. In these studies, we show that endogenous Runx1 binds to three Runx consensus sites upstream of the c-Myc transcriptional start site in Jurkat T cells and murine primary splenocytes. Retroviral transduction of Jurkat T cells with Runx1.d190 results in the increased transcription of c-Myc as determined by microarray analysis. In order to monitor c-Myc expression in response to early-acting and transient Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Treatment of murine primary splenocytes with TAT-Runx1.d190 protein results in a transient increase in the transcription of c-Myc and a corresponding increase in c-Myc protein levels. This effect is dependent on the ability of Runx1.d190 to bind to DNA. These data demonstrate that Runx1 directly regulates c-Myc expression in a C-terminally and DNA-binding dependent manner. In these studies, we also investigate the effects of the truncation of Runx1 C-terminus on hematopoietic stem cells (HSCs). We found that treatment of bone marrow cells enriched for HSCs with TAT-Runx1.d190 results in a 12.5 fold increase in hematopoietic precursors compared to untreated precursors in vitro as determined by Colony Forming Cell assays. We also show that hematopoietic precursors treated with TAT-Runx1.d190 are able to able to differentiate normally both in vitro and in vivo and thus represent functional hematopoietic precursors. Our findings show that we are able to transiently expand hematopoietic precursors ex vivo by treating the cells with a Runx1 construct that lacks the C-terminus. Collectively, this work demonstrates that Runx1 regulates the expression of c-Myc and the expansion of hematopoietic precursors in a C-terminally dependent manner.

Notch1 specifically upregulates expression of the cytokine interferon-γ in peripheral T cells through activation of NF-κB. However, how Notch mediates NF-κB activation remains unclear. NF-κB activation occurs within minutes of TCR engagement and this activation is sustained for at least 48 hours following TCR signaling. We used either γ-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members or siRNA against Notch1 to reduce endogenous expression of Notch1 specifically. We demonstrate that GSI blocked the later, sustained NF-κB activation, but did not affect the initial activation of NF-κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF-κB and competes with IκBα, leading to retention of NF-κB in the nucleus, and that N1IC can directly regulate IFN-γ expression through complexes formed on the IFN-γ promoter. Additionally, within the immunological synapse, cytosolic Notch1 associates with CARMA1 and BCL10, mediating a direct interaction among them. Upon TCR and CD28 stimulation, Notch1 directly interacts with PKC&thetas; and the IKK complex, leading to IKK-mediated activation of NF-κB. In the absence of Notch1, there is no formation of CARMA1/BCL10/Malt1 complex, which is required for IKK activation. Taken together, these data suggest that Notch1 plays two roles during T cell activation: as an activation scaffold in the cytoplasm and as a transcriptional activator in the nucleus.

Notch signaling is critical for the regulation of differentiation, proliferation and apoptosis in many cell types. Notch receptors and various Notch ligands have been shown to have a regulatory effect on cell cycle progression during the processes of development and differentiation. Cyclins are proteins that regulate cell cycle check-points, thereby controlling cell cycle progression. The D-cyclins, specifically, are required for overcoming the G1/S checkpoint. It has been shown previously (Ronchini et al) that NotchIC regulates the expression of Cyclin D1, one member of the Cyclin-D family. It has also been reported (Sicinska et al.) that Cyclin D3-/- mice display impaired thymocyte development and do not develop Notch1-induced leukemia. Based on these observations, we hypothesized that cyclin D3 may be a downstream target of Notch signaling in T cells. We observed that T cell receptor signaling increases Cyclin D3, cdk4 and cdk6 expression in peripheral T cells and inhibiting Notch activity reduces Cyclin D3, cdk4 and cdk6 expression in activated T cells. Using reporter assays, as well as chromatin immunoprecipitation, we show that the transactivation domain of Notch1 is critical for regulating the Cyclin D3 promoter. We demonstrate that cyclin D3-cdk4 and cdk6 is also responsible for cell cycle progression in Notch-dependent human T-ALL cell lines. We show that Cyclin D3 and cdk4/6 are important targets in constitutively active Notch signaling in leukemic T cells, as they can partially override the G1 arrest observed with GSI treatment. Together, our data indicate that Notch signaling controls peripheral and leukemic T cell proliferation. Furthermore, we have begun to outline a possible mechanism for the regulated expression of Cyclin D3 and cdk4/6 in leukemic T cells, and through our future experiments we hope to reveal the oncogenic potential of Cyclin D3 as a target of dysregulated Notch1 signaling.