Cytotoxic T lymphocytes (CTL) are differentiated CD8+ T cells that eliminate virally infected cells and tumor cells. CTL lyse target cells by at least two distinct mechanisms: degranulation of cytolytic molecules and cell surface expression of Fas ligand (FasL), which induces apoptosis of Fas-expressing target cells. In addition to their defense function, these two cytolytic mechanisms also play crucial roles in homeostatic regulation and contribute to pathogenesis in many different model systems. To fully exploit killer cells in tumor and virus elimination, or dampen the immune response in, for example, autoimmune diseases, it is essential to understand the mechanisms that CTL employ to destroy target cells. In contrast to the well-characterized degranulation mechanism, the regulation of FasL expression on the CTL cell surface remains elusive and even controversial. The prevailing model at the time I initiated my studies was that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. In this thesis, I revealed for the first time that there are two waves of FasL cell surface expression upon target cell engagement, which are differentially regulated by TCR signaling and perform distinct roles in CTL mediated responses. I demonstrated that CTL degranulation and FasL lytic mechanisms are fully independent with respect to stored component localization and regulation. Finally, based on cell fractionation and imaging studies, I suggested that FasL is stored in a recycling endosome associated compartment, which is located in a special niche between the ER and mitochondria and uses a novel microtubule-independent secretory mechanism to translocate to the cell surface. Together, these findings provide important insight into the regulation and role of FasL in CTL mediated responses. / Immunology

Graduation date: 1998

Thesis.

The I-V characteristics of Tin films in the superconducting transition have been measured when ac current was applied. The experimental results suggest that the electrical response in ac is not satisfied with the I-V equation in dc. A new equation was suggested to describe the vortex motion and the vortex pair separation in the two dimensional superconducting transition with ac current, which is satisfied with our experimental results. The excess noises of Tin films in the superconducting transition have been found to depend strongly on the temperature and ac current. An empirical expression of voltage noise density in term of resistance has been used to fit the data. The peak of voltage noise density follows closely but always shifted down from dR/dT. Comparison with the dc noise measurement shows the voltage noise density with ac current is much larger than with dc current. The excess noises with ac appear earlier than the noises with dc. The difference of excess noises between ac and dc can be explained by the fluctuation of vortex pair separation process which dominates the noises generation in ac. I-V characteristics and voltage noises are measured simultaneously to reveal the nature of the excess noises. The coincidence of the excess noise and the third harmonic voltage suggests that the fluctuation of vortex pair separation process is one of the main contributions to excess noises in the two dimensional superconducting transition.

Neuroendocrine cell populations were systematically studied and characterized in the thymus, an avian primary immune organ. The expression of the specific mRNAs for both Chromogranin A (CgA) and Carboxypeptidase E (CpE) in the thymus was first verified by RT-PCR. Additional evidence using immunofluorescent dual labeling, has demonstrated for the first time the co-existence of CgA and CpE in identical neuroendocrine cells at the protein level in a vetebrate primary lymphoid organ. These CpE- and CgA-positive cells were primarily found in the transition zone between the cortex and the medulla of the thymic lobules, an area known to contain numerous arterioles and to be heavily innervated by the autonomic nervous system, suggesting that these cell population can potentially receive input from each other, from the autonomous nervous system, from the circulation, or all of the above. (Neuro)endocrine messenger molecules produced by the thymic microenvironment, such as somatostatin (SST), seem to play a potentially important immunomodulatory role with regard to cell proliferation, differentiation, and migration, as well as cytokine production. The results showed that both SST and its receptor, SSTR2, are expressed locally within chicken thymus. The in vitro study showed that SST significantly inhibits IL-2 and concanavalin A (ConA) induced proliferation of thymocytes. In comparison with controls (medium containing IL-2 and ConA but without SST), addition of SST at 10-9 M and 10-6 M resulted in a nearly 20% decrease in proliferation (P < 0.01). The effects of somatostatin (SST) on the immune system, the role of SST on the gene expression of cytokines (IL-1, TGF, INF), chemokine receptors (CXCR4) as well as MHC-I components was assessed by real-time PCR. The question as to exactly which stimuli trigger the release of mediators such as somatostatin remains for future study. In addition, a complete inventory of all substances stored in the thymic LDCV and their effects on the developing T-cells when released in the microenvironment of the thymus are also questions that warrant further investigation.

In addition to their activity against intracellular pathogens, it is now clear that CD8+ T-lymphocytes also mediate anti-tissue responses. In order to manipulate these responses in the setting of tumor immunity or autoimmunity, it is necessary that we understand the parameters that promote CD8+ activation. In the first section of this thesis, a transgenic mouse model was used to explore the effectiveness of peptide/adjuvant-based and dendritic cell (DC)-based vaccination techniques at eliciting CD8-mediated anti-pancreatic responses. It was found that, while peptide vaccines were unable to stimulate autoimmunity, the transfer of DCs promoted autoimmune diabetes in a manner that was dependent upon the toll-like receptor (TLR)-based maturation of the DCs. Furthermore, the diabetes induction was dependent upon the engagement of the immunodominant CD8+ population and a second T-cell specificity, indicating that polyclonal responses may be required for effective tissue destruction. In the second section of this thesis, I explored the requirements for CD28-signaling during the activation of naïve self-reactive CD8+ T-cells. The transfer of mature DCs was insufficient to promote diabetes in CD28-deficient animals, whereas infection with lymphocytic choriomeningitis virus could induce diabetes in the same animals. Anti-tissue responses were further explored in tumor-bearing mice following DC transfer and demonstrated that a critical determinant of the induction of anti-tissue immunity in the absence of CD28-derived costimulatory signals, was the persistence of antigen presentation. In the final section of this thesis, I explored the role of nuclear factor kappa B 1 (NF-κB1) in DC maturation using the DC transfer model described above. Surprisingly, NF-κB1-deficient DCs were capable of inducing diabetes without the need for external stimulation. Furthermore, the absence of NF-κB1 in unstimulated DCs was associated with dysregulated production of tumor necrosis factor alpha (TNF-α), and this cytokine was required for the proper upregulation of the cytotoxic effector molecule granzyme B in CD8+ T-cells that infiltrated the pancreatic islets. This work therefore presents a novel model of autoimmune tissue destruction, in which defined genes and pathways that contribute to DC-T-cell interactions can be explored in an in vivo non-TCR transgenic setting.

The temperature differential of chilled water is an important factor used for evaluating the performance of a chilled water system. A low delta-T may increase the pumping energy consumption and increase the chiller energy consumption. The system studied in this thesis is the chilled water system at the Dallas/Fort Worth International Airport (DFW Airport). This system has the problem of low delta-T under low cooling loads. When the chilled water flow is much lower than the design conditions at low cooling loads, it may lead to the laminar flow of the chilled water in the cooling coils. The main objective of this thesis is to explain the heat transfer performance of the cooling coils under low cooling loads. The water side and air side heat transfer coefficients at different water and air flow rates are calculated. The coefficients are used to analyze the heat transfer performance of the cooling coils at conditions ranging from very low loads to design conditions. The effectiveness-number of transfer units (NTU) method is utilized to analyze the cooling coil performance under different flow conditions, which also helps to obtain the cooling coil chilled water temperature differential under full load and partial load conditions. When the water flow rate drops to 1ft/s, laminar flow occurs; this further decreases the heat transfer rate on the water side. However, the cooling coil effectiveness increases with the drop of water flow rate, which compensates for the influence of the heat transfer performance under laminar flow conditions. Consequently, the delta-T in the cooling coil decreases in the transitional flow regime but increases in the laminar flow regime. Results of this thesis show that the laminar flow for the chilled water at low flow rate is not the main cause of the low delta-T syndrome in the chilled water system. Possible causes for the piping strategy of the low delta-T syndrome existing in the chilled water system under low flow conditions are studied in this thesis: (1) use of two way control valves; and (2) improper tertiary pump piping strategy.

A critical step in mounting an immune response is antigen recognition by T cells. This step proceeds by productive interactions between T cell receptors (TCR) on the surface of T cells and foreign antigen, in the form of peptide-major-histocompatibility-complexes (pMHC), on the surface of antigen-presenting-cells (APC). Antigen recognition is exceedingly difficult to understand because the vast majority of pMHC on APCs are derived from self-proteins. Nevertheless, T cells have been shown to be exquisitely sensitive, responding to as few as 10 antigenic pMHC in an ocean of tens of thousands of self pMHC. In addition, T cells are extremely specific and respond only to a small subset of pMHC by virtue of their specific TCR. To explain the sensitivity of T cells to pMHC it has been proposed that a single pMHC may serially bind multiple TCRs. Integrating present knowledge on the spatial-temporal dynamics of TCR/pMHC in the T cell-APC contact interface, we have constructed mathematical models to investigate the degree of TCR serial engagements by pMHC. In addition to reactions within clusters, the models capture the formation and mobility of TCR clusters. We find that a single pMHC serially binds a substantial number of TCRs in a TCR cluster only if the TCR/pMHC bond is stabilized by coreceptors and/or pMHC dimerization. In a separate study we propose that serial engagements can explain T cell specificity. Using Monte Carlo simulations, we show that the stochastic nature of TCR/pMHC interactions means that multiple binding events are needed for accurate detection of foreign pMHC. Critical to our studies are estimates of TCR/pMHC reaction rates and mobilities. In the second half of the thesis, we show that Fluorescence Recovery After Photobleaching (FRAP) experiments can reveal effective diffusion coefficients. We then show, using asymptotic analysis and model fitting, that FRAP experiments can be used to estimate reaction rates between cell surface proteins, like TCR/pMHC. Lastly, we use FRAP experiments to investigate how the actin cytoskeleton modulates TCR mobility and report effective reaction rates between TCR and the cytoskeleton.

The objective of this study is to design and manufacture a tube hydroforming test machine with axial feeding, which includes a forming apparatus¡Bhydraulic system and control system. Using feedback control and fuzzy control criterion to conduct T-Shape hydraulic bulging experiments. Using annealed AA6063-T5 aluminum alloy tubes, experiments are carried out with different working path and different feeding distance, analyze the influences of these parameters on the formability of the tubes.

Abstract¡G TAL1 gene was originally discovered as a result of its activation by chromosome rearrangements in T cell acute lymphoblastic leukaemia(T-ALL). Further studies have shown that TAL1 expression is aberrantly activated by several mechanisms including chromosome translocations, interstitial deletion and transactivation without detectable chromosomal alteration. TAL1 gene encodes a bHLH transcription factor, that is essential for the development of all haematopoietic lineages and its expression is maintained during differentiation along erythroid, mast and megakaryocytic cell lineages, but not in normal peripheral T-lymphocytes. The bHLH motif of these protein is responsible for DNA binding and dimerization with other bHLH proteins involved in transcription regulation. TAL1 protein is able to form heterodimers with the ubiquitously expressed E2A gene products, E47 and E12, and the heterodimers bind to E-box motif with the general sequence CANNTG. But the target genes for TAL1 oncoprotein have not yet been identified. We have previously isolated TAL1/E12 heterodimer bound genomic fragments by chromatin immunoprecipitation from K562 cells, and selected 6 fragments with one to four E-box CANNTG sequences. In order to determine if these fragments could be the regulatory elements of potential target genes of TAL1 oncoprotein, we inserted these 6 DNA fragments individually into pGL3 to generate recombinant reporter plasmids. The transfection experiments indicated that K34 and K94 DNA fragments behaved as a transcriptional transactivating sequence, and TAL1 and E12 proteins are required for efficient transcriptional activity. We also showed that transfection of these two recombinant constructs into K562 cells generated positive transcriptional activity, in a level similar to that in TAL1 and E12 co-transfected COS1 cells. These results established that both K34 and K94 DNA fragments are likely to contain a promoter of potential TAL1 target genes.