To study hybrid sterility our laboratory uses mouse strains PWD/Ph (PWD), derived from Mus musculus musculus wild mice and the common laboratory strain C57BL/6J (B6) mostly of Mus musculus domesticus origin as a model. Crossing between PWD female and B6 male results in sterile male progeny. F1 hybrid males carry defects in the repair mechanisms of asymmetric double-strand DNA breaks (DSBs). Functional interplay of SPO11 and PRDM9 proteins in the meiotic prophase I is necessary for repairs. Its defect leads to incorrect synapse formation between homologous chromosomes, leading to halt in spermatogenesis and thus male sterility. The formation of DSBs and their subsequent repair is essential for first meiotic division. The working hypothesis stems from the findings in yeast model, where supposed antirecombinatorial mechanism of mismatch repair genes Msh6 and Msh2 prevents DSBs repairs during meiosis. Despite the functional mechanism of these two genes is not explicitly known, existence of similar repair system in mice is presumed. Variety of methods was implemented in this thesis. The effects of Msh6 deletion on meiotic prophase I and sperm maturation were performed by designing guide RNAs for CRISPR/Cas9 for creation of three knock-outs in B6 mice. The PCR was used to amplify regions adjacent to the...

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We examined the influence of the host genetic background on the pathogenesis of tick-borne encephalitis. We determined virus titers in organs and serum in different time intervals post-infection for different ways of inoculation. We also stated mean survival times and antibody production in different strains of mice infected with tick-borne encephalitis virus. Moreover, differences in expression of immunologicaly important genes in brains of infected mice were compared.

O emprego de \"Total Genome Scan\" em modelos genéticos de doenças complexas tem sido fundamental para seleção de regiões cromossômicas envolvidas com traços complexos. Em nosso laboratório, identificamos cinco regiões cromossômicas associadas ao traço quantitativo pressão arterial (BP-QTL) que explicam 43% da variação da pressão arterial numa progênie obtida a partir de animais espontaneamente hipertensos (SHR) e \"Brown Norway\" (BN). Os BP-QTLs foram, então, validados por desenvolvimento de linhagens congênicas, incluindo uma para o cromossomo 4 (SHR.BN4) cuja substituição das sequências SHR pelo do animal BN levou a redução da pressão arterial sistólica basal (~14 mm Hg). O objetivo deste trabalho foi identificar as variantes genéticas candidatas neste intervalo cromossômico com base em diferenças no padrão de expressão gênica e na presença de alterações genéticas não sinônimas \"missense\" ou em regiões regulatórias conservadas que possam estar envolvidas na gênese da hipertensão. Identificamos 533 genes com expressão renal, dentre os 682 do intervalo, sendo que 28 apresentaram padrão de expressão diferente entre amostras de animais adultos (congênico vs. SHR) e seis apresentaram alterações não sinônimas \"missense\". É importante salientar que dos genes diferentemente expressos, encontramos alterações estruturais em regiões conservadas com potencial de participar na regulação em 11. Em conjunto, utilizamos uma plataforma integrada para selecionar 34 genes candidatos no cromossomo 4, dos quais 17 genes serão priorizados, para ser investigados quanto sua contribuição na hipertensão arterial do SHR e na hipertensão primária humana / Total genome scan in genetic models of complex diseases have been instrumental to select candidate genes underlying complex traits. We previously mapped 5 blood pressure related quantitative trait loci (BP-QTLs) that explain about 43% of the BP variance in a progeny derived from Spontaneous Hypertensive Rat (SHR) and Brown Norway (BN) rats. The BP-QTLs were then validated by derivation of congenic strains, including one for chromosome 4 (SHR.BN4) in which a segment from BN replaced the SHR sequences reducing basal systolic BP (~14 mm Hg). The aim of this project is to identify the candidate genetic variants within the chromosome interval based on differences in renal gene expression patterns and structural changes in both non-synonymous missense or within adjacent regulatory sequences that may contribute to hypertension. We identified 533 genes with renal expression, out of 682 in the interval, in which 28 presented differences in expression pattern in adult samples (congenic vs. SHR) and six presented non-synonymous missense alterations. In addition, 11 out of 28 differentially expressed genes showed structural alterations in adjacent conserved regions that potentially contribute to gene regulation. Taken together, using the proposed combination of strategies, we selected 34 hypertensive candidate genes in chromosome 4, in which 17 will be prioritized, to be further explored to assess their contribution to hypertension in the SHR and to essential hypertension in humans

O emprego de \"Total Genome Scan\" em modelos genéticos de doenças complexas tem sido fundamental para seleção de regiões cromossômicas envolvidas com traços complexos. Em nosso laboratório, identificamos cinco regiões cromossômicas associadas ao traço quantitativo pressão arterial (BP-QTL) que explicam 43% da variação da pressão arterial numa progênie obtida a partir de animais espontaneamente hipertensos (SHR) e \"Brown Norway\" (BN). Os BP-QTLs foram, então, validados por desenvolvimento de linhagens congênicas, incluindo uma para o cromossomo 4 (SHR.BN4) cuja substituição das sequências SHR pelo do animal BN levou a redução da pressão arterial sistólica basal (~14 mm Hg). O objetivo deste trabalho foi identificar as variantes genéticas candidatas neste intervalo cromossômico com base em diferenças no padrão de expressão gênica e na presença de alterações genéticas não sinônimas \"missense\" ou em regiões regulatórias conservadas que possam estar envolvidas na gênese da hipertensão. Identificamos 533 genes com expressão renal, dentre os 682 do intervalo, sendo que 28 apresentaram padrão de expressão diferente entre amostras de animais adultos (congênico vs. SHR) e seis apresentaram alterações não sinônimas \"missense\". É importante salientar que dos genes diferentemente expressos, encontramos alterações estruturais em regiões conservadas com potencial de participar na regulação em 11. Em conjunto, utilizamos uma plataforma integrada para selecionar 34 genes candidatos no cromossomo 4, dos quais 17 genes serão priorizados, para ser investigados quanto sua contribuição na hipertensão arterial do SHR e na hipertensão primária humana / Total genome scan in genetic models of complex diseases have been instrumental to select candidate genes underlying complex traits. We previously mapped 5 blood pressure related quantitative trait loci (BP-QTLs) that explain about 43% of the BP variance in a progeny derived from Spontaneous Hypertensive Rat (SHR) and Brown Norway (BN) rats. The BP-QTLs were then validated by derivation of congenic strains, including one for chromosome 4 (SHR.BN4) in which a segment from BN replaced the SHR sequences reducing basal systolic BP (~14 mm Hg). The aim of this project is to identify the candidate genetic variants within the chromosome interval based on differences in renal gene expression patterns and structural changes in both non-synonymous missense or within adjacent regulatory sequences that may contribute to hypertension. We identified 533 genes with renal expression, out of 682 in the interval, in which 28 presented differences in expression pattern in adult samples (congenic vs. SHR) and six presented non-synonymous missense alterations. In addition, 11 out of 28 differentially expressed genes showed structural alterations in adjacent conserved regions that potentially contribute to gene regulation. Taken together, using the proposed combination of strategies, we selected 34 hypertensive candidate genes in chromosome 4, in which 17 will be prioritized, to be further explored to assess their contribution to hypertension in the SHR and to essential hypertension in humans

Le but de cette thèse est premièrement d’évaluer l’effet du vieillissement sur les fonctions psychomotrices des souches de souris sélectionnées génétiquement en fonction de leur tension artérielle (TA); deuxièmement, de localiser les déterminants génétiques des phénotypes psychophysiologiques à partir de souches recombinantes congéniques (RCS). Ces travaux ont mené à la publication de 4 articles. Le premier article décrit l’évaluation des fonctions psychomotrices des souches avec une tension artérielle élevée (HBP), basse (LBP) et normale (NBP). La performance aux épreuves d’exploration, d’habiletés motrices et d’apprentissage spatial, a été mesurée sur deux cohortes âgées respectivement de 12 mois et de trois mois. Indépendamment de l’âge, les HBPs sont hyperactives dans l’open-field (OF), mais pas dans le test d’exploration de trous. Inversement, les LBP explorent moins d’espaces que les NBP et, à trois mois seulement, sont hypoactives dans l’OF. Par ailleurs, les HBPs et les LBP présentent des déficits précoces de coordination motrice et des fonctions visuo-motrices. Le second article concerne l’évaluation longitudinale de la coordination motrice, de l’anxiété et de l’apprentissage spatial des souches HBP, LBP et NBP, à l’âge de deux mois et de 12 mois. Le vieillissement accentue l’hyperactivité des HBPs dans l’OF. Par contre, l’hypoactivité des souris LBP est détectable seulement à l’âge de deux mois. Indépendamment de l’âge, les souris HBP et LBP montrent une perception réduite du danger dans l’épreuve d’anxiété et des dysfonctions visuo-motrices au labyrinthe aquatique. Enfin, des déficits précoces de coordination motrice se manifestent seulement chez les HBPs. Il reste à déterminer si les déficits observés sont liés à des déterminants génétiques indépendants ou secondaires aux altérations de la tension artérielle. Le troisième article présente la comparaison entre les souches consanguines A/J et C57Bl/6J (B6) aux épreuves de l’OF, de la planche à trous, du labyrinthe aquatique et du cintre (coordination motrice). Les B6 explore d’avantage l’OF et la planche à trous. Les B6 sont moins rapides sur le cintre, mais supérieurs aux A/J dans le labyrinthe aquatique, avec une plate-forme invisible ou visible. Ces résultats démontrent l’implication de déterminants génétiques. Cette thèse se termine par un quatrième article sur la localisation des déterminants génétiques de la susceptibilité au stress dans les RCS, dérivées de A/J et B6, et présentant un agencement spécifique de 12.5% du génome. La réactivité émotionnelle est évaluée dans l’OF et le plus-maze; la réponse de stress est mesurée par radio télémétrie de la température interne pendant le stress d’immobilisation (SI) sous diète régulière et riche en sel; l’excrétion des électrolytes urinaires est dosée après 24 heures de diète salée. Les loci les plus significatifs sont situés dans les régions suivantes: de l’émotionalité dans l’OF (Emo1) sur le chr. 1 (LOD=4.6) correspondant à la région homologue impliquée dans la cohorte d’hypertension familiale du Saguenay; de la dopa décarboxylase (ddc) sur le chr. 11 pour l’émergence du plus-maze (LOD=4.7); de la protéine liant l’endotoxine (lbp) sur le chr. 2 pour l’hypothermie initiale en réponse au SI (LOD=4); et de HSP90 sur le chr. 12 pour l’excrétion de Ca++ (LOD=4.6). Des banques de données sont ensuite interrogées pour recenser les polymorphismes des régions régulatrices ou codantes des gènes candidats chez les souches ancestrales A/J et B6, dont les séquences sont disponibles pour le génome entier. Des utilitaires web permettent de dévoiler les changements dans la structure secondaire de l’ARNm, l’interférence avec des microARN ou avec d’autres motifs de liaison. Plusieurs SNPs fonctionnels ont été identifiés pour le QTL du chr. 1, particulièrement dans les éléments de régulation; ceux-ci impliquant des gènes reliés avec les réponses inflammatoire/immunitaire ou avec le système cardiovasculaire. La quantification par la PCR confirme une régulation à la baisse d’atp1a2 dans le cœur et le cerveau des souches susceptibles à l’anxiété. Ces résultats confirment l’intrication des altérations de la susceptibilité au stress et de la régulation de la TA. / Our studies in this thesis, which led to 4 publications, are divided in two parts. The first part describes the neuropsychological effects of aging in strains of mice genetically selected for high (HBP), low (LBP) or normal blood pressure (NBP). The second part focuses on the genetic determinants of these neuropsychological phenotypes in recombinant congenic strains (RCS) of mice. The first manuscript compares HBP or LBP mice to normotensive controls in tests of exploration, motor coordination, and spatial learning at two age levels: 3 and 12 months. At either age, HBPs were hyperactive in an open field (OF) but not in terms of hole-poking responses. On the contrary, LBPs were hypoactive in the OF and in the hole-board, with the effect on the former measure being limited to the younger cohort. In either cohort, HBP and LBP mice were deficient in subtle aspects of motor coordination, and visuomotor function. These strains may serve as experimental models for the evaluation of beneficial early antihypertensive or antihypotensive treatments on brain function. The second study uses a longitudinal design to compare either HBP or LBP mice to normotensive controls at 2 and 12 months of age for motor coordination, anxiety, and spatial learning. Hyperactivity of HBPs in the OF increased with aging; whereas LBP mice were hypoactive only at 2 months of age. At both age, HBP and LBP mice displayed reduced levels of anxiety in the elevated plus-maze (EPM), abnormal coordination and visuomotor guidance. It remains to determine if these strain-, age-, and test-specific abnormalities are genetically related or secondary to uncontrolled hypertension or hypotension. The following article compares the C57BL/6J (B6) to the A/J inbred mouse strain in exploration of the OF and the hole-board, in the coat-hanger coordination test, and in spatial learning of a water maze. B6 mice displayed a higher number of segment crossings in the open-field and of hole-poking responses than A/J mice. By contrast to their hypo activity, A/J strain were faster in the coat-hanger motor test, but deficient in the submerged but also in the visible platform version of the water maze. These results indicate the considerable potential of genetic models derived from B6 and A/J mice for discerning the determinants of several behavioural phenotypes. In the last manuscript, the genomic loci bearing stress-related phenotypes were dissected by genome wide analysis of linkage in the recombinant congenic strains (RCS), resulting from a cross of B6 and A/J progenitors, each strain bearing 12.5% of specific parts of one progenitor on the background of the other. Adult male mice from 14 A/J and 22 B6 background lines were evaluated for emotional reactivity in the OF and the EPM. Core temperature was monitored by radio-telemetry during immobilization (IM), under standard and salt-enriched diets. In addition, urinary electrolytes were measured. The highest LOD scores strengthen the evidence for a previously reported locus for emotionality in the open-field on Chr 1 (LOD=4.6), in the Ddc region encoding dopa decarboxylase, on Chr 11 in the EPM (LOD=4.7), near Lbp (lipopolysaccharide binding protein), on Chr 2 for initial hypothermia during IM (LOD=4), as well as in the region of Hspca, encoding heat shock protein 1 alpha (48.0 cM) on Chr 12 for Ca++ excretion after a 24 hr-salt load (LOD=4.6). RCS stress QTL overlapped with several candidate loci for cardiovascular disease. In silico evidence of functional polymorphisms by comparative sequence analysis of progenitor strains assisted to ascertain this convergence, then further tested using quantitative PCR for releant genes mRNA. The anxious BcA70 strain showed down regulation of the Atp1a2 gene expression in the heart (P < 0.001) and brain (P < 0.05) compared to its parental B6 strain, compatible with the enhanced emotionality described in knock out animals for this gene, also involved in the salt-sensitive component of hypertension. Functional polymorphisms in regulatory elements of candidate genes of the cardiovascular / inflammatory / immune systems support the hypothesis of genetically-altered environmental susceptibility in cardiovascular disease development.

Le but de cette thèse est premièrement d’évaluer l’effet du vieillissement sur les fonctions psychomotrices des souches de souris sélectionnées génétiquement en fonction de leur tension artérielle (TA); deuxièmement, de localiser les déterminants génétiques des phénotypes psychophysiologiques à partir de souches recombinantes congéniques (RCS). Ces travaux ont mené à la publication de 4 articles. Le premier article décrit l’évaluation des fonctions psychomotrices des souches avec une tension artérielle élevée (HBP), basse (LBP) et normale (NBP). La performance aux épreuves d’exploration, d’habiletés motrices et d’apprentissage spatial, a été mesurée sur deux cohortes âgées respectivement de 12 mois et de trois mois. Indépendamment de l’âge, les HBPs sont hyperactives dans l’open-field (OF), mais pas dans le test d’exploration de trous. Inversement, les LBP explorent moins d’espaces que les NBP et, à trois mois seulement, sont hypoactives dans l’OF. Par ailleurs, les HBPs et les LBP présentent des déficits précoces de coordination motrice et des fonctions visuo-motrices. Le second article concerne l’évaluation longitudinale de la coordination motrice, de l’anxiété et de l’apprentissage spatial des souches HBP, LBP et NBP, à l’âge de deux mois et de 12 mois. Le vieillissement accentue l’hyperactivité des HBPs dans l’OF. Par contre, l’hypoactivité des souris LBP est détectable seulement à l’âge de deux mois. Indépendamment de l’âge, les souris HBP et LBP montrent une perception réduite du danger dans l’épreuve d’anxiété et des dysfonctions visuo-motrices au labyrinthe aquatique. Enfin, des déficits précoces de coordination motrice se manifestent seulement chez les HBPs. Il reste à déterminer si les déficits observés sont liés à des déterminants génétiques indépendants ou secondaires aux altérations de la tension artérielle. Le troisième article présente la comparaison entre les souches consanguines A/J et C57Bl/6J (B6) aux épreuves de l’OF, de la planche à trous, du labyrinthe aquatique et du cintre (coordination motrice). Les B6 explore d’avantage l’OF et la planche à trous. Les B6 sont moins rapides sur le cintre, mais supérieurs aux A/J dans le labyrinthe aquatique, avec une plate-forme invisible ou visible. Ces résultats démontrent l’implication de déterminants génétiques. Cette thèse se termine par un quatrième article sur la localisation des déterminants génétiques de la susceptibilité au stress dans les RCS, dérivées de A/J et B6, et présentant un agencement spécifique de 12.5% du génome. La réactivité émotionnelle est évaluée dans l’OF et le plus-maze; la réponse de stress est mesurée par radio télémétrie de la température interne pendant le stress d’immobilisation (SI) sous diète régulière et riche en sel; l’excrétion des électrolytes urinaires est dosée après 24 heures de diète salée. Les loci les plus significatifs sont situés dans les régions suivantes: de l’émotionalité dans l’OF (Emo1) sur le chr. 1 (LOD=4.6) correspondant à la région homologue impliquée dans la cohorte d’hypertension familiale du Saguenay; de la dopa décarboxylase (ddc) sur le chr. 11 pour l’émergence du plus-maze (LOD=4.7); de la protéine liant l’endotoxine (lbp) sur le chr. 2 pour l’hypothermie initiale en réponse au SI (LOD=4); et de HSP90 sur le chr. 12 pour l’excrétion de Ca++ (LOD=4.6). Des banques de données sont ensuite interrogées pour recenser les polymorphismes des régions régulatrices ou codantes des gènes candidats chez les souches ancestrales A/J et B6, dont les séquences sont disponibles pour le génome entier. Des utilitaires web permettent de dévoiler les changements dans la structure secondaire de l’ARNm, l’interférence avec des microARN ou avec d’autres motifs de liaison. Plusieurs SNPs fonctionnels ont été identifiés pour le QTL du chr. 1, particulièrement dans les éléments de régulation; ceux-ci impliquant des gènes reliés avec les réponses inflammatoire/immunitaire ou avec le système cardiovasculaire. La quantification par la PCR confirme une régulation à la baisse d’atp1a2 dans le cœur et le cerveau des souches susceptibles à l’anxiété. Ces résultats confirment l’intrication des altérations de la susceptibilité au stress et de la régulation de la TA. / Our studies in this thesis, which led to 4 publications, are divided in two parts. The first part describes the neuropsychological effects of aging in strains of mice genetically selected for high (HBP), low (LBP) or normal blood pressure (NBP). The second part focuses on the genetic determinants of these neuropsychological phenotypes in recombinant congenic strains (RCS) of mice. The first manuscript compares HBP or LBP mice to normotensive controls in tests of exploration, motor coordination, and spatial learning at two age levels: 3 and 12 months. At either age, HBPs were hyperactive in an open field (OF) but not in terms of hole-poking responses. On the contrary, LBPs were hypoactive in the OF and in the hole-board, with the effect on the former measure being limited to the younger cohort. In either cohort, HBP and LBP mice were deficient in subtle aspects of motor coordination, and visuomotor function. These strains may serve as experimental models for the evaluation of beneficial early antihypertensive or antihypotensive treatments on brain function. The second study uses a longitudinal design to compare either HBP or LBP mice to normotensive controls at 2 and 12 months of age for motor coordination, anxiety, and spatial learning. Hyperactivity of HBPs in the OF increased with aging; whereas LBP mice were hypoactive only at 2 months of age. At both age, HBP and LBP mice displayed reduced levels of anxiety in the elevated plus-maze (EPM), abnormal coordination and visuomotor guidance. It remains to determine if these strain-, age-, and test-specific abnormalities are genetically related or secondary to uncontrolled hypertension or hypotension. The following article compares the C57BL/6J (B6) to the A/J inbred mouse strain in exploration of the OF and the hole-board, in the coat-hanger coordination test, and in spatial learning of a water maze. B6 mice displayed a higher number of segment crossings in the open-field and of hole-poking responses than A/J mice. By contrast to their hypo activity, A/J strain were faster in the coat-hanger motor test, but deficient in the submerged but also in the visible platform version of the water maze. These results indicate the considerable potential of genetic models derived from B6 and A/J mice for discerning the determinants of several behavioural phenotypes. In the last manuscript, the genomic loci bearing stress-related phenotypes were dissected by genome wide analysis of linkage in the recombinant congenic strains (RCS), resulting from a cross of B6 and A/J progenitors, each strain bearing 12.5% of specific parts of one progenitor on the background of the other. Adult male mice from 14 A/J and 22 B6 background lines were evaluated for emotional reactivity in the OF and the EPM. Core temperature was monitored by radio-telemetry during immobilization (IM), under standard and salt-enriched diets. In addition, urinary electrolytes were measured. The highest LOD scores strengthen the evidence for a previously reported locus for emotionality in the open-field on Chr 1 (LOD=4.6), in the Ddc region encoding dopa decarboxylase, on Chr 11 in the EPM (LOD=4.7), near Lbp (lipopolysaccharide binding protein), on Chr 2 for initial hypothermia during IM (LOD=4), as well as in the region of Hspca, encoding heat shock protein 1 alpha (48.0 cM) on Chr 12 for Ca++ excretion after a 24 hr-salt load (LOD=4.6). RCS stress QTL overlapped with several candidate loci for cardiovascular disease. In silico evidence of functional polymorphisms by comparative sequence analysis of progenitor strains assisted to ascertain this convergence, then further tested using quantitative PCR for releant genes mRNA. The anxious BcA70 strain showed down regulation of the Atp1a2 gene expression in the heart (P < 0.001) and brain (P < 0.05) compared to its parental B6 strain, compatible with the enhanced emotionality described in knock out animals for this gene, also involved in the salt-sensitive component of hypertension. Functional polymorphisms in regulatory elements of candidate genes of the cardiovascular / inflammatory / immune systems support the hypothesis of genetically-altered environmental susceptibility in cardiovascular disease development.