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Innate immunity has received impressive attention in the past decade owing to the discovery of the Toll like receptors (TLRs) and the NOD-like receptors (NLRs). While the TLRs specialize in fighting microbes at the cell surface, the NLRs complement by detecting and responding to intracellular microbes. Recently, the non-microbe sensing NLR called inflammasomes, have been identified, which senses metabolic stress as well as certain pathogenic microbes and elicits host’s inflammatory response. The NLR, NALP3 (formerly known as cryopyrin) forms a large cytoplasmic complex called the ‘inflammasome’ when NALP3, activated by a stimuli, associates with the adaptor proteins ASC and CARD-8. This interaction leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the gene encoding NALP3, termed NLRP3 can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under cryopyrin associated periodic syndromes (CAPS). This thesis describes a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was found to be a heterozygous carrier of two common polymorphisms Q705K in NLRP3 and C10X in the CARD-8. Experimental studies showed elevated levels of caspase-1 and IL-1β in the patient, and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms combined, were found to occur in approximately 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. Therefore, a cohort of rheumatoid arthritis (RA) patients, where elevated IL-1β could be one of the reasons behind chronic inflammation, was investigated. We found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup of patients might benefit from IL-1β blockade. Additional studies are warranted to elucidate the functional effects of the two polymorphisms and to determine whether they identify a subgroup of patients that could benefit from IL-1 targeted therapy. Given the structural similarity of NALP3 to other NALPs, the possibility of involvement of the alternative, homologous genes cannot be eliminated.

Thesis (M. A.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 118-130). Also available via the World Wide Web

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Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Bovine tuberculosis (BTB) is a chronic, infectious disease found in domestic livestock and wildlife, and has serious biodiversity, economic and public health implications. African buffalo act as a wildlife reservoir of BTB, maintaining and transmitting the disease within the environment. The research presented in this thesis addresses the role of host genetic variation in resistance to BTB infection in African buffalo, and reviews the possible practical application of such information. Annual BTB prevalence within the African buffalo population in Hluhluwe iMfolozi Park, South Africa, was evaluated over a seven year period in order to define the extent of M. bovis infection. Prevalence changes over time suggest that the test and cull operation currently in place is performing successfully with respect to the original aims of the programme. A review of genetic studies of BTB in livestock and wildlife collated previous findings in this field and provided a collection of possible candidate genes and variants. It also highlighted a lack of research in wildlife, and the limitations of working with species with insufficient genetic data. To overcome the absence of whole-genome data, next-generation sequencing was performed on nine African buffalo, in order to identify novel genetic variants in this species. Upwards of 76 000 novel SNPs within gene regions were identified, and subsequent fluorescent genotyping of 173 SNPs showed a 57% validation rate. From the validated set, 69 SNPs located in genes related to the immune system were selected for association testing with BTB status in African buffalo, and were fluorescently genotyped in 868 individuals. Three SNPs, in the Solute Carrier family 7, member A13 (SLC7A13), Deleted in Malignant Brain Tumour-1 (DMBT1) and Interleukin 1 alpha (IL1α) genes, were identified as significantly associated with BTB status. Very little sequence information of the NRAMP1 (SLC11A1) gene was obtained from the next-generation sequencing performed, and this gene has been associated with brucellosis, salmonella and paratuberculosis in other animal species, making it an excellent candidate for BTB resistance. To characterise this gene in African buffalo, Sanger sequencing was performed to generate the complete coding region, and partially sequence the 5’UTR, intronic and 3’UTR regions. Fifteen novel polymorphisms and three microsatellites were identified within the gene. Finally, a review was prepared to assess the applicability of genetic information on BTB resistance to selective breeding programmes for African buffalo. Phenotypic, marker-assisted and genomic breeding strategies were discussed, with particular emphasis on their suitability to African buffalo. Identifying genes and variants involved in BTB resistance in African buffalo provides potential targets for drug or vaccine development, as well as information that could be incorporated into selective breeding programmes. This may support new management options for controlling the BTB epidemic in the game parks of South Africa, as an alternative to, or in conjunction with, lethal control / AFRIKAANSE OPSOMMING: Beestuberkulose (BTB) is ‘n chroniese, aansteeklike siekte wat in vee en wild voorkom en wat ernstige gevolge vir die ekonomie, biodiversiteit en openbare gesondheid inhou. Die Kaap-buffel is ‘n wild reservoir vir BTB wat die siekte onderhou en versprei in die omgewing. Die navorsing wat in hierdie tesis aangebied word fokus op die rol van gasheer genetiese variasie in die weerstand teen BTB infeksie in Kaap-buffels en gee ‘n oorsig van die moontlike praktiese toepassing van die resultate. Die jaarlikse BTB voorkomsyfer in die Kaap-buffel bevolking in die Hluhluwe iMfolozi Park in Suid-Afrika is oor ‘n tydperk van sewe jaar geëvalueer om die omvang van M. bovis infeksie te bepaal. Die verandering in voorkomsyfer oor tyd dui daarop dat die toets-en-slag operasie wat tans gebruik word die oorspronklike doelwitte van die program suksesvol bereik. ‘n Oorsig en vergelyking van vorige genetiese studies van BTB in vee en wild het ‘n versameling van moontlike kandidaatgene en –variante verskaf. Dit het ook die gebrek aan navorsing in wildediere uitgewys en die navorsingsbeperkinge wanneer ‘n spesie met onvoldoende genetiese data bestudeer word benadruk. Aangesien daar nie heel genoom data beskikbaar is nie, is volgende-generasie volgordebepaling van 9 Kaap-buffels gedoen om nuwe genetiese variasies in hierdie spesie te identifiseer. Meer as 76 000 nuwe enkel-nukleotied polimorfismes (ENPs) binne geen-areas is geïdentifiseer en die daaropvolgende genotipering van 173 ENPs het ‘n bevestigingskoers van 57% gehad. Vanuit die bevestigde stel ENPs is 69 gekies vir assosiasietoetse met BTB status in die Kaap-buffel en genotipering van 868 individue is gedoen. Drie ENPs, in die Solute Carrier family 7, member A13 (SLC7A13), Deleted in Malignant Brain Tumour-1 (DMBT1) en Interleukin 1 alpha (IL1α) gene, was beduidend geassosieer met BTB status. Baie min volgorde inligting van die NRAMP1 (SLC11A1) geen is verkry uit die volgende-generasie volgordebepaling. Aangesien hierdie geen voorheen met brucellose, salmonella en paratuberkulose in ander dierespesies geassosieer is, is dit ‘n uitstekende kandidaat vir BTB weerstand. Hierdie geen is in Kaap-buffels gekarakteriseer deur Sanger volgordebepaling van die volledige koderende, gedeeltelike 5’UTR, introniese en 3’UTR areas te doen. Vyftien nuwe polimorfismes en drie mikrosatelliete is geïdentifiseer. Ten slotte is ‘n oorsigstudie gedoen om die toepaslikheid van BTB genetiese weerstandsdata in selektiewe telingsprogramme van Kaap-buffels te evalueer. Fenotipiese, merkerbemiddelde en genomiese teling strategieë is bespreek, met spesifieke klem op die geskiktheid van die metodes vir Kaap-buffels. Identifisering van gene en variante wat betrokke is by BTB weerstand in die Kaap-buffel bied potensiële teikens vir medikasie of entstof ontwikkeling, sowel as inligting wat in selektiewe telingsprogramme gebruik kan word. Dit kan nuwe bestuursopsies vir die beheer van die BTB-epidemie in die parke van Suid-Afrika bied as 'n alternatief vir, of in samewerking met, dodelike beheermetodes.

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression. My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

Wolfram Syndrome (WFS) is a debilitating autosomal recessive neurodegenerative disorder characterized by juvenile onset insulin dependent diabetes mellitus (DM) and optic atrophy (OA) as well as a number of neurological and endocrine complications that result in early death due to respiratory complications. Previous research has mapped Wolfram syndrome to chromosome 4p16.1 and the disease has been attributed to mutations in the WFS1 gene affecting the WFS1 protein (wolframin), an ER membrane glycoprotein that plays an important role in the unfolded protein response (UPR) and in intracellular Ca2+ homeostasis. An additional locus for WFS on chromosome 4q22-24 was identified by linkage studies of four Jordanian Bedouin families with 16 affected individuals (El-Shanti et al., 2000). In this study, we attempted to identify the causative gene for the second WFS locus and identified a single missense mutation in a novel highly conserved iron-sulfur binding domain gene, CISD2, in the three consanguineous families of Jordanian descent from the El-Shanti et al. (2000) study (Amr et al., 2007). A G→C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the missense mutation was found to disrupt messenger RNA splicing by eliminating exon 2 which results in the introduction of a premature stop codon. CISD2 is expressed in a wide variety of tissues, including those affected in WFS, the brain and pancreas. The CISD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein) localizes to the endoplasmic reticulum but does not appear to interact directly with wolframin. Furthermore, lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium. To understand the underlying pathogenesis in WFS2 patients, we examined cell death as well as known stress pathways. Cisd2 was knocked down in three cell lines derived from tissues most affected by the disease, namely rat pancreatic insulinoma cells (INS1), mouse neuroblastoma cells (N1E115), and mouse embryonic stem cell like cells (P19) which could be differentiated into neuronal cells. Cisd2 knockdown in INS1 cells shows an increase in apoptotic cell death and in the expression of the apoptotic markers CHOP and BAX, but no increase in the autophagic marker LC3-II. Assessment of the UPR in CISD2 deficient cells shows no activation of the UPR response, while Cisd2 expression in wild-type INS1 and N1E115 cells did not increase under conditions of ER stress. These findings indicate that there is an increase in apoptosis in WFS2 similar to WFS1 but the pathogenesis involves a molecular mechanism that is different than that in WFS1. Investigation of markers of oxidative stress, another major contributor to diabetes and neurodegeneration, show an increase in expression of the antioxidant enzymes Sod1 and Sod2 as well as an increase in global tyrosine nitration in INS1 Cisd2 knockdown cells compared with controls. Cell death in those cells was exacerbated with addition of known oxidative stressors, thapsigargin and paraquat compared with controls. These findings indicate that oxidative stress is a contributor to WFS2 pathogenesis, but it is not clear whether it is the primary causative factor. A recent article implicated ERIS in the BCL-2 associated inhibition of autophagy (Chang et al., 2009) and showed an increase in levels of autophagy in response to starvation in Cisd2 knockdown in a human epithelial carcinoma cell line (H1299). Starvation of INS1 Cisd2 knockdown cells did not elicit a greater autophagic response compared with controls, but did show an increase in expression of Cisd2. P19 Cisd2 knockdown cells that were differentiated into neurons by retinoic acid treatment did not show an inhibition in differentiation markers, but Cisd2 levels returned to levels similar to pre-differentiation wildtype P19 cells, which indicates that Cisd2 is upregulated during neuronal differentiation. In conclusion, the pathogensis of WFS2 can be attributed to apoptotic death of cells in affected tissues, with oxidative stress and not endoplasmic reticulum stress contributing to the development of disease, while ERIS’ relationship with BCL-2-mediated autophagy and neuronal differentiation suggest its important role in cell differentiation and survival.

The reexpression of the fetal γ-globin gene in adult erythrocytes is of therapeutic interest due to its ameliorating effects in β-hemoglobinopathies. We recently showed that Methyl CpG Binding Domain Protein2 (MBD2) contributes to the silencing of the chicken embryonic ρ-globin and human fetal γ-globin genes. We further biochemically characterized an erythroid MeCP1 complex that is recruited by MBD2 to mediate the silencing of these genes. These observations suggest that the disruption of the MeCP1 complex could augment the expression of the fetal/embryonic globin genes. In the studies presented in chapter 2, we have pursued a structural and biophysical analysis of the interaction between two of the six components of the MeCP1 complex: MBD2 and p66α. These studies show that the coiled coil regions from MBD2 and p66α form a highly stable heterodimeric complex. Further, overexpressing the p66α coiled coil domain in adult erythroid cells can augment the expression of the chicken ρ-globin and human γ-globin genes, by disrupting the assembly of a functional MeCP1 complex. This indicates that the exogenously expressed p66α coiled coil peptide competes with the endogenous p66α for the interaction with the coiled coil domain of MBD2. These studies show that the coiled coil interaction between MBD2 and p66α could serve as a potential targets for the therapeutic induction of fetal hemoglobin. The laboratory showed that knockout of MBD2 in transgenic mice carrying the human β-globin gene cluster, results in an elevated expression of γ-globin in adult erythrocytes. However, MBD2 does not directly bind to the γ-globin gene to mediate its silencing. In the work presented in chapter 3, we have tested the hypothesis that MBD2 may suppress γ-globin gene transcription in adult erythrocytes indirectly, by binding to and repressing transcription of intermediary gene/s which may be involved in γ-globin gene regulation. Employing microarray technology, we have identified Gab1 and ZBTB32 as candidate genes that may be involved in the MBD2 mediated silencing of γ-globin.

Molecular chaperones, commonly known as heat shock proteins (HSPs), are essential for mammalian cells to maintain homeostasis, and HSPs function by inducing an ATPase-coupled structural change, followed by interactions with diverse co-chaperones and over 200 client proteins implicated in many critical signaling networks. These highly expressed HSPs participate in the onset and progression of several human diseases including cancer, and their connection with tumorigenesis has facilitated research and clinical trials related to targeting HSPs as a novel anti-tumor therapy. The predominant mechanism of chaperone inhibition is through either disruption of the HSP association with client protein or an altered binding state that ultimately leads to proteasome-mediated degradation. Importantly, chaperone inhibition results in the degradation of several client proteins that play critical roles in many of the pathways known as the Hallmarks of Cancer, such as proliferation, angiogenesis, invasion, metastasis, and drug resistance. Here, we discuss: (1) the current knowledge of HSPs, particularly studies related to Hsp90-targeted cancer therapy, (2) the targeting of Hsp90-mediated signaling interactions to prevent emergence of core Hallmarks of Cancer, (3) the recent progression of Hsp90 inhibitors in clinical trials. Finally, we propose combinatorial therapy, additional inhibitor discovery, and location-specific inhibition of HSPs as necessary next steps in chaperone-targeted research relevant to cancer therapy.

Induced Pluripotent Stem cells (iPScs) are artificially generated cells that demonstrate multilineage differentiation potential. These cells demonstrate similar morphology and high differentiation potential to Embryonic Stem Cells (ESCs). Generation of these cells from a terminally differentiated cell line requires activation of the core pluripotency genes Nanog, Oct4, and Sox2 as well as an oncogenic stimulus such as c-Myc. Here we examine the effect of the Human Pappiloma Virus derived proteins E6 and E7 on the ability of a terminally differentiated fibroblast cell line to a more primitive state and examine its multilineage differentiation capacity. In this paper, we attempt to differentiate BJ hTERT fibroblasts into adipogenic and osteogenic lineages with and without the core pluripotency factors Nanog, Oct4, Sox2 and also c-Myc using non-integrative adenoviral infections. We review the potential mechanisms through which changes in differentiation capacity changes occur through examination of the effects of E6 on the tumor suppressor protein p53. We determined that the proteins E6 and E7 when stably infected into BJ hTERT fibroblasts increase induced differentiation into adipogenic and osteogenic lineages. E6 and E7 can be considered components for generating cells with multipotent capacity with the addition of as little as one core pluripotency factor.