"Major Subject: Laboratory Animal Medicine" Title from author supplied metadata (automated record created 2010-03-12 12:08:51). Includes bibliographical references.

"Major Subject: Laboratory Animal Medicine" Title from author supplied metadata (automated record created 2010-03-12 12:08:51). Includes bibliographical references.

"Major Subject: Laboratory Animal Medicine" Title from author supplied metadata (automated record created 2010-03-12 12:08:51). Includes bibliographical references.

This master thesis focuses on proper workbook elaboration regarding to the subject Specific laboratory methods, which is taught on medical high school, program Laboratory assistant.

Magister Public Health - MPH / The Zimbabwean government recognizes the critical role laboratories play in ensuring the health of the nation. Well-resourced and functioning laboratories are the sine qua non of effective diagnosis, treatment and clinical monitoring of medical problems such as HIV/AIDS, tuberculosis and malaria. In Zimbabwe, frequent unavailability of essential laboratory reagents and consumables have been reported but less well-reported are the factors associated with these "stockouts" at medical laboratories. Applying qualitative research methodologies, this study sought to explore the bottlenecks to the availability of laboratory consumables at state-owned medical laboratories in Harare Province. Semi-structured interviews were used to elicit stakeholders' perspectives and experiences with regard to the availability of laboratory consumables. These were complemented by observation of procurement, supply and distribution processes and individual follow-up interviews in 7 facilities where medical laboratory scientists were purposively selected. Rigour was ensured through data-source triangulation, provision of thick descriptions of the setting, maintaining an audit trail and transcribing data verbatim. Data analysis identified recurring themes and key suggestions made by respondents. A complex web of economic, human resources and supply chain factors affect laboratory commodity availability in Harare. Salient factors negatively affecting commodity availability included inadequate funding, human resources, poor communication and coordination among stakeholders, lack of transport, long lead times and limited inventory management skills. A comprehensive approach to resolving the challenge is warranted through advocating for more funding, complementing donor efforts on staff retention, improved coordination and collaboration among stakeholders and re-designing the laboratory supply chain. Further research would assist in determining ways of efficiently utilizing the limited available resources.

Previous in vitro studies have shown that shark cartilage extracts stimulate human leukocytes to release significant levels of TNFα, a cytokine typical of a Th1 immune response. The purpose of this study was to investigate further the effects of shark cartilage on cellular immune function, particularly cell proliferation, apoptosis, and IL-4 and INFγ production. The viability and proliferation of cell cultures grown in the presence of shark cartilage extract was not significantly different from unstimulated control cultures or those stimulated with mitogens (Con A, PMA, LPS), respectively. The effect of shark cartilage on apoptosis was determined by microscopic analysis of morphological apoptotic changes and by the detection of DNA fragmentation observed as characteristic ladder formation in agarose gel electrophoresis. While DNA fragmentation could not be demonstrated for cartilage-stimulated cells, characteristic morphological changes, indicative of apoptosis, were observed in leukocytes following incubation with shark cartilage extract. A statistically significant difference was not noted in the number of apoptotic cells present in cartilage-stimulated leukocytes and those stimulated with 0.5 μM/ml of staurosporine, suggesting that apoptosis is induced in the presence of cartilage extract. Culture supernatants of cartilage-stimulated leukocytes were assayed for IL-4 and IFN-γ by enzyme-linked immunosorbent assay (ELISA). Although a low level of IL-4 and INFγ was detected in culture supernatants of Con A and PMA stimulated cells it was not significantly different from that of unstimulated control cultures. Thus the significance of the absence of detectable IL-4 or INFγ in supernatants of cartilage- stimulated cultures could not be determined. However, as previously shown, supernatants did contain TNFα. Results of the study did not show a definitive pattern of cytokine production, characteristic of either a Th1 or Th2 type immune response.

Includes abstract. / Includes bibliographical references (leaves 93-109). / Nearly 130 years have elapsed since the discovery of Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, yet today it is estimated that 1 in every 3 of the worldʼs population is infected with this pathogen. In South Africa alone there were approximately 1000 new TB cases per 100 000 population in 2007, ranking the country second in incidence rate, globally. Hence research into new vaccine strategies to control the epidemic is vital. Current vaccines under development are prophylactic and designed to boost pre-existing immunity induced by the only licensed TB vaccine, BCG. A new approach is the development of a post-infection vaccine aimed at inducing an immune response that prevents progression to TB disease when administered to individuals latently infected with M.tb. This vaccine would have a dramatic impact on the worldwide TB burden. Our objective was to address 2 areas in TB vaccinology, firstly a novel postinfection TB vaccine strategy, and secondly, optimal measurement of vaccineinduced responses using a new immunological assay. The aim of the first study was to investigate human T cell responses to antigens that have been associated with M.tb latency. Rv2660 and Rv2659 were investigated, as these antigens are candidate antigens for a postinfection vaccine based on findings from in vitro models of M.tb suggesting preferential expression during latency in vivo. No information exists on the immune response to these antigens in M.tb infected or TB diseased individuals. Hence, we investigated the immune recognition of Rv2660 and Rv2659 in these 2 groups, and further characterised the nature of these antigen-specific T cell responses. We observed that (i) these antigens are significantly more likely to be recognised during M.tb infection compared with TB disease as shown by measurement of soluble IFN-γ in response to the specific antigens, (ii) M.tb infected persons had greater Rv2660- and Rv2659- specific CD4+ T cell proliferation and associated cytokine expression compared, with TB diseased persons. We propose that Rv2660 and Rv2659 may be candidates for incorporation into a post-infection vaccine.

Includes abstract. / Includes bibliographical references (leaves 101-111). / Interleukin-4 receptor alpha (IL-4Ra) signaling, mediated by the ligands IL-4 and IL- 13, is important for effective host protection during murine Nippostrongylus brasiliensis (N. brasiliensis) and Schistosoma mansoni (S. manson!) infection. Among other cell types, IL-4Ra responsive smooth muscle cells influence immunological responses and are needed for host protection during N. brasiliensis and S. mansoni infection.

Includes bibliographical references (leaves 75-91).

Lymphoblastic leukemia 1 (Lyl1) is a well-studied transcription factor known to exhibit oncogenic potential during various forms of leukemia. Since its discovery in 1989, many reports have been published describing its relationship with cancer as well as demonstrating its function during hematopoiesis. Lyl1 has been shown to serve a significant role during thymopoiesis by contributing to T-cell development. However, it has been recently reported that irrespective of its significance during T-cell development, mature comparable single positive T-cells are observed in mouse models. The use of murine models has been crucial in identifying potential targets for host-directed therapies (HDT) which has been shown to provide great potential in treating tuberculosis (TB). It is evident that Mycobacterium tuberculosis (Mtb), the causative agent for TB, is capable of developing resistance to various treatments that target the bacterium itself. Therefore, by designing therapies that directly target host factors could assist in circumventing Mtb resistance. By analyzing Mtb-infected bone marrow-derived macrophages (BMDM) that have been subjected to genome-wide transcriptional deep sequencing of total RNA using a single molecule sequencer in conjunction with the cap analysis gene expression (CAGE) technique, various differentially expressed genes were identified, including the oncogenic transcription factor, Lyl1. With the use of murine models, we investigated whether Lyl1 is important for various immunological responses at steady state, the regulation of Lyl1 in response to various immune stimulants including LPS and whether this transcription factor is relevant in bacterial infections including Listeria monocytogenes (Lm) and Mtb. The data in this thesis demonstrate comparable immunological responses, including cellular recruitment by means of flow cytometry and cytokine responses by means of ELISA, between naïve littermate control and Lyl1-deficient mice. Further evaluation of Lyl1 regulation revealed the influence of MAPk and NFκB signaling on Lyl1 expression upon LPS stimulation by significantly downregulating this transcription factor in immune stimulated macrophages. A role for Lyl1 during bacterial infections was observed in Lm-infected mice whereby Lyl1-/- mice succumbed earlier to listeriosis compared to the littermate controls. We further established a functional role for this transcription factor during Mtb infection in vitro and in vivo. The early surrender of Lyl1-deficient mice to Mtb HN878 infection, accompanied by increased bacterial burden during chronic Mtb infection, demonstrated enhanced susceptibility in the absence of Lyl1. We show that Lyl1-deficient host susceptibility is a consequence of enhanced inflammatory responses and increased bacterial growth. This is demonstrated by increased neutrophilic inflammation, pro-inflammatory cytokine and chemokine secretion along with a reduction in anti-inflammatory cytokine release during chronic Mtb infection. Here, we demonstrate the first non-leukemia role for Lyl1 by suggesting a role and requirement for this transcription factor during bacterial infections. Given the significant role during Mtb infection, our studies suggest the use of Lyl1 associated pathways as a potential HDT target for TB.