Objective.Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and disease progression. This heterogeneity is attributed to the interplay between the environment, viral diversity, immune response and host genetics. This study focused on host genetics. We studied the association of single nucleotide polymorphisms (SNPs) in peptidyl prolyl isomerase A (PPIA), transportin 3 (TNPO3) and PC4 or SFRS1 interacting protein 1 (PSIP1) genes with HIV-1 infection and disease progression. These genes code for Cyclophilin A (CypA), Transportin-SR2 (TRN-SR2) and Lens epithelium derived growth factor/p75 (LEDGF/p75) proteins respectively, which are all validated HIV replication cofactors in vitro. Methods. One SNP A1650G in the PPIA gene was genotyped in 168 HIV-1 negative and 47 acutely infected individuals using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 6 intronic and 2 exonic haplotype tagging (ht) SNPs (rs13242262; rs2305325; rs11768572; rs1154330; rs35060568; rs8043; rs6957529; rs10229001) in the TNPO3 gene, 4 intronic ht SNPs (rs2277191, rs1033056, rs12339417 and rs10283923) and 1 exonic SNP (rs61744944, Q472L) in the PSIP1 gene were genotyped in 195 HIV-1 negative and 52 acutely infected individuals using TaqMan assays. The rs1154330, rs2277191, rs12339417 and rs61744944 were further genotyped in 403 chronically infected individuals. CypA and LEDGF/p75 messenger RNA (mRNA) expression levels in peripheral blood mononuclear cells (PBMCs) were quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The impact of the Q472L mutation on the interaction of LEDGF/p75 with HIV-1 integrase (IN) was measured by AlphaScreen. Results. The minor allele (G) of SNP A1650G (1650G) in the promoter region of PPIA was significantly associated with higher viral load (p<0.01), lower CD4+ T cell counts (p<0.01) and showed a possible association with rapid CD4+ T cell decline (p=0.05). The 1650G was further associated with higher CypA expression post HIV-1 infection. The minor allele (G) of rs1154330 in the intron region of TNPO3 was associated with faster HIV-1 acquisition (p<0.01), lower CD4+ T cell counts, higher viral load during primary infection (p<0.05) and rapid CD4+ T cells decline (p<0.01). The minor allele (A) of rs2277191 (rs2277191A) in the intron region of PSIP1 was more frequent among seropositives (p=0.06). Among individuals followed longitudinally, rs2277191A was associated with higher likelihood of HIV-1 acquisition (p=0.08) and rapid CD4+T cell decline (p=0.04) in the recently infected (primary infection) cohort. In contrast, the minor allele (C) of rs12339417 (rs12339417C) also in the intron region of PSIP1 was associated with higher CD4+ T cell counts during primary infection. The rs12339417C was also associated with slower rate of CD4+ T cell decline (p=0.02) and lower mRNA levels of LEDGF/p75 (p<0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 compared to nonseroconverters (p<0.01) and these levels decreased after HIV-1 infection (p=0.02). The Q472L mutation showed approximately 2-fold decrease in the association constant (Kd), suggesting stronger binding to HIV-1 integrase. Our findings demonstrate, for the first time, that genetic polymorphisms in the TNPO3 and PSIP1 genes may be associated with susceptibility to HIV-1 infection and the disease progression. These data provide in vivo evidence that TRN-SR2 and LEDGF/p75 are important host cofactors for HIV-1 replication. This is also the first study to show the association of genetic polymorphisms in the PPIA gene with disease outcome in a population (South African) with high burden of HIV-1 infection. Conclusions. Genetic variation in HIV-1 replication cofactors may be associated with disease outcome in a South African population. These data strongly support the role of these HIV replication cofactors in disease pathogenesis in vivo and suggest that these factors are possible targets for therapeutic interventions. However, these data will need to be replicated in larger cohorts to confirm the effect of these genetic variants. Further studies on how to target these factors in antiviral strategies are needed. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Health in archaeological populations needs to be investigated using a holistic approach. Molecular techniques, particularly multiplex PCR, can be used with paleopathology and dietary analysis to understand aspects of population health. This thesis demonstrates how spina bifida, a multi-factorial disease, can be investigated using this paleoepidemiological approach. Based on skeletal evidence, spina bifida was present in a pre-Columbian Cuban population from the archaeological site of Canimar Abajo. Molecular techniques were employed to examine disease potential, examining individuals for five single nucleotide polymorphisms associated with spina bifida. It is postulated that the combined effect of these polymorphisms, as well as dietary factors, determines the risk of the population for spina bifida, and that these factors came together to create the observed high disease prevalence. Therefore, this thesis demonstrates how the methods of molecular paleopathology, corroborated by dietary analyses, can be used within a paleoepidemiological framework to understand population health and disease.

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Health in archaeological populations needs to be investigated using a holistic approach. Molecular techniques, particularly multiplex PCR, can be used with paleopathology and dietary analysis to understand aspects of population health. This thesis demonstrates how spina bifida, a multi-factorial disease, can be investigated using this paleoepidemiological approach. Based on skeletal evidence, spina bifida was present in a pre-Columbian Cuban population from the archaeological site of Canimar Abajo. Molecular techniques were employed to examine disease potential, examining individuals for five single nucleotide polymorphisms associated with spina bifida. It is postulated that the combined effect of these polymorphisms, as well as dietary factors, determines the risk of the population for spina bifida, and that these factors came together to create the observed high disease prevalence. Therefore, this thesis demonstrates how the methods of molecular paleopathology, corroborated by dietary analyses, can be used within a paleoepidemiological framework to understand population health and disease.

Metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer. Unlike other solid tumors that are associated with osteolytic bone metastases, prostate cancer bone metastases stimulate osteoblastic activity with sclerosis in the bone lesions as a consequence. Osteoprotegerin (OPG) is part of a system with three proteins that play a key role in bone remodeling; namely OPG, RANK and RANKL. RANKL regulates osteoclast activity by binding to RANK on the osteoclasts surface, and this interaction is interrupted by OPG. OPG also plays a role in the lifecycle of tumor cells by blocking TNF-related apoptosis-inducing ligand (TRAIL) making it possible for them to evade cell death. The aim of this thesis was to investigate the interaction between the OPG/RANK/RANKL system and prostate cancer. Data showed that there was production of OPG from prostate cancer cell lines in vitro. This expression was under the influence of cytokines that are present in the microenvironment of bone. Further, there was documented a previously unnoticed cell surface expression of RANKL. Co-culturing the prostate cancer with human osteoblasts increased the expression of RANKL. To connect these findings with in vivo studies, OPG-gene single nucleotide polymorphisms (SNP) were investigated. To evaluate OPG SNPs association with bone, a cohort of elderly men was used. OPG SNPs was shown to be correlated to bone mineral density at hip and spine. There was also an association to fragility fractures. Then there was examined the association of the same SNPs to the incidence of prostate cancer but after a four-year follow-up there was no association to the genetic variants. To summarize this research, we hereby present data that the OPG/RANK/RANKL system might be relevant for prostate cancer growth in bone, and for the skeletal related morbidity in this disease. Future in vitro and in vivo studies will demonstrate the relative importance of this crosstalk, and whether pharmacological interference with the system might be used as a therapeutic tool aiming to decrease skeletal morbidity and possibly also prolong survival in prostate cancer.

Includes 6 reprints of papers by the author / Includes bibliographical references. / 147 leaves + [51] leaves : ill. (some folded) ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1969

The metabolism of vitamin D commences with exposure of the skin to sunlight. The growing recognition of its role in insulin resistance, autoimmune disorders, infections, cancer, as well as the health of cells that influence physical and mental function have profound implications on how we define vitamin D requirements and why we should care whether they are met or not. Most of the actions of vitamin D are mediated by the vitamin D receptor (VDR), a protein whose gene sequence can vary, giving rise to polymorphic forms which are potent enough to affect the binding capacity of this protein to vitamin D. Some of these polymorphic forms of VDR gene may be associated with reduced effectiveness of vitamin D and hence predispose individuals to diseases such as type 2 diabetes and insulin resistance. An earlier study, the Surya Study, looked at the responsiveness of the South-Asian women living in Auckland to vitamin D. The research described here is an extension of this study and its focus was to identify the associations/linkages between certain polymorphic forms of the VDR gene and the disease conditions and intervention responsiveness in the same women. The first objective was to compare two well known techniques for genotyping single nucleotide polymorphisms (SNPs) of the VDR gene at the 3’ end, namely BsmI, ApaI and TaqI: the newer real-time polymerase chain reaction (qPCR) and the traditional restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) techniques. This comparison was performed to evaluate alternative methods for genotyping which consumed less time than RFLP-PCR. When the presence of each polymorphism by both the techniques was compared in this cohort of South-Asian women, it was found that RFLP-PCR proved to be a more reliable technique than qPCR for genotyping the VDR gene. Another objective of this project was to investigate the prevalence of the above three polymorphisms along with Cdx-2 and FokI SNPs which are present at the 5’ end of the VDR gene, in the population under study and their possible association with phenotypes such as vitamin D responsiveness and insulin resistance. These women were screened and biochemical data was collected during the earlier Surya Study. Of these, eighty-one women were then selected for intervention based on them having high insulin resistance (HOMA-IR>1.93) and serum 25(OH)D<50 nmol/L. Out of these eighty-one women, forty-two were given vitamin D supplement and thirty-nine were given a placebo for six months. Baseline and endpoint measurements included insulin resistance (HOMA-IR), insulin sensitivity (HOMA2%S) etc. How each individual responded to treatment in the intervention group was analysed in the context of the polymorphisms that they had. An association of insulin resistance with BsmI, ApaI and TaqI SNPs was observed in this cohort of 239 women. The response to insulin resistance in the vitamin D supplemented group significantly differed for FokI genotype compared to other genotypes. This explained why certain women responded to treatment better than the others. When the frequencies of the genotypes of these five SNPs of the VDR gene were compared to other studies of different ethnicities, the results of this study were consistent with few studies but contradictory to others. The possible reasons for these differences could be because of small sample size and different ethnicities under study due to which the frequency of alleles and hence the genotypes differed.

The metabolism of vitamin D commences with exposure of the skin to sunlight. The growing recognition of its role in insulin resistance, autoimmune disorders, infections, cancer, as well as the health of cells that influence physical and mental function have profound implications on how we define vitamin D requirements and why we should care whether they are met or not. Most of the actions of vitamin D are mediated by the vitamin D receptor (VDR), a protein whose gene sequence can vary, giving rise to polymorphic forms which are potent enough to affect the binding capacity of this protein to vitamin D. Some of these polymorphic forms of VDR gene may be associated with reduced effectiveness of vitamin D and hence predispose individuals to diseases such as type 2 diabetes and insulin resistance. An earlier study, the Surya Study, looked at the responsiveness of the South-Asian women living in Auckland to vitamin D. The research described here is an extension of this study and its focus was to identify the associations/linkages between certain polymorphic forms of the VDR gene and the disease conditions and intervention responsiveness in the same women. The first objective was to compare two well known techniques for genotyping single nucleotide polymorphisms (SNPs) of the VDR gene at the 3’ end, namely BsmI, ApaI and TaqI: the newer real-time polymerase chain reaction (qPCR) and the traditional restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) techniques. This comparison was performed to evaluate alternative methods for genotyping which consumed less time than RFLP-PCR. When the presence of each polymorphism by both the techniques was compared in this cohort of South-Asian women, it was found that RFLP-PCR proved to be a more reliable technique than qPCR for genotyping the VDR gene. Another objective of this project was to investigate the prevalence of the above three polymorphisms along with Cdx-2 and FokI SNPs which are present at the 5’ end of the VDR gene, in the population under study and their possible association with phenotypes such as vitamin D responsiveness and insulin resistance. These women were screened and biochemical data was collected during the earlier Surya Study. Of these, eighty-one women were then selected for intervention based on them having high insulin resistance (HOMA-IR>1.93) and serum 25(OH)D<50 nmol/L. Out of these eighty-one women, forty-two were given vitamin D supplement and thirty-nine were given a placebo for six months. Baseline and endpoint measurements included insulin resistance (HOMA-IR), insulin sensitivity (HOMA2%S) etc. How each individual responded to treatment in the intervention group was analysed in the context of the polymorphisms that they had. An association of insulin resistance with BsmI, ApaI and TaqI SNPs was observed in this cohort of 239 women. The response to insulin resistance in the vitamin D supplemented group significantly differed for FokI genotype compared to other genotypes. This explained why certain women responded to treatment better than the others. When the frequencies of the genotypes of these five SNPs of the VDR gene were compared to other studies of different ethnicities, the results of this study were consistent with few studies but contradictory to others. The possible reasons for these differences could be because of small sample size and different ethnicities under study due to which the frequency of alleles and hence the genotypes differed.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 79-88). Also avialable via the World Wide Web.