Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2005. / Dr. Cheng Zhu, Committee Chair ; Dr. Periasamy Selvaraj, Committee Co-Chair ; Dr. Timothy Wick, Committee Member ; Dr. Lyle Sinor, Committee Member ; Dr. Raymond Vito, Committee Member ; Dr. Robert Nerem, Committee Member.

Thesis (Ph.D.) - University of St Andrews, May 2008.

M.Sc. / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). TB is a multifactorial disease, influenced by both environmental and genetic factors. Susceptibility varies among individuals and is likely explained by differential environmental exposures and genetic factors. For example, Africans are more susceptible to TB than non-Africans, likely attributed to their generally lower socioeconomic status and possible higher frequencies of ‘susceptible’ genetic variants. Similarly, males are more susceptible to TB than females, presumably as a consequence of gender-based sociocultural differences as well as biological and/or genetic differences. From a host genetic perspective, TB is a complex disease associated with variants from several genes. The Vitamin D Receptor (VDR) gene, coding for the VDR protein, has received much attention as a candidate gene; the VDR mediates vitamin D functions, of which one is to restrict M. tuberculosis survival in macrophages. Several studies attempting to associate VDR single nucleotide polymorphisms (SNPs) with TB susceptibility have given inconsistent results. Factors suggested to contribute to these inconsistencies include confounding environmental factors as well as higher VDR genetic/haplotypic diversity and less linkage disequilibrium (LD) in African populations compared to non-African populations. However, epigenetic variation has not yet been considered as an additional confounding factor leading to inconsistencies in genetic association studies for TB and VDR. vi Epigenetic factors are heritable and pivotal to regulate gene transcription. Moreover, epigenetic factors are highly susceptible to environmental influences and have been shown to be the underlying factor in certain disease aetiologies. Not only are epigenetic factors susceptible to environmental influences, but also to genetic factors acting in cis or in trans. An example is the formation or elimination of a methylatable CpG by a SNP. On the other hand, epigenetic factors may influence the genotype through formation of methylation-induced SNPs. The synergistic effect of genetic variants, epigenetic variants and the environment on disease is known as the common disease genetic epigenetic (CDGE) hypothesis. The CDGE hypothesis supports the study of both genetic and epigenetic variants to provide a better understanding of disease aetiologies and to increase the power of association studies.

The cellular degradation of the low density lipoprotein (LDL) receptor, and its ligand, LDL, were investigated in order to clarify certain mechanistic aspects of these important processes. Long-term lymphoblastoid cell lines and cultured human skin fibroblasts were used to examine the fate of ¹²⁵I-LDL subsequent to its uptake via receptor-mediated endocytosis. In both cases, binding activity was saturable, depended on the presence of calcium ions in the medium, and was calculated to have an equilibrium dissociation constant at 4ᵒC of 2 μg ¹²⁵I-LDL/ml. No high-affinity binding was detected when the ligand was modified by acetylation. After incubating the monolayers at 37°C LDL/LDL receptor complexes were internalized, and the receptors were recycled back to the surface within about 10 minutes. Apolipo-protein B in the LDL particles was largely degraded to the amino acid level: chloroquine, a lysosomotropic agent, inhibited the formation of the ¹²⁵I-LDL degradation products. Cells obtained from a number of heterozygous and homozygous familial hypercholesterolemic patients, as expected, bound markedly reduced amounts of ligand. The half-life of ¹²⁵I-LDL was measured after it had been introduced into cultured fibroblasts by one of the following processes: (i) uptake via receptor-mediated endocytosis in human skin fibroblasts with normal LDL receptors, or (ii) incorporation via scrape-loading into fibroblasts defective in LDL receptor content. The half-lives obtained were about 1 hour and 50 hours, respectively, indicating that efficient degradation of LDL occurred only when it was deIivered to lysosomes via receptor-mediated endocytosis.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Receptor-activated phosphatidylinositol (PtdIns) hydrolysis was examined in AR42J rat pancreatic acini. Cholecystokinin-octapeptide (CCK₈) and bombesin induced a dose-dependent accumulation of [³H] inositol monophosphate ([³H]InsP₁). Manganese (Mn²⁺), a known calcium channel blocker, did not alter basal PtdIns hydrolysis. In contrast, when added 5 minutes prior to the addition of agonists for 60 minutes, Mn²⁺ markedly inhibited secretagogue-mediated [³H]InsP1 formation. Mn²⁺ also attenuated the CCK₈-mediated increase in biologically active inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. These inhibitory effects of Mn²⁺ were mimicked by lanthanum and by EGTA. Addition of calcium to EGTA-treated cells abolished the inhibitory effects of extracellular calcium depletion. Mn²⁺, La³⁺ and EGTA exerted similar inhibitory effects on PtdIns hydrolysis in pancreatic acini. These findings suggest that receptor-activated calcium influx is required for full activation of the CCK₈-mediated signal transduction pathway that is coupled to PtdIns hydrolysis.

A number of polymorphisms in the human VDR gene (located on chromosome 12q12-14) have previously been associated with TB susceptibility, of which most commonly, FokI, BsmI, ApaI and TaqI. The active metabolite of vitamin D, 1,25 dihydroxyvitamin D3, is an important immunoregulatory hormone. Its effects are exerted via the VDR, which is present on human monocytes and activated T and B-lymphocytes. Considering the role of the VDR and it specific immunological functions, including activation of monocytes, stimulation of cell-mediated immunity, suppression of lymphocyte proliferation, immunoglobulin production and cytokine synthesis, variation in the VDR genes may contribute to disease susceptibility. Hypothesis and objectives: In this study it was hypothesized that polymorphisms in the vitamin D receptor (VDR) gene influence TB susceptibility in the Venda population of South Africa. The role of VDR polymorphisms in TB susceptibility was investigated by setting the following objectives: 1. Typing of VDR gene polymorphisms (FokI, BsmI, ApaI and TaqI) in controls and TB cases as well as family samples from a Venda population of the Northern Province of South Africa. Comparing VDR allele and genotype frequencies between the Venda population and other populations as reported in the literature. 2. Analysis of possible associations in the case-control and family-based study 3. Studying Linkage disequilibrium (LD) in the case-control and the family samples. 4. Screening VDR haplotypes in the case-control and family-based study for possible association with TB. Patient and laboratory methods: VDR polymorphisms were studied in controls (n=110), TB cases (n=113) and 25 families in the Venda population inhabiting the Northern Province of South Africa. Case-control studies are vulnerable to false positive results caused by inadequate matching of the two groups. For this reason, a withinfamily association test was also implemented (the transmission disequilibrium test, TDT), as this is robust to this possible source of error. ARMS-PCR was used to type the VDR allele polymorphisms. The Statistical Package for Social Sciences (SPSSv10) was used to analyze possible association in case-controls, while TRANSMIT was used to analyze transmission of VDR alleles to affected offspring. LD between the single nucleotide polymorphisms (SNPs) of the VDR gene was investigated using the software “Graphical Overview of Linkage Disequilibrium” (GOLD) in both case-control and family studies. Possible association of TB with haplotypes comprising the VDR SNPs, ApaI and TaqI, were determined using SPSSv10 and TRANSMIT respectively for case-controls and the family based study. Results: 1. The comparative analysis indicated that the allele frequencies of the VDR SNPs related to the African American population. Genotype frequencies for FokI, BsmI and TaqI were similar to the West African population, whereas ApaI did not relate to any of the populations investigated. 2. No significant differences existed concerning the allele frequencies or genotype distribution between controls and TB cases with regard the VDR SNPs; FokI, BsmI and TaqI. The ApaI allele ‘A’ differed between cases and controls (p=0.041) but, no significant differences were obtained for genotype distribution between cases and controls for this SNP. All SNPs were in Hardy-Weinburg equilibrium except for ApaI. No differences between controls and cases were observed concerning allele presence or absence. In the family-based study, no significant difference was observed between observed and expected numbers of transmission for neither alleles nor genotypes, although, a borderline association was observed between TaqI ‘T’ allele and TB susceptibility (p=0.084). 3. LD in the case-control study was found between the VDR SNPs: BsmI and ApaI (p=0.051, D’=0.478), BsmI and TaqI (p=0.007, D’=0.222) and ApaI and TaqI (p=0.003, D’=0.479). In the family-based study, LD was only observed between ApaI and TaqI (p=0.049, D’=0.001) 4. Haplotype analysis in the case-control study indicated that no significant difference between controls and TB cases was observed. The family-based study indicated that transmission of the VDR haplotype ‘AT’ and ‘at’ differed from the expected transmission, but was of borderline significance. ‘AT’ was transmitted more than expected (P=0.090) and ‘at’ was transmitted less than expected (P=0.075), indicating that ‘AT’ may play a role in TB susceptibility and ‘at’ may play a protective role. Conclusion: The current study supports a possible role for VDR polymorphisms, ApaI (‘A’, p=0.041), or nearby yet unidentified markers in TB susceptibility in African populations (FokI-ApaI-BsmI-TaqI) (p=0.049). A larger sample size may clarify associations found to be of borderline significance. Results obtained may contribute to an improved understanding of genetic susceptibility factors in Africans. / Prof. L. Bornman

In the developing CNS, presynaptic neurons often have exuberant overgrowth and form excess (and overlapping) postsynaptic connections. Importantly, these excess connections are refined during circuit maturation so that only the appropriate connections remain. This synaptic rearrangement phenomenon has been studied extensively in vertebrates but many of those models involve complex neuronal circuits with multiple presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila melanogaster (the giant fiber circuit), we developed tools that enabled us to study the molecular development of this circuit; which consists of a bilaterally symmetrical pair of presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral, postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned their terminals and refined their connectivity such that only a single postsynaptic connection remained with the ipsilateral target. Furthermore, if we ablated one of the two giant fibers during development in wildtype animals, the remaining giant fiber often retained excess connections with the contralateral target that persisted into adulthood. After demonstrating that the giant fiber circuit was suitable to study synaptic rearrangement, we investigated two proteins that might mediate this process. First, we were able to prevent giant fibers from refining their connectivity by knocking out highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining excess connections, while exclusively presynaptic (or postsynaptic) expression of either protein had no effect. We further showed that by simultaneously reducing (Slit-Robo) midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we significantly enhanced the proportion of giant fibers that formed excess connections. Our findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in refining synaptic circuits and shaping giant fiber circuit connectivity. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Translocation of viral integrase into nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). It has been considered as an important target for the drug development to treat AIDS. In order to understand the detailed mechanisms of integrase-host cell protein complex interactions, we cloned HIV-1 integrase-EGFP into pTRE2hyg as visible tag to monitor the translocation process. When transiently transfected this vector into Tet-off ready HeLa cells, the EGFP: integrase is mainly localized in the nucleus. It has been hypothesized that any drugs that can inhibit the translocation process are novel class of drugs for AIDS treatment. More than 30000 synthetic compounds and 80000 natural products were screened by virtual screening. A total of 34 compounds were obtained and screened for their ability to block the nuclear entry of HIV-1 integrase by monitoring the EGFP fluorescence in the cells by high-throughput live cell imaging. Eight synthetic compounds (DW-IN4, DW-IN5, DW-IN6, DW-IN9, DW-IN15, DW-IN16, DW-IN17, DW-IN21) and one natural product (DW-IN719) were found to block integrase translocation significantly. According to our screening result, six compounds (INNB-1, INNB-2, INNB-3, INNB-4, INNB-5, INNB-6) were designed and synthesized. INNB-1 and INNB-2 had significant inhibition on integrase nuclear translocation. DW-IN6, DWIN719, INNB-1, INNB-2, INNB-3 and INNB-4, showed significant inhibition on P24 production in live virus assay. DW-IN6, INNB-1, INNB-2, INNB-3 and INNB-4 showed significant syncitia formation inhibition in live virus assay. Six compounds (KM7, KM8, KM14, KM30, KM37, KM79) from Kunming were screened as integrase nuclear translocation inhibitors. Using similar cell imaging techniques, we have cloned the GFP-tagged chemokine receptor CXCR4 using the lentivirus transfection system. CXCR4 receptor is a critical co-receptor in CD4 positive lymphocytes mediating the fusion of HIV into the CD4 positive cells. CXCR4-GFP was over-expressed in 293T cells and the results showed that GFP:CXCR4 receptor is expressed at the plasma membrane of the cells. These cells have been used to monitor the blockage of CXCR4 receptor internalization for drug development. Four compounds (KX128, KX166, KX171, KX180) from Kunming showed CXCR4 internalization blockage in imaging assay. The interaction of these compounds with CXCR4 was predicted by molecular docking. KX128 showed significant HIV inhibition in live virus assays. / Gu, Wangang. / Advisers: Pang Chui Shaw; David Chi Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.