Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010. / Thesis presented in partial fulfillment of the requirements for the degree Master of Science in Medical Biochemistry at the University of Stellenbosch. / Bibliography / ENGLISH ABSTRACT: Tuberculosis (TB) is an infectious disease which has plagued society for thousands of years. Despite public health programs, anti-TB drugs and a vaccine, the absolute numbers of people infected with TB each year continue to rise as populations grow. The high TB-burden areas are also plagued by other debilitating factors including HIV/AIDS infection, poverty and malnutrition. Nutrition has been implicated in TB susceptibility in a number of studies. While most are observational reports made during times of war, famine or natural disaster, multiple studies provide convincing evidence for poor nutritional status increasing the morbidity and mortality of TB. Numerous approaches are currently utilized in TB research, and there has been convincing evidence to support the role of host genetics in TB susceptibility. Based on previous linkage studies and a search of current literature, three genes were selected for this case-control study. Subsequently, variations located in cathepsin Z (CTSZ), melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) were genotyped in the South African Coloured (SAC) population to determine the existence of an association with TB disease. CTSZ is a lysosomal cysteine protease expressed in cells of the immune system. Interaction between this 303 amino acid protein and β2 integrin receptors lymphocyte function-associated antigen-1 (LFA-1) and macrophage antigen-1 (MAC-1) leads to altered lymphocyte proliferation. As a result, a single exonic variant in CTSZ, rs34069356, the same identified in a previous linkage study, showed strong evidence for association with TB susceptibility in cases (n = 410) and controls (n = 301) in the SAC population (p< 0.0001). MC3R and MC4R are two of 5 melanocortin receptors. MC3R has been found to be a key regulator in energy expenditure and host metabolism while activation of MC4R leads to a decrease in food intake. Activation of these two receptors is regulated by leptin, a hormone released by adipose tissue. A variant located upstream of the MC3R gene, rs6127698, also showed evidence of disease association with the less frequent allele, T, being under-represented in cases (n = 540) compared to controls (n = 541) (genotypic frequency, p = 0.0039), suggesting a possible resistance phenotype. Functional analysis of this variant revealed an increase in MC3R expression when stimulated with BCG, with individuals homozygous for the T allele exhibiting an even larger upregulation of MC3R expression than individuals homozygous for the G allele, though this difference was not statistically significant. A single haplotype in MC3R was found to be associated with TB susceptibility (p = 0.0008) and this association remained after permutation testing to correct for multiple testing (p = 0.0061) Three variants were selected for genotyping in MC4R and while none of these showed a statistically significant difference between cases (n = 510) and controls (n = 487), this gene should not be ruled out as both MC3R and MC4R have been found to work closely though not redundantly and double knockout experiments result in exacerbated obesity, suggesting that these proteins have a synergistic effect. The results of this study support both a role of host genetics and nutritional status in TB and strongly motivate further research in both of these fields. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is ‘n aansteeklike siekte wat reeds vir eeue die gesondheid van die publiek bedreig. Ten spyte van publieke gesondheidsprogramme en verskeie anti-TB medikasie middele, groei die aantal van mense wat hiermee ge-infekteer word steeds jaarliks. Dit is veral in areas waar TB steeds groei, waar ook ander neerdrukkende faktore soos HIV/Vigs, armoede en wanvoeding hoogty vier. Na aanleiding van verskeie verslae tydens oorloë, hongersnood en ander natuulike rampe is dit veral duidelik dat swak nutriënt inname morbiditeit en sterftes wat met TB gepaard gaan verhoog. Talle benaderings word tans gebruik in TB-navorsing, Bewyse is oortuigend om die rol van genetika van die gaheer met vatbaarheid vir TB te verbind. Op grond van vorige studies en die huidige literatuur, het ons drie gene gekies vir hierdie pasiënt-kontrole studie. Variante geleë in cathepsin Z (CTSZ), melanocortin 3 receptor (MC3R) en melanocortin 4 receptor (MC4R) is ge-genotipeer in die Suid-Afrikaanse Kleurling bevolking (SAK) (540 gevalle en 540 kontrole) om sodoende die assosiasie met TB te bepaal. CTSZ is ‘n lisosomale sisteïen protease wat uitgedruk word in immuunselle. Interaksie tussen hierdie 303 aminosuur protein en β2 integrin reseptore nl. LFA-1 en MAK-1 bring veranderde limfosiet proliferasie mee. ‘n Enkele eksoniese variant in CTSZ, rs34069356, dieselfde soos ge-identifiseer in ‘n vorige studie, verskaf sterk bewys vir assosiasie met TB vatbaarheid in gevalle (n = 410) en kontrole (n = 301) in die SAK bevolking. MC3R en MC4R is twee van 5 melanokortien reseptore. Daar is gevind dat MC3R 'n sleutelrol speel in die energie regulering van gasheer metabolisme, terwyl die aktivering van MC4R eindelik lei tot 'n afname in voedsel inname. Aktivering van hierdie twee reseptore word gereguleer deur Leptien, 'n hormoon wat vrygestel word deur adipose weefsel, ‘n Variant, stroomop geleë vanaf MC3R, rs6127698, is ook bewys om met TB ge-assosieer te wees, met die T-alleel meer seldsaam in gevalle (n = 540) as in kontroles (n = 541) wat dui op 'n moontlike weerstandsfenotipe. Funksionele analise van hierdie variant onthul 'n toename in MC3R uitdrukking wanneer gestimuleer met BCG, met individue homosigoties vir die T-alleel wat selfs groter opregulation veroorsaak wanneer vergelyk word met individue homosigoties vir die G allele. Hierdie resultaat was egter nie statisties beduidend nie. 'n Enkele haplotiepe in MC3R is ge-assosieer met TB vatbaarheid en die assosiasie is onveranderd nadat ‘n permutasie korreksie aangebring is (p = .0061). Voorts is drie variante gekies vir genotipering in MC4R en ten spyte daarvan dat nie een daarvan 'n statisties beduidende verskil getoon het tussen pasiënte (n = 510) en kontroles (n = 487) nie, behoort hierdie geen nie uitgesluit word nie, Die rede hiervoor is dat beide MC3R en MC4R verskeie kere gevind is om in samewerking ‘n rol te speel om vetsug te voorkom of te vererger. Die resultate van hierdie studie beaam beide 'n rol van gasheer genetika en voedingstatus in TB en motiveer veral verdere navorsing in beide van hierdie vakgebiede.

Ce travail a porté sur l'identification et la caractérisation de nouveaux facteurs de l'hôte nécessaires au cycle infectieux des virus à ARN (genre Potyvirus) chez Arabidopsis thaliana, ces facteurs pouvant constituer des cibles pour la lutte génétique contre les phytovirus. L'exploration de la diversité génétique naturelle d'Arabidopsis a conduit à l'identification d'un gène récessif de résistance au Watermelon mosaic virus (WMV) chez l'accession Cvi-0. Il s'agit du gène rwm1 qui agit à un stade précoce de l'interaction en empêchant l'accumulation du virus au niveau des premières cellules infectées. Les résultats de clonage positionnel de rwm1, de séquençage d'allèles de résistance et de sensibilité et de validation fonctionnelle argumentent en faveur d'un rôle de la phosphoglycérate kinase chloroplastique (cPGK), une enzyme clé de la photosynthèse, dans la résistance conférée par rwm1. En parallèle, une stratégie gène candidat a été mise en œuvre pour étudier le rôle de la protéine kinase Target Of Rapamycin (TOR) dans l'interaction plante-potyvirus. La caractérisation de lignées RNAi pour le gène TOR d'Arabidopsis montre une diminution voire une suppression de l'accumulation du WMV, ainsi qu'une réduction de l'accumulation du Turnip mosaic virus. L'étude comparative de la composition des complexes de liaison à la coiffe entre une lignée sauvage et une lignée RNAi TOR en conditions saines et infectées par le WMV suggère un rôle de TOR dans l'interaction avec les potyvirus par le biais d'une régulation de l'activité du facteur d'initiation de la traduction eIF3. / This work describes the use of forward and reverse genetics in Arabidopsis thaliana to identify and characterize new host factors required for the infectious cycle of plant RNA viruses (genus Potyvirus). The exploration of the Arabidopsis natural genetic diversity led to the identification of the recessive rwm1 gene for resistance to Watermelon mosaic virus (WMV). Rwm1-mediated resistance was identified in the Cvi-0 accession where it acts at an early stage of the infection process by impairing virus accumulation in initially infected tissues. Map-based cloning results, allelic sequence analysis and functional validation experiments, strongly suggest the involvement of chloroplast phosphoglycerate kinase (cPGK), a key photosynthetic enzyme, in rwm1-mediated resistance. In parallel, a candidate gene approach was developed to investigate the potential role of the TOR kinase (Target Of Rapamycin) in the control of susceptibility to potyviruses. Arabidopsis RNAi lines displaying partial TOR inactivation showed a significant decrease of susceptibility to the infection by two potyviruses, namely WMV and Turnip mosaic virus. The results from a comparative proteomic analysis of cap-binding complexes between wild type and TOR RNAi infected and healthy lines suggest that TOR could play a role in plant-potyvirus interactions by regulating the activity of the translation initiation factor eIF3. Overall, this work opens a challenging research area to provide novel insights on the molecular principles underlying viral infection processes and opportunities to improve and optimize the way we tackle plant resistance to viruses through a diversification of genetic targets.