The JAK/STAT pathway is a non-receptor tyrosine kinase signaling pathway that is well conserved and highly re-utilized in many mammalian and Drosophila developmental processes. Compared to dozens of ligands and receptors in mammalian JAK/STAT, Drosophila JAK/STAT pathway is simpler with one receptor and three ligands, Upd, Upd2 and Upd3, which have similar amino acid sequences. Previous literature shows that upd and upd2 exhibit the same dynamic striped expression pattern in embryos and have semi-redundant functions during embryogenesis. Do Upd and Upd3 also have redundant functions? To answer this question, the functions of Upd3 in Drosophila development were investigated in this dissertation. In addition, the coordinate expression mechanism of upd and upd3 in eye discs was also analyzed. To study the functions of Upd3 in development, the expression pattern of upd3 was examined and detected in larval eye discs, wing discs, haltere discs, lymph glands and adult ovaries with in situ hybridization to upd3 mRNA and an upd3 reporter line. Consistent with the expression pattern, the loss of function mutants of upd3 exhibit small eyes, outstretched wings, downward extended halteres and reduced circulating blood cell concentration, demonstrating the roles of Upd3 in these tissues’ development. However, functions of Upd3 in other aspects of immune response were not detected. To investigate the mechanism of the coordinate expression of upd and upd3, the genetic and molecular relationship of upd, upd3 and os was dissected. The os alleles, oso, oss and os1, are a group of classical alleles which display outstretched wings, small eyes, or both, respectively. The genetic complementation tests of upd, upd3 and os showed that both upd and upd3 failed to complement os while upd complemented upd3, suggesting functions of both upd and upd3 are affected in os alleles. Consistent with the genetic tests, the expression of upd and upd3 in eye discs is lost in os allele. Molecularly, putative enhancer regions are deleted at the 5’ end of upd3 in os alleles. Hence, a transcriptional co-regulation model of upd and upd3 is proposed in which upd and upd3 share a common cis-regulatory region, lesions of which cause the os phenotype.

Epidemiological studies suggest that both retinoid and carotenoid intakes are inversely correlated with the incidence of human cancers. Animal studies show that both retinoids and carotenoids inhibit tumor cell growth. Both retinoids and carotenoids activate the cytotoxicity function of macrophages in animal experiments. The purpose of this study is to evaluate the molecular mechanism for 13-cis retinoic acid (13-cRA) and beta-carotene (BC) induced immunomodulation which could explain their anti-cancer affects. The effects of 13-cRA and BC were studied on various subpopulations of T-lymphocytes both in vitro and in vivo. For in vitro studies, peripheral blood mononuclear cells (PBMC) were incubated with test compounds at clinically achievable concentrations (10⁻⁸M) for three days. Then the cells were stained with monoclonal antibodies followed by the analysis of flow cytometer. For in vivo studies, PBMC were collected from Barrett's esophagus or oral leukoplakia patients during treatment with 13-cRA (1mg/kg/day) or BC (30 mg/day), respectively. Then the cells were analyzed with monoclonal antibodies and flow cytometry. Both compounds showed the capability of stimulating different subpopulations of T-lymphocytes. 13-cRA predominantly increased the number of T-helper cells, their interleukin 2 (IL-2) receptors and their response to mitogens. Whereas, BC elevated the number of Natural Kill (NK) cells, their IL-2 receptors and their cytotoxicity against K562 target cells. Though these immunomodulatory effects appeared to be unaffected by the presence and cytotoxic functions of macrophages, cytokines seemed to have an important role in the retinoid- and carotenoid-induced immunomodulation. Plasma levels of IL-2 and tumor necrosis factor (TNF) measured by ELISA procedures were increased in patients treated for two months with 13-cRA and BC respectively. Anti-IL-2 and anti-TNF antibodies blocked the retinoic- and carotenoid-induced immunomodulation in in vitro studies. These results indicate that 13-cRA, activating T-helper cells with IL-2 production, and BC, activating NK cells with TNF release, induced immunostimulation which might be able to provide the anti-cancer affects in part seen in epidemiological studies.

No description available.

No description available.

Clostridium difficile is an increasingly common cause of nosocomial infection. C. difficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage to mild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. It is not yet fully understood why this spectrum is seen, however, it is believed that the immune response mounted by an individual plays an important role in determining the outcome of infection. This thesis comprises three studies. Firstly, a comparative study of immune cell populations within the lamina propria of colonic tissue not exhibiting pathological changes and taken from individuals with symptomatic CDI (cases); asymptomatic carriers; and non-colonised controls. Effector T cells, B cells, plasma cells and macrophages were enumerated by means of immunohistochemical staining of tissue sections. Secondly, a study to establish the prevalence within these three study groups of specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8 genes by PCR genotyping and to determine whether an association existed between these genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms of moxifloxacin resistance in a collection of clinical isolates. This study also sought to determine whether the competitive advantage conferred by resistance to moxifloxacin influenced the fitness of C. difficile isolates, in particular growth and the expression of the virulence factors toxins A and B. Carriers were found to have fewer of all four immune cell types quantified than both cases and controls. However, in only one instance, that of plasma cells, was this difference statistically significant. Cases had fewer of all cell types than controls but these differences were not significant. These findings suggest that individuals who become infected, both symptomatically and asymptomatically, with C. difficile display altered mucosal immune cell populations when compared with those of uninfected individuals. The data regarding host polymorphisms are suggestive of an association between the presence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2 genotypes were carried by cases and carriers while the variant TLR5 genotype was carried by cases only. No variant genotypes were present in control subjects. All moxifloxacin resistant isolates characterised in this study, with the exception of an isolate with intermediate resistance and a third-generation mutant with reduced susceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Efflux pumps are known to play a role in multi-drug resistance in many bacterial species. Semiquantitative PCR analysis of expression of the putative efflux pumps cme and cdeA found no correlation between overexpression and moxifloxacin resistance, suggesting that these genes do not play a role. Three novel mutations in the putative promoter region of CD3197, a MerR family transcriptional regulator found immediately upstream of cme, were identified. No association between the presence of these mutations and overexpression of cme or resistance or sensitivity to moxifloxacin was found. The competitive advantage conferred by resistance to moxifloxacin does not influence the fitness of C. difficile isolates, as measured in terms of growth and toxin production.

Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.

Earthworms, Lumbricus terrestris, were challenged orally and intracoelomically with two bacterial species, Aeromonas hydrophila and Pseudomonas aeruginosa, and mortality rates were observed. Neither were found to be particularly pathogenic at injected doses of up to 108 bacteria per earthworm. The influence of Cu++ (as CuSO4) on the earthworm's response to bacterial challenge was investigated by exposing earthworms to sublethal levels of Cu++ prior to bacterial challenge. Exposure at sublethal concentrations up to 3 m g/cm2 did not have a pronounced influence on host resistance to challenge as measured by earthworm mortality. Cu++ increased the earthworm's ability to agglutinate rabbit erythrocytes, indicating that Cu++ exposure caused coelomocyte death, autolysis and release of agglutinins into the coelom, possibly explaining resistance to bacterial challenge.

Interleukin-l0 (IL-10) helps to limit the duration of potentially harmful inflammatory responses but has also been implicated in the persistence of a number of chronic viral infections. This thesis aimed to investigate the phenotype and function of mv -specific IL-l0-producing cells in chronic HIV-I infection, and the effect of IL-10 blockade on responses to candidate HIV -I vaccines. A cytokine capture assay was used to determine the HIV -specific cellular sources of IL- 10 in PBMC from 55 chronically infected individuals. A rare subset of CD8+ T cells was found to be the major HIV -I Gag-specific IL-10-producing population; these cells were restricted to ART-naive individuals and did not express the regulatory T cell markers CD25 or FoxP3 but could co-express IFN-y. A proportion of the population (median 48% and 9% respectively) expressed the P7 chain of the gut-homing integrin a4p7 and the chemokine receptor CXCR3, which mediates lymphocyte migration to sites of inflammation. Experimental depletion of Gag-specific IL-10+ CD8+ T cells did not affect T cell activation, or the production of cytokines such as IL-2 or IFN-y during short-term culture. However, depletion was associated with a significant increase in CD38 expression on CDI4+ monocytes, a trend towards increased HLA-DR expression on the same cells, and a significant increase in the concentration of the pro-inflammatory cytokine IL-6 in culture supernatants. There was also a significant increase in the number of HIV-infected (p24 antigen+) CD4+ T cells in cultures depleted of Gag- specific IL-10+ CD8+ T cells after 3 days, indicating that this population may contribute to control of viral replication. In order to determine the effect of IL-10 blockade on vaccine immunogenicity, IL-10R blocking antibody was administered to BALB/c mice prior to immunisation with two mV-I candidate vaccines, HIVA and HIVconsv. IL-10R blockade resulted in a trend towards increased IFN-y production by CD8+ T cells in response to the dominant H (Env) and P (Pol) epitopes of HIV A, and a significant increase in IFN-y ELISPOT responses to the subdominant Gl (Gag) epitope of HIV consv in vitro. Collectively, these data suggest that IL-10 producing cell populations may play critical but different roles in chronic infection and vaccination. Further research into how the timing of IL-10 responses affects disease outcome may allow IL-IO blockade to be explored as a therapeutic strategy in humans

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Insects are confronted by a large variety of potentially harmful microorganisms to which they are resistant as they are able to build up an efficient innate defense system that relies on three tightly interconnected reactions. One of the reactions is the transient and rapid synthesis of a battery of antimicrobial peptides (AMPs). Development of antimicrobial therapeutic drugs and vaccines is very crucial due to factors such as the emergence of multiple-drug resistance. AMPs have been termed natural antibiotics because of their large spectrum of activity. The current study focused on the isolation and identification of cationic antimicrobial peptides and the analysis of immune response in the South African Euoniticellus intermedius embryonic (SAEIE08) cell line upon exposure to pathogens. E. intermedius is of the Coleopteran order in the Scarabaeoidea superfamily. Liquid growth inhibition assay showed higher antimicrobial activity in SAEIE08 that was treated with heat-killed E. coli compared to untreated. Further evidence for antimicrobial activity was seen as a clear zone of inhibition in solid growth inhibition assay when a gel run with protein extracts was plated and overlayed with live E. coli. Changes in protein expression patterns that were analysed in SDS-PAGE and 2-D PAGE indicated the most intense bands and spots at low molecular weight sizes around 10 kDa and/or 16 kDa which implicated increased induction of AMP expression upon exposure to pathogen. Homologues of Saccharomyces cerevisiae proteins were found in some of the 5′/3′ RACE sequences. Possible explanation for matches to these homologues could be that short sequences were used for database searches. The proteins were identified as flavin-containing monooxygenase, long-chain fatty acyl-CoA synthetase, severe depolymerization of actin protein and serine/threonine protein kinase. Interestingly, these proteins play roles in metabolism, cell proliferation and/or molecular pathways which do occur when cells are exposed to stress. There was also an insect peptide allatotropin from Spodoptera frugiperda. The results show that there is inducible antimicrobial activity in embryonic E. intermedius cell line.