This thesis examines the genetic factors controlling host response to malaria. Recombinant congenic strains AcB55 and AcB61 were identified as uniquely resistant to malaria despite a susceptible genetic background. These mice display splenomegaly, anemia, reticulocytosis and extra-medullary erythropoiesis. Genome wide mapping led to the identification of a mutation in pyruvate kinase (PklrI90N) that is associated with increased survival and decreased peak parasite levels. We found a significant malaria-protective effect with a second Pklr mutation (PklrG338D) on a distinct genetic background (CBA/N), thus validating our initial findings. The mechanism of protection was linked to increased erythrocyte uptake and compensatory erythropoiesis. Subsequently, we showed that PK-deficiency in humans was protective against P.falciparum replication in vitro. A second genetic locus in AcB55, Char9, was identified and localized to a 14Mb C57Bl/6-derived congenic segment. The Vnn1/Vnn3 genes map within the minimal interval and encode a pantetheinase enzyme; they show cis-regulated gene expression A/J-like alleles associated with an absence of mRNA. In addition, no enzymatic activity was detected in A/J tissues. Administration of cysteamine, the product of pantetheinase, to susceptible A/J mice resulted in increased survival and decreased parasite levels. The inhibition of parasite replication by cysteamine suggests a potential for this compound to be used as a novel anti-malarial monotherapy or in combination with current therapeutics. Finally, a third recombinant congenic strain, AcB62, shows rapid parasite replication with an early peak of infection, despite the presence of the PklrI90N mutation. AcB62 mice do display PK-deficiency associated anemia, erythropoiesis, tissue iron overload, and concomitant increases in iron-regulated proteins. We identified a novel locus controlling peak parasite levels in an [AcB6 / Cette thèse examine les facteurs génétiques contrôlant la réponse de l'hôte au paludisme. Les souches recombinantes congéniques (RCS) AcB55 et AcB61 ont été identifiées comme présentant une résistance unique au paludisme malgré un fond génétique de susceptibilité. Ces souris développent une splénomégalie, de l'anémie, de la réticulose et une érythropoïèse extra médullaire. Un génome scan a conduit à l'identification d'une mutation de la pyruvate kinase (PklrI90N), laquelle est associée à une survie accrue et à une diminution des niveaux de pic de parasitémie. Un effet protecteur significatif contre le paludisme, du à une seconde mutation de la pyruvate kinase (PklrG338D) sur un fond génétique différent (CBA/N), a également été mis en évidence, validant ainsi nos premiers résultats. Le mécanisme de protection est associé à une augmentation de la phagocytose érythrocytaire et de l'érythropoïèse compensatoire. Nous avons également montré, chez l'humain, qu'un déficit en pyruvate kinase protégeait contre la réplication de P. falciparum in vitro. Un second locus, Char9, a été identifié sur la RCS AcB55 et a été localisé sur un segment congénique de 14Mb dérivé de C57Bl/6. Les gènes Vnn1/Vnn3 sont situés à l'intérieur de l'intervalle minimum et codent pour une enzyme pantéthéinase. Leur expression est régulée en cis et les allèles de type A/J ne codent pour aucun ARNm. Par ailleurs, aucune activité enzymatique n'a été détectée dans les tissus A/J. L'administration de cystéamine (un produit de la pantéthéinase) à des souris susceptibles A/J est responsable d'une survie accrue et d'une diminution des niveaux de parasitémie. L'inhibition de la réplication du parasite par la cystéamine suggère que ce composé pourrait potentiellement être utilisé comme nouvel antipaludéen, en monothérapie ou en combinaison avec des drogues actue

Background. Tuberculin skin testing is an important tool for diagnosis and control of tuberculosis. The role of host genetics in responsiveness of pediatric tuberculosis patients to purified protein derivatives (PPD) has rarely been investigated. / Objective. To determine the impact of the candidate genes - Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Vitamin D Receptor (VDR) and Interleukin 4 Receptor (IL4R) with other covariates on PPD reaction size in pediatric tuberculosis patients. / Methods. A family-based association study was conducted using eighty-three parent-child trios from a database of tuberculosis patients and their families from Texas, USA. / Results. Marker IL4Rrs1805010_GA was in linkage disequilibrium with PPD size (R2 = 16%; p = 0.0017). PPD size was a predictor of transmission of allele 1 ("G") of IL4Rrs1805010_GA with an OR (95% CI) of 1.9 (1.16, 3.14) per five millimeter average increase in PPD size adjusted for age of diagnosis. / Conclusions. This thesis provides evidence for the role of host genetics in PPD responsiveness in pediatric tuberculosis patients.

The importance of host factors in determining susceptibility to systemic Candida albicans infections is evident in both humans and mice. We have used a mouse model to study the genetic basis of susceptibility, using the inbred strains A/J and C57BL/6J, which are susceptible and resistant to infection, respectively, based on different parameters of response to infection. To identify genes responsible for this differential host response, brain and kidney fungal load were measured in 128 [A/J x C57BL/6J]F 2 mice 48 hours after infection with 5x104 C. albicans blastospores. Segregation analysis in this informative population identified complement component 5 (C5/Hc) as the major gene responsible for this differential susceptibility (LOD of 22.7 for kidney, 19.0 for brain), with a naturally-occurring mutation that causes C5 deficiency leading to enhanced susceptibility. C5 was also found to control heart fungal load, survival time, and serum TNF-alpha levels during infection. Investigation of the response to C. albicans challenge in a series of AcB/BcA recombinant congenic strains validated the importance of C5 in determining the host response, although some strains with discordant phenotypes were identified, suggestive of alternate mechanisms of genetic control.

Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. Segregation analyses using macrophages from susceptible and resistant inbred mice previously indicated that a single genetic locus, named Lgn1 , could determine permissiveness to intracellular replication of L. pneumophila. A positional cloning strategy was undertaken, which makes use of genetic and molecular biology techniques to identify the gene responsible for a particular phenotype, based mostly on its location within a chromosome. The work described in this thesis covers three aspects of Lgn1: (1) Building upon the work of others, the Lgn1 genetic interval was narrowed to 0.32 cM within distal mouse chromosome 13. The corresponding 140 Kb Lgn1 physical interval contains only two known transcripts: the Neuronal Apoptosis Inhibitor Protein ( Naip) genes Naip2 and Naip5. (2) The expression profile of the Lgn1 candidates was investigated both at the mRNA and protein levels. Expression of both Naip2 and Naip5 in mouse macrophages strengthened their candidacy for the Lgn1 locus. (3) Transfer of BAC clones from the critical interval into transgenic mice was successfully used to functionally complement the Lgn1 susceptibility phenotype of A/J mice with cloned DNA from non-permissive 129X1 or C57BL/6J origins. Two independent rescuing BAC clones were identified, with a 56-Kb overlap where the entire Lgn1 transcript must lie. The only known full-length transcript coded in this reduced genomic region is Naip5. / Thus, in our last publication we have proposed that Naip5 (recently named Birc1e) is the gene within the Lgn1 locus responsible for differential permissiveness to intracellular L. pneumophila replication in mice.

Gene expression during adipocyte differentiation is known to be regulated by transcriptional and post-transcriptional events. Few studies have examined the roles of RNA metabolism or posttranscriptional regulation during adipogenesis. Sam68, the Src associated substrate during mitosis of 68kDa, has been shown to be involved in several aspects of RNA metabolism. The Richard lab has generated Sam68-/- mice, which were observed to be leaner than their wildtype counterparts, and are protected from dietary induced obesity. In addition, pre-adipocyte 3T3-L1 cells depleted of Sam68 also exhibited impaired differentiation. Using a microarray approach, a gene expression profile was obtained from Sam68-depleted 3T3-L1 pre-adipocytes during differentiation, giving insight into which key players may be affected by Sam68. In addition, using a splicing sensitive exon array, I identified a number of potential alternative splicing gene targets which may be regulated by Sam68 during differentiation. Together these findings shed light on possible roles played by Sam68 during adipogenesis. / L'expression génique pendant la différentiation d'adipocytes est contrôlée par des évènements de transcription et post-transcription d'ARN. Il y a présentement un manque d'études sur les rôles des évènements post-transcriptionelles et de métabolisme d'ARN au cours du processus d'adipogenèse. Sam68 (Src associated substrate during mitosis of 68kDa) est impliqué dans plusieurs aspects de métabolisme d'ARN. Des souris ont été générées dont l'expression du gène Sam68 a été supprimé (Sam68-/-). Ces souris apparaissent plus maigres que les souris de phénotype sauvage, et ils sont protégés contre le développement de l'obésité. De plus, les cellules pré-adipocyte 3T3-L1, dont l'expression de Sam68 est réduite, démontrent un défaut de différentiation. A l'aide d'une méthode d'analyse à base de puce d'ADN 'microarray' nous avons obtenu un profil d'expression génique pendant le processus de différentiation pour ces cellules 3T3-L1 qui n'expriment pas de Sam68. Cela nous a donné une liste de gènes qui sont possiblement sous le contrôle de Sam68. De plus, nous avons utilisé une analyse à base de puce d'ADN 'exon array' pour identifier des évènements d'épissage qui peuvent être contrôlés par Sam68 pendant la différentiation d'adipocytes. Ensemble, ces trouvailles nous ont donné une idée des rôles possibles joués par Sam68 pendant le processus d'adipogenèse.

Kinase suppressor of Ras (KSR) is a scaffold protein that is required to localize components of the Ras/ERK pathway to the plasma membrane. In C. elegans, two genes, ksr-1 and ksr-2, encode KSR proteins which are redundantly required for development. Less than 1% of ksr-1(-) animals die in L1 larval stage in a distinct rod-like manner, but additional loss of ksr-2 or other positive regulators of the pathway enhance the rod-like lethal phenotype. To identify addition genes that might function with KSR, we performed a forward genetic screen to isolate genes that enhance ksr-1 rod-like lethality. From the screen, six enhancers were isolated, three of which are alleles of an E2 ubiquitin conjugating enzyme, ubc-25. Additionally, three alleles of an unknown gene on chromosome X were isolated but are not enhancers of ksr-1 rod-like lethality. These new enhancers as well as the mutations on chromosome X might define new regulators of Ras/ERK signaling. / Kinase suppressor of Ras (KSR) est une protéine d'échafaudage requise pour la localisation des composants du chemin de signalisation Ras/ERK vers la membrane cytoplasmique. Chez C.elegans, deux gènes codent pour les protéines KSR, ksr-1 et ksr-2, requises pour le développement. Moins d'un pourcent des nématodes ksr-1(-) meurent en adoptant un phénotype de tige, mais la perte additionnelle de ksr-2 ou d'autres régulateurs de ce chemin de signalisation augmentent la létalité avec ce phénotype tige. Pour découvrir davantage de gènes qui peuvent fonctionner avec KSR, nous avons fait un crible génétique pour isoler des mutations qui augmentent la létalité avec un phénotype tige. Nous avons ainsi trouvé six mutants, parmis lesquels trois sont des allèles du gène ubc-25, une enzyme activatrice de l'ubiquitine. De plus, trois allèles d'un gène non identifié ont été isolés sur le chromosome X, mais ceux-ci n'augmentent pas la létalité avec un phénotype tige de ksr-1. Ces six mutants pourraient être de nouveaux régulateurs du chemin de signalisation Ras/ERK.

Fetal branching determines final nephron number. Mild nephron deficit causes hypertension in "normal" adults. Thus, mechanisms that control branching are important. PAX2 and retinoic acid (RA) have profound effects on final nephron number, although the mechanism remains unknown. / We hypothesised that common variations in PAX2 cause subtle reduction in nephrons. We associated haplotype tagging single nucleotide polymorphisms (htSNPs) in PAX2 with a reduction in kidney mass in "normal" newborns and identified a haplotype on which an ancestral polymorphism is presumed to lie. We identified a SNP in linkage disequilibrium with this haplotype that caused alterations in PAX2 expression, linking reduction in expression with reduced nephrons. / We hypothesized that Lgl1, a pulmonary branching morphogen, is stimulated by RA to affect renal branching. We characterized Lgl1 expression in the kidney and its response to RA. Finally, we showed that Lgl1+/- embryos have reduction in kidney branching, implicating Lgl1 in nephron formation.

Valproic acid (VPA) during pregnancy leads to congenital anomalies, possibly by disrupting folate metabolism. Mild deficiency of methylenetetrahydrofolate reductase (MTHFR), an enzyme of folate-dependent homocysteine metabolism, is common due to a polymorphism at bp 677, and may influence response to VPA. To examine interactions between VPA and MTHFR in vivo and in vitro, VPA-induced teratogenicity was studied in Mthfr -deficient mice, and the effects of VPA on MTHFR expression in HepG2 cells were investigated. Mthfr+/+ and Mthfr+/- pregnant mice were injected with VPA on gestational day 8.5; resorption rates and occurrence of neural tube defects (NTDs) were examined on gestational day 14.5. MTHFR expression in HepG2 cells was studied by promoter assays, quantitative RT-PCR and Western analysis. Mthfr+/+ mice had increased resorption rates (36%) after VPA treatment, compared to saline treatment (10%), whereas resorption rates were similar with Mthfr+/- mice with the 2 treatments (25-27%). NTDs were only observed in one group (VPA-treated Mthfr+/+). VPA increased activity of both MTHFR promoters (1.8- and 3.5-fold), and levels of MTHFR mRNA and protein (2.5- and 3.7-fold, respectively) in HepG2 cells. Consistent with MTHFR upregulation in vitro, plasma homocysteine decreased in mice one hour after VPA injection. VPA increases MTHFR expression and may have lower teratogenicity in MTHFR deficiency. These phenomena underscore the importance of folate interconversion in VPA teratogenicity.

Skeletal muscle wasting can be a fatal complication of many diseases, such as cancer, AIDS and neuromuscular disorders. During wasting, the ubiquitin-proteasome system (UPS) is the primary pathway for the catabolism of myofibrillar proteins. Many studies have explored the importance of enzymes mediating the conjugation of ubiquitin to muscle proteins. However, the role of deubiquitinating enzymes in skeletal muscle wasting is poorly understood. Our laboratory previously identified USP19 as a deubiquitinating enzyme which is upregulated in response to atrophic stimuli in vivo and is capable of indirectly regulating the expression of myofibrillar proteins in muscle cells in vitro. However, the role of USP19 during skeletal muscle wasting in vivo remains unexplored. To address this question, I have characterized the phenotype of USP19 KO mice and determined the effects of denervation induced wasting of hindlimb muscles of USP19 KO mice by measuring various anatomical and structural parameters. Denervation stimulus was chosen to minimize number of animals used since control and treated limbs are within the same animal. USP19 KO mice had slightly heavier gastrocnemius muscle (GAS) mass than WT mice, with a trend towards a larger fiber size and higher protein content. After denervation, the skeletal muscle mass of KO mice is 30% heavier than that of denervated WT mice in both the tibialis anterior (TA) and GAS muscle. This significant sparing is correlated with KO having 30% more protein than WT in GAS muscles, after denervation. The level of the myofibrillar protein tropomyosin was significantly higher in denervated KO GAS than in denervated WT GAS, consistent with previous USP19 siRNA studies in rat skeletal muscle cells. Although, structural analysis of denervated GAS fiber reveals a small but significant sparing in the fiber size of KO mice as compared to WT mice, this difference only accounts for 5% of the mass sparing observed in KOs. Loss of neural stimulation / L'atrophie musculaire est une grave complication qui peut être fatale dans plusieurs maladies comme le cancer, le SIDA ou les maladies neuromusculaires. Le système ubiquitine-protéasome (UPS) est le mécanisme le plus impliqué dans la dégradation des protéines myofibrillaires durant l'atrophie. Plusieurs études ont démontré l'importance de certaines enzymes catalysant la conjugaison de l'ubiquitine aux protéines musculaires. Parcontre, le rôle des enzymes de déubiquitination durant l'atrophie musculaire est encore très peu connu. Notre laboratoire a précédemment identifié USP19, une enzyme de déubiquitination, qui est régulée à la hausse en réponse à plusieurs stimuli d'atrophie musculaire in vivo et qui est aussi capable de réguler indirectement des protéines myofibrillaires dans des cellules musculaires in vitro. Toutefois, le rôle de USP19 dans l'atrophie musculaire in vivo est encore inconnu. Afin d'aborder cette question, j'ai caractérisé le phénotype de souris dont le gène USP19 a été inactivé (KO) et j'ai déterminé les effets d'une dénervation des muscles de la patte postérieur sur ces souris KO en mesurant divers paramètres anatomiques et structuraux. Les souris USP19 KO possèdent une masse du muscle gastrocnemius (GAS) légèrement plus grande que les souris contrôles (WT) avec une tendance vers des fibers plus grosses et un contenu protéique plus élevé. Après dénervation, les muscles squelletiques des souris KO étaient 30% plus lourds que ceux des souris WT et ce dans le GAS ainsi que dans le tibialis postérieur (TA). Cette économie significative corrèle bien avec une augmentation de 30% du contenu protéique dans les muscles KO (GAS) comparé aux muscles WT, après dénervation. Aussi, les niveaux de la protéine myofibrillaire tropomyosine étaient significativement plus élevés dans le GAS KO que dans que dans le GAS WT, après dénervation, ce qui est consistant avec des études postérieures de siRNA

The kidney filters blood through nephrons, to recycle water and solutes in order to maintain homeostasis in the body. A reduction in nephron formation during fetal development renders the affected individual susceptible to hypertension and end stage renal disease. Nephrogenesis relies on the iterative branching of the ureteric bud epithelium, which triggers the metanephric mesenchyme to condense and form nephrons at its tips. / A critical question during kidney development is to identify the genes that are responsible for nephron formation. The culture of mouse embryonic kidneys has been an important in vitro model to examine the effects of candidate genes during nephrogenesis. By culturing embryonic kidneys in the presence of growth factors, blocking antibodies to specific proteins, or by blocking transcription using RNA interference, gene function can be ascertained. Microinjection and electroporation has also been used to overexpress DNA constructs in mouse embryonic kidneys. We have reviewed the method of microinjection and determined the optimal conditions for introducing DNA into embryonic kidneys. We examined high voltage/short pulse parameters and compared them to previously reported low voltage/long pulse settings. The use of a high voltage with a short pulse led to 100 fold more transgene expression and a longer duration of expression, when compared to a low voltage/long pulse protocol. Furthermore, we can target either the ureteric bud or the mesenchyme of the developing kidney, by selectively microinjecting the transgene in the organ. / We have examined the expression pattern of Alk6 and members of the claudin gene family during kidney development. Their expression pattern along with their function has not been fully characterized in the embryonic kidney. In the future we plan to employ our novel electroporation protocol to study the function of these candidate genes in the developing kidney.