Brain damage, occuring in the perinatal period, are associated with several neurodevelopmental pathology, for example cerebral palsy. Those brain lesions arise from hypoxic-ischemic (HI) and infection/inflammation aggressions occuring at a crucial step during the neurodevelopmental period. There are currently no treatment designed to protect the newborn brain and alleviate the neurodevelopmental outcome. The common factor that is induce by both type of aggression is inflammation, and especially the production of pro-inflammatory cytokine, mainly interleukin-1 (IL-1). The association between IL-1 expression and the modulation of brain development is well-known but the causal link between the two is still a controversial manner. This is mainly due to the use of several different experimental model which are not always representative of the clinical reality. To get a better understanding of the role of IL-1 in perinatal brain damage we first characterized, both anatomically and histologically, an experimental model which combined both insults most often encounter in humans, to establish the reliability of the model to the human pathology (i.e. cerebral palsy). Using this model, we then demonstrated the central role of the IL-1 system in the genesis of brain damage. We also showed that the developmental stage corresponding to the preterm human newborn was more susceptible than their adult counterpart since the pro-inflammatory imbalance, in the agonist/antagonist ratio induced by the aggressions, was more obvious at this particular stage of development. This shift of the IL-1 system towards a pro-inflammatory state found in the premature brain was also detected in the placenta after maternal exposure to LPS at the end of gestation. The causal link between the IL-1 system and the neonatal brain damage was confirmed with the use of the IL-1 receptor antagonist (IL-1Ra) administered maternally which increased pups survival and protected them against microgliosis and motor deficits. We showed the therapeuthic potential of the IL-1Ra against both placental and neurodevelopmental defects when administered maternally, at the end of gestation. The implication of the IL-1 system in brain lesions was also studied directly on human newborn brain tissu presenting white matter damages reminiscent of cerebral palsy. Those data correlated with what was obtained using the experimental model showed the pro-inflammatory orientation of the IL-1 system, particularly in lesioned areas of the brain. In conclusion, the work presented in this thesis demonstrated the central role of the IL-1 system in the genesis of perinatal brain damage after exposure to HI and/or infection/inflammation and the therapeuthic potential of the prenatal administration of IL-1Ra.

Les dommages cérébraux survenant durant la période périnatale sont associés à plusieurs pathologies neurodéveloppementales, dont les encéphalopathies néonatales. Ces lésions résultent d’agressions hypoxiques-ischémiques (HI) et infectieuses survenant durant une étape cruciale du développement cérébral. Le seul traitement disponible pour diminuer l’impact de ces agressions sur le neurodéveloppement de l'enfant est l’hypothermie. Les deux types d’agressions ont un facteur commun, l’inflammation, et surtout la production de cytokines pro-inflammatoires telle que l’interleukine (IL)-1. Bien que l'association entre l'expression d'IL-1 et la modulation du développement cérébral soit connue, le lien de cause à effet entre les deux est encore méconnue. Ceci est dû, entre autres, à l'utilisation de nombreux modèles expérimentaux manquant souvent de pertinence avec ce qui est observé en clinique. Dans le but de comprendre le rôle de l’IL-1 dans les dommages cérébraux périnataux du nouveau-né à terme, nous avons fait la caractérisation anatomique et fonctionnelle d’un modèle animal combinant les deux types d’agressions les plus fréquemment rencontrées chez l’humain, afin d’en établir la concordance par rapport à la pathologie humaine. À l’aide de ce modèle, nous avons ensuite démontré que le système de l’IL-1 était au cœur de la pathophysiologie. De plus, nous avons montré que le stade de développement cérébral correspondant au nouveau-né à terme comporte des différences par rapport à l’adulte et au nouveau-né prématuré dans les composantes inflammatoires exprimées et par le type cellulaire exprimant ces composantes. En inhibant le système de l’IL-1, nous avons démontré le lien causal entre l’expression de l’IL-1 et la survenue de dommages cérébraux ainsi que son impact sur la motricité des animaux via l'administration de l'antagoniste du récepteur de l'IL-1 (IL-1Ra). Nous avons démontré le potentiel thérapeutique post-natal de l’IL-1ra administré directement aux animaux lors de l’agression. En conclusion, les travaux présentés dans cette thèse démontrent le rôle central de l’IL-1 dans la genèse des lésions cérébrales périnatales suite à une agression de type HI et infectieuse/inflammatoire ainsi que le potentiel thérapeutique de l'administration post-natal de l'IL-1Ra dans un modèle animal correspondant au nouveau-né à terme.

Denna forskningsöversikt behandlar digitaliseringen av undervisningen utifrån koncepten 1:1 (en dator per elev) och ASL (att skriva sig till läsning). Vi har via databassökning och manuell sökning tagit del av relevant litteratur som vi sedan gjort en kvalitativ innehållsanalys på. Vi behandlar vilka effekter koncepten enligt forskningen har för elever och lärare. Det råder ingen allmän konsensus om detta, då forskningsresultat pekar mot både positiva och negativa effekter. Vi söker därför även i litteraturen efter faktorer som visat sig gynna goda resultat. De faktorer vi funnit är struktur av fortbildning och stöd, av både teknisk och didaktisk karaktär, samt ett sociokulturellt arbetssätt.Genomgående i den litteratur vi läst efterfrågas mer forskning på området. Detta inkluderar både kritisk granskning av den forskning som nu finns och ny forskning som tar hänsyn till fler aspekter av de begrepp som berörs och som innefattar en vidgad kunskapssyn.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

HIV-1, the virus that causes AIDS, is spreading at an alarming rate. Subtype C, which accounts for approximately 50% of infections worldwide, and 98% of infections in Southern Africa, is by far the most prevalent form of the virus. Most molecular and biochemical studies have been performed on HIV-1 subtype B isolates and products, however, and there is a relative scarcity of corresponding data on subtype C. It is therefore of crucial importance to study subtype C HIV-1 strains in order to understand their characteristic pathogenic effects and to develop effective treatment strategies. The aim of research in our laboratory is the development of novel treatment strategies, with particular focus on identifying novel Subtype C Env-binding peptide ligands. This necessitates the development of reagents for use in the discovery and testing of these compounds. In line with this, the aim of this project was the production and characterisation of recombinant Subtype C gp120s generated from a recently compiled HIV-1 virus cohort. To this end, the gp160-coding regions of 20 South African Subtype C HIV-1 strains isolated from AIDS patients presenting at the Johannesburg General hospital in 2005 were amplified by PCR and sequenced. The gp160 amplicons were used to amplify and clone the gp120-encoding regions of these isolates. Two clones, pTriEx- FV3 and pTriEx-FV5, originating from CXCR4- and CCR5-utilising strains respectively, were selected for further use. These clones were cotransfected into insect cells together with a baculoviral DNA backbone in order to generate gp120-expressing baculoviruses by homologous recombination. Recombinant baculoviruses were used to infect Sf9 insect cell cultures for expression of recombinant gp120, which was then purified using a combination of lectin affinity chromatography and ion exchange chromatography. In order to determine the functionality and conformational integrity of the recombinant gp120, the ability of these purified gp120s to bind CD4 and a panel of well-characterised monoclonal antibodies against various epitopes on gp120 (F425 A1g8, 2G12, F425 B4a1, F425 B4e8, 48d, 17b, IgG1 b12, 5F7, 4G10, 9301, ID6, Chessie 13-39.1, 654-30D and 670-30D) was assessed. Gp120 from the CXCR4-using isolate, FV3, appeared to have an intact, functional CD4 binding site as measured by its ability to bind to CD4 and the CD4 binding site antibody 654-30D. It showed low binding to the monoclonal antibody 654- 30D, moderate binding to 2G12, Chessie 13-39.1 and 9301, and high binding to ID6, but did not show binding to any of the other antibodies used in the recognition profile. Gp120 from the CCR5-using isolate, FV5, showed low binding to the monoclonal antibodies F425 B4a1 and Chessie 13-39.1, moderate binding to 2G12, and showed good binding to 9301and ID6. FV5 gp120 could not, however, bind to CD4. This is likely to be related to a D368G substitution, a mutation affecting a critical structural determinant of CD4 binding. The lack of CD4-binding activity of this gp120 highlights the importance of Asp368 for CD4 binding and hints at a region vulnerable for therapeutic targeting. Our results also highlight the challenges of developing broadly therapeutic drugs for HIV-1, as well as the importance of investigating the specific biochemical and pathogenic properties associated with subtype C HIV-1.

MSc (Med), Virology, Faculty of Health Sciences, University of the Witwatersrand / Introduction High resolution melting analysis (HRMA) accurately, rapidly and cost effectively detects single nucleotide polymorphisms by monitoring DNA dissociation kinetics. This technology was applied to HIV samples to assess whether it could be used to detect clinically relevant drug resistance mutations. Methods HRMA-PCR assays incorporating unlabeled probes were designed to genotype 12 mutation codons in the HIV-1 p66/p51 of engineered plasmids and 116 HIV-1 samples. Results HRMA correctly genotyped 63%-88% of the K103N, Y181C, M184V, Q151M and G190A mutations. Each assay had a 1.7%-3.4% discordance, most of which was due to the increased analytical sensitivity of HRMA (~5-20%). Only mutant K65R and V106M were correctly identified while the 41, 67, 70, 215 and 225 codons could not be genotyped. Assay modifications had some success in masking the affects of polymorphisms. Conclusion These assays can be used for genotyping selected major HIV-1 resistance mutations and should be further developed as a resistance surveillance tool.

A research report submitted to the Faculty of Health Sciences, School of Public Health, University of the Witwatersrand, Johannesburg In partial fulfillment for the requirement for the degree Master of Public Health 25 August 2014 / BACKGROUND Tuberculosis is the leading opportunistic infection among HIV-infected adults, including pregnant women, globally. Accurate screening tools are needed to identify those requiring further laboratory testing and to initiate isoniazid preventive therapy in a timely manner. This study determined the accuracy of symptom-based screening and in particular the performance of the WHO recommended TB symptom screening algorithm in HIV-infected pregnant women. METHODS A cross-sectional study was conducted among consenting HIV-infected pregnant women attending routine antenatal clinics in Matlosana, South Africa recruited >1 week after first HIV diagnosis between June 2010 and February 2011. Sputum was collected from all women followed by a systematic TB symptom screen. The performances of each symptom (cough, fever, weight loss and night sweats) alone and in combination were assessed with TB confirmed by sputa using microscopy and liquid culture (MGIT), as reference or gold standard. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were calculated for each of the four symptoms (cough, fever, weight loss and night sweats) and their combination. Logistic regression was carried out to find associations between patient characteristics and TB. RESULTS Overall, Mycobacteria Growth Indicator Tube (MGIT) confirmed prevalence of TB was 2.4% (35/1456) in this sample group. Only 11/38 (29%) women with confirmed TB reported any symptoms. Cough, fever, weight-loss and night sweats, individually and in combination had sensitivities ranging from 2.7-27% and specificities ranging from 84-97%. The positive predictive and negative predictive values for any symptoms of cough, fever, night sweats, or weight loss were 4.2% and 98%, respectively. TB was associated with decreasing CD4 count, close TB contact, cough, and night sweats. DISCUSSION The remarkable number of asymptomatic TB in HIV-infected patients, including in the cohort included in this study highlights the limitation of symptom-based screening. The low sensitivity of the symptom screen would incorrectly stratify patients who are being considered for Isoniazid Preventive Therapy (IPT). However, one could argue that the high negative predictive value of the symptom screen would justify its use in resource-limited settings as the initial step in identifying patients who should receive IPT. Although household TB and the father of the baby having TB were found not to have statistically significant associations with active TB, they are of public health importance as they play a role in the spread of the infection. CONCLUSION The WHO 4-symptom screen had low sensitive among HIV-infected pregnant women but negative predictive value was high. Few women with TB disease reported symptoms on direct questioning; the high rate of subclinical/ asymptomatic TB is concerning. There is an urgent need for more sensitive screening tools for TB in HIV-infected pregnant women

Low affinity Fcγ receptors (FcγR) mediate key immune effector mechanisms through the engagement of the Fc portion of immunoglobulin G (IgG). These receptors are involved in multiple biological processes, including clearance of antigen/antibody immune complexes, enhancement of antigen presentation, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, regulation of antibody production, and activation of inflammatory cells. FcγR phenotypic variability modulates these processes through altering receptor IgG subclass binding affinity (FcγRIIa-H131R and FcγRIIIa-F158V), subcellular localization (FcγRIIb-I232T), post-translational modification (FcγRIIIb-HNA1a/b/c), expression of an otherwise pseudogene (FcγRIIc), and receptor surface density (gene copy number variability and promoter haplotypes). Accumulating data suggest that FcγR-mediated effector functions play a significant role in HIV-1 protective immunity, which is substantiated by the association of FcγR phenotypic variants with HIV-1 disease outcome. This study set out to characterize FcγR functional variability in the South African population, and to investigate the potential role thereof in HIV-1 transmission and disease progression in South African Black individuals. Since the only known determinant of FcγRIIIa surface density – FCGR3A gene copy number – is rare, this study investigated novel genetic determinants of FcγRIIIa expression by flow cytometry and nucleotide sequencing. FcγRIIIa expression on peripheral blood mononuclear cells was characterized for 32 South African Caucasian individuals and 22 South African Black individuals (Chapter 3). Significant differences in the proportion of FcγRIIIa-positive monocytes and FcγRIIIa expression levels on natural killer (NK) cells were observed between the population groups. A novel four-variant FCGR3A intragenic haplotype that associated with increased surface expression of FcγRIIIa on NK cells was detectable in Caucasian individuals, but not Black individuals and may account for the observed population differences. Further exploration of genetic diversity at the low affinity FCGR gene locus was extended to include all currently known functional variants of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb using a commercial multiplex ligation-dependent probe amplification assay (Chapter 4). Thirty-two South African Caucasian individuals and 131 South African Black

M.Sc. (Nursing), Faculty of Health Sciences, University of the Witwatersrand, 2011