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.

<p> COPD is characterized by an abnormal regulatory T cell (Treg) response with a shift towards a Th1 and Th17 cell responses. However, it is unclear if the function of Treg cells is impaired by smoking and in COPD. In addition, the miRNA profile of Treg cells in COPD is unknown and whether miRNA deregulation contributes to COPD immunopathogenesis. We set the objective to study Treg cell function isolated from peripheral blood of patients with COPD versus controls and to compare their miRNA profiles. We also were interested in exploring the function of some of the differentially expressed Treg cell miRNAs. We assessed the Treg cell function by observing their suppressive activity on autologous effector T cells and analyzed their miRNA expression initially by microarray analysis then conducted real time RT-PCR validation for selected miRNAs. In Silico target gene analysis for the validated miRNAs suggested that miR-199-5p is particularly relevant to Treg cell physiology so its function was investigated further using CCD-986Sk and MOLT-4 cells. We found no difference in Treg cell function between COPD and controls but we were able to identify 6 and 96 miRNAs that were differentially expressed in COPD versus control Treg cells. We confirmed that miR-199a-5p was repressed by approximately 4 fold in Treg cells of COPD patients compared to cells in healthy smokers. Importantly, miR-199a-5p had significant overrepresentation of its target genes in the Treg cell transcriptome, with many targets associated with the TGF-&beta; activation pathway. We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan&reg; arrays of the Human TGF-&beta; Pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favor of a Th1 and Th17 response.</p>

It is widely accepted that activating mutations of genes encoding the Notch family of transmembrane receptors, specifically Notch1, are associated with oncogenic transformation. Previous data from our lab has shown that an active form of Notch1 (Nic) provides a protective effect against apoptosis in D011.10 T cells, and that this effect may be attributed to Nic binding the pro-apoptotic protein Nur77. Nur77 is an immediate early gene that is upregulated during negative selection of thymocytes and activation-induced apoptosis in D011.10 T cells. Nur77 upregulation is tightly regulated and requires MEF2D, NFAT, and the co-activator, p300, to effectively respond to apoptotic stimuli. In this report we show that Nic has the ability to interfere with the induction of transcription of Nur77, and that this interference is directly related to the inability of p300 to bind the Nur77 promoter in the presence of Nic. We also show that blocking Notch activation through gamma secretase-inhibitors or siRNA directed against Notch1 in T cell acute lymphoblastic leukemia (T-ALL) cell lines restores Nur77 upregulation in response stimuli. These observations support a model in which during thymocytes negative selection activating mutations of Notch1 inhibit the upregulation of a crucial proapoptotic molecule. Studies to determine the mechanism by which Nur77 induces apoptosis have indentified a unique translocation of Nur77 from the nucleus to the cytosol. It has been determined that once in the cytosol Nur77 interacts with members of the Bcl-2 family of proteins at the mitochondrial membrane. This interaction induces a conformational change of Bcl-2 so that is becomes pro-apoptotic instead of protective. Of similar interest is the role that Nur77 itself plays during the induction of activation-induced apoptosis which may be independent of Bcl-2 conformational change. In an effort to describe possible functions of Nur77, DO11.10 cells that have Nur77 under a tet-inducible promoter were observed for changes IP3R. Initial results from our lab suggest that Nur77 alone has the ability to induce cell death in DO11.10 cells using this tet-inducible system. Interestingly we have been able to identify distinct changes in IP3R isoforms during stimulation induced apoptosis and Nur77-dependent apoptosis. Current experiments are focused on a mechanism beyond the known function of the Nur77/Bcl-2 interaction; that Nur77 may also be acting as a physical barrier between the known anti-apoptotic interaction of IP3R and Bcl-2, leading to sustained calcium flux.

Biology is the study of life and life systems. Until recently, biologists have concentrated in examining how cells proliferate, differentiate, and survive in biological systems. Although such studies reveal extremely interesting insights into the complexity of living organisms, there is much more to this story. Just as cells live, cells must also eventually die. The study of how and why cells die has become the focus much scientific research over the past decade. Because the regulation of the number and specificity of cells in the immune system is critical to the life of a mammalian organism, researchers began to investigate how this strict control was accomplished. It was found that large quantities of immature T cells die in the course of their development by a specific type of cell death process coined apoptosis. This Ph.D. dissertation was directed towards examining the mechanisms involved in the regulation of thymocyte apoptosis in a murine model system. The first portion of the project involved isolating differentially regulated genes using either a plus/minus or subtractive hybridization screening strategy The second component of this dissertation investigated possible roles molecular oxygen and/or free radicals play during thymocyte apoptosis. Results from these studies both identified numerous putative death transcripts as well as revealed the requirement for oxygen during cell death in thymocytes induced by specific stimuli.

Studies were undertaken to examine immunoglobulin repertoire diversification in cattle. Diversification was examined in a number of organs from late first trimester bovine fetuses and from the ileal Peyer’s patch (IPP) follicles of young calves. To investigate the diversification in IPP follicles, individual IPP follicles were isolated by microdissection and diversification of the lambda variable region was examined by RT-PCR and subsequent cloning and sequencing. When intrafollicular sequences from a 4 week old calf were determined and compared, two major groups could be delineated. An examination of these groups revealed clear genealogical relationships that implicated both gene conversion and untemplated somatic hypermutation as the mechanisms responsible for diversification of VX within the IPP follicles. Diversification of Vλ was also examined in early (95–110 gestational day) fetal organs. The organs examined included fetal spleen, blood, liver, thymus, ileum and bone marrow. Sequences obtained from the various organs revealed that while Vλ sequences were highly diversified in spleen, very little VλX diversification was seen in the blood, liver, ileum or bone marrow. The sequences obtained from spleen indicated that both gene conversion and untemplated somatic hypermutation could be taking place in fetal spleen. Evidence for diversification in fetal spleen was also obtained by examining expression of recombination activating genes (RAG). An examination of fetal tissues for the expression of RAG-1 found that RAG-1 transcripts were present only in fetal thymus, bone marrow and spleen. The presence of both RAG-1 transcripts and a highly diversified population of Vλ sequences implicates the fetal spleen as an organ where both Vλ rearrangement and diversification might take place in cattle.

Cattle represent an economically important species whose immune system seems to depart from the standard human and murine models. Little has been documented about bovine lymphocyte activation in vitro, such work is needed for comparative immunology and for us to understand bovine immunology. In general, lymphocyte activation is accompanied by many molecular changes at the mRNA level. The unique characteristics of lymphocyte activation imply that a unique set of genes is associated with this biological process. However, there is very little known (in any species) about lymphocyte-specific genes that are differentially up-regulated after activation. In this thesis, bovine lymphocyte activation was first stimulated in vitro. Secondly, representational difference analysis (RDA) method was used to clone mRNAs that are exclusively present in activated bovine lymphocytes. Subsequently, the cDNAs were analyzed by DNA sequencing and homology search against the Genbank database. Clone E8 was identified as a potential G-protein-coupled receptor. E8 is up-regulated in activated bovine lymphocytes at 2 hours post stimulation. When up-regulated, E8 mRNA level remains constant from 2 hours to at least 72 hours post stimulation. Similar kinetic expression of E8 is observed following either LPS or Con A stimulations. Expression of E8 was also detected in murine lymphocytes upon activation with LPS or Con A and with similar kinetic expression. E8 showed increased level of expression when human T and B lymphocytes were activated by cross-linking of antigen receptors along with costimulatory molecules. E8 expression was found not to be associated with resting non-lymphoid tissues, activated non-lymphoid cell lines, nor activated macrophages and neutrophils. Therefore, E8 represents an early gene specific to lymphocyte activation. The size of the full-length transcript of clone E8 was estimated at about 2.2 kb. A full-length cDNA was obtained by the RACE procedure. Sequence alignment revealed that E8 is homologous to EB11, a human gene induced by EBV; CXCR1, as well as human CCR4 and CCR5 genes. Potential biological functions of this gene are discussed.