Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol related problems and abuse continue to be serious problems in the U.S. today affecting nearly 17.6 million Americans. Understanding of the specific genes and related behaviors associated with alcohol use may provide substantial preventative measures for those who are at an increased risk. Genetically selected lines such as the high-alcohol preferring (HAP) and low-alcohol preferring (LAP) mice have been created to examine which endophenotypes co-segregate with alcohol preference. One behavioral trait that has been commonly associated with alcohol related problems is impulsivity. Impulsivity is the inability to withhold a response (motor impulsivity) or to act without forethought (cognitive impulsivity). The latter comprises much of the research and literature today using delay discounting models to tease out differences in subject’s wiliness to discount larger reinforcers for smaller immediate reinforcers. This study utilized relatively two newer paradigms associated with motor impulsivity in attempt to test differences in response disinhibition between two independent replicate HAP and LAP lines. It is hypothesized that the genes responsible for alcohol preference would be genetically correlated with motor impulsivity as HAP mice would display a greater degree of response disinhibition. Two independent replicates consisting of 48 mice (24 HAP II and 24 LAP II, representing the 37th generation; 24 HAP III and 24 LAP III, representing the 13th generation) were tested in two separate identical experiments. Each experiment was comprised of three phases. Phase I utilized a fixed interval (FI) 120s procedure for 30 days. After the 30 days of FI exposure mice were immediately moved to phase II for 10 days which implored a differential reinforcement of low rate procedure (DRL) at a time interval of 20s. Phase III used the same procedures as Phase II except the DRL was increased to 32s. As hypothesized, there was a moderate genetic correlation between alcohol preference and impulsivity as the HAP II mice displayed greater response disinhibition throughout all three phases compared to the LAP II mice. No differences were observed amongst the replicate III mice in any of the three phases. The findings from this study provide additional support that a genetic correlation between alcohol preference and impulsivity exists as seen in the delay discounting literature. Though this was observed in only one of the two replicates, interpretations must be taken at caution as the replicate III mice are still in the early stages of selection. It is possible at this stage in the selection process that increases in alcohol over successive generations are associated with selecting for taste until a threshold is met where selection shifts to pharmacologic drinking relevance. Until later generations of replicate III mice are studied where pharmacologic drinking occurs, conclusions from this study provide a moderate genetic correlation between alcohol preference and impulsivity.

Um componente considerado crítico para o manejo bem sucedido de populações silvestres é a manutenção da variação genética. No intuito de avaliar a magnitude e a distribuição da variabilidade genética existente em populações de Caiman latirostris, nove populações distribuídas ao longo do eixo latitudinal da distribuição da espécie no Brasil e uma população em cativeiro foram estudadas com auxílio de onze locos microssatélites. A diversidade gênica média (He=h) e a heterozigosidade média observada (Ho) apresentaram valores elevados, 0,628 e 0,567, respectivamente, denotando existência de elevada variabilidade genética para esta espécie nas regiões de estudo. A população paulista mesmo estando na zona intermediária de distribuição geográfica no Brasil não possui a maior variabilidade genética. O valor FST estimado foi 0,270 e o RST foi 0,342. Ambas as medidas de diferenciação entre as populações foram significativas (P<0,05). As altas estimativas de FST e RST sugerem a ausência ou fluxo gênico restrito entre essas populações, exceção feita entre as populações de Natal (RN) e João Pessoa (PB), onde não houve diferenciação significativa entre as populações, sugerindo assim que há fluxo gênico entre elas, fato confirmado pelo coeficiente de parentesco. Pôde-se concluir neste trabalho que a distância genética entre a população do litoral é afetada pela existência da Serra do Mar como barreira geográfica pelo fato desta população apresentar as maiores diferenciações genéticas e não se agrupar a nenhuma população pelo método de agrupamento UPGMA, mesmo estando próxima das populações paulista. A distância genética entre as populações parece não acompanhar a distância geográfica, em termos de gradiente latitudinal (r=0,206). Entretanto quando retiramos a população da Ilha do Cardoso esta correlação aumenta significativamente (r=0,540), indicando haver um certo padrão espacial da variabilidade genética entre as populações. O coeficiente médio de parentesco foi baixo entre e dentro das populações estudadas. Com estes resultados podemos começar a entender a dinâmica e estrutura social de populações de Caiman latirostris, e quanto mais se compreende sobre a biologia destes animais mais precisa serão decisões visando condições que permitam a existência continua da espécie. / A component considered critical for the managment well succeed of wild populations it is the maintenance of genetic variation. In the intention of evaluating the extend and the distribution of the existent genetic variability in populations of Caiman latirostris, nine populations distributed along the latitudinal axis of the distribution of the species in Brazil and a population in captivity they were studied with aid of eleven locos microsatellite. The genic mean diversity (He=h) and the observed mean heterozygosity (Ho) across all loci for all populations ranged from 0,628 and 0,567, respectively, denoting existence high genetic variability. The population from São Paulo being in the intermediate zone of geographical distribution in Brazil doesn't possess the largest genetic variability. The value dear FST was 0,270 and RST it was 0,342. Both differentiation measures among the populations were significant (P < 0,05). The higher estimates of FST and RST suggested a absence or low gene flow among those populations, exception done between the populations of Natal (RN) and João Pessoa (PB), where there was not significant differentiation among the populations, suggesting a gene flow pattern among them, fact confirmed by the related coefficient. It could be concluded in this work that the genetic distance among the population of the coast is affected by the existence of the Mountain of the Sea as geographical barrier for the fact of this population to present the largest genetic differentiations and not to group the any population for the grouping method UPGMA, same being close of the populations from São Paulo. The genetic distance among the populations seems not to accompany the geographical distance, in terms of latitudinal gradient (r=0,206). However when we removed the population of Cardoso's Island this correlation it increases significantly (r= 0,540), indicating there to be a certain space pattern of the genetic variability among the populations. The medium coefficient of related was low among and inside of the studied populations. With these results we can begin to understand the dynamics and it structures social of populations of Caiman latirostris.

Les petits ARN interférents (siRNA) sont encore rarement utilisés chez les vertébrés transgéniques pour inhiber l'expression de gènes. En effet, les vecteurs contenant un promoteur de type ARN polymérase III comme ceux des gènes U6 et H1 qui permettent une expression élevée des gènes codant pour des ARNi dans des cellules sont souvent silencieux in vivo. Dans cette thèse, divers vecteurs exprimant des petits ARN double brins (shRNA) ont été testés dans des cellules en culture et chez des souris transgéniques pour inhiber l'ARN m du gène précoce IE du virus de la pseudo rage porcine responsable de la maladie d'Aujeszky. La quantité et la séquence des si RNA produits ont été étudiées par qPCR. Dans des cellules CHO transfectées pour une expression transitoire, les vecteurs contenant les gènes U6-shRNA ont été de loin les plus efficaces pour inhiber le gène IE en raison du niveau élevé de siRNA produit. Par ailleurs, deux constructions contenant le promoteur de type ARN polymérase II, le promoteur du gène eF1-α etune séquence de shRNA bordée par 5T ou introduite dans un gène de microARN (miRNA) le miR30 ont permis d'obtenir une inhibition significative mais limitée de l'ARNm du gène IE. Ceci parait être du au niveau relativement faible de siRNA produit. Le siRNA produit par le gène du miRNA s'est avéré aussi efficace que ceux obtenus à partir des constructions U6-shRNA bien que ces derniers soient un peu plus longs. Ces diverses constructions ont été utilisées pour obtenir des souris transgéniques. Des souris contenant la séquence du shRNA n'ont pu être obtenues qu'à partir de la construction miRNA. Ceci peut être du au fait que les siRNA produits par les autres constructions ont exercé un effet inhibiteur sur des cibles aspécifiques (off-targeting) qui ne s'est pas produit avec le siRNA provenant de la construction miRNA car il contient quelques nucléotides en moins. Les souris transgéniques contenant la construction miRNA ont été soumises à une infection par le virus de la pseudo rage porcine. Bien que les souris exprimaient le gène shRNA qu'à un faible niveau. Quelques souris transgéniques ont résisté à l'infection. La seconde partie de la thèse a consisté à sélectionner d'autres séquences de shRNA capables d'inhiber l'expression du gène IE sans exercer des effets aspécifiques. Deux séquences de shRNA ont permis une telle inhibition. L'une est dirigée contre la région 5'UTR du gène IE et l'autre contre la région 3'UTR. Ces données suggèrent que (1) l'efficacité d'un shRNA n'est pas déterminée par sa séquence d'une manière totalement prévisible (2) l'efficacité d'un siRNA est d'autant plus élevé que sa séquence cible dans l'ARNm est en structure double brin (3) un effet inhibiteur intense et optimum peut être obtenu avec des concentrations faibles d'un siRNA (4) les effets secondaires et en particulier le off-targeting peuvent avoir lieu à faible concentration du siRNA mais ils ont d'autant plus de chance de se produire que la concentration du si RNA est plus élevée (5) un siRNA destiné à être utilisé chez des animaux transgéniques devrait être choisi pour sa capacité à inhiber efficacement un gène à faible concentration pour réduire ses effets secondaires.

Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T18:58:55Z : No. of bitstreams: 1 limaneto_jf_me_botfmvz.pdf: 482694 bytes, checksum: 8e409b567c057a2c7915beaa9c6e8077 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O primeiro sucesso na clonagem de eqüídeos através do uso de transferência nuclear foi relatado em maio de 2003 após o nascimento de uma mula clonada a partir de células fetais. Acredita-se que a utilização de células diplóides em fase do ciclo celular G0 ou G1 facilite a coordenação entre o ciclo celular e a reprogramação nuclear após a transferência de núcleo. Sendo assim, este experimento objetivou estudar o comportamento de fibroblastos de eqüinos adultos em cultura, analisando o estado do ciclo celular e a viabilidade das células em cultivo pré- e pós-descongelação. Fragmentos de pele de 6 eqüinos adultos, sendo 3 machos e 3 fêmeas, foram divididos em pedaços de aproximadamente 1mm3 e acondicionados (10 fragmentos/garrafa) no fundo de duas garrafas de cultivo (25cm2) umedecidas com 1mL SFB. As garrafas foram inclinadas para a posição vertical e adicionado 5ml de meio DMEM alta glicose + 10% SFB, 100UI/mL Penicilina / 100æg/mL Estreptomicina e 3,0æg/mL Anfotericina B. Após 30 minutos em estufa com 5% CO2 em ar, as garrafas foram dispostas em posição horizontal, para que o meio de cultivo entrasse em contato com os fragmentos teciduais. Todas as garrafas permaneceram por sete dias em estufa com 5% CO2 em ar até o início do crescimento celular. Com 70% de confluência, foi realizada a tripsinização e a primeira passagem. O mesmo procedimento foi repetido para a segunda passagem. Para os grupos de privação de soro (0,5% de SFB) foram produzidas garrafas de cultivo em duplicata para a análise da viabilidade e do ciclo celular nos períodos de 24, 48, 72, 96, 120, 144 e 168 horas de cultivo. Para os grupos de confluência foram produzidas duas garrafas de cultivo para a análise da viabilidade e do ciclo celular no período em que o tapete celular atingisse a confluência... / The first success in equine cloning, using nuclear transfer, was reported in May of the 2003 after the birth of a cloned mule obtained from fetal cells. Nowadays there is an agreement, in the literature, that diploid cells in phase G0 or G1 of the cell cycle facilitate coordination between the cell cycle and the nuclear reprogramming after the nuclear transfer. This experiment aimed to study the behavior of equine fibroblasts in culture though the analysis of the cellular cycle and the viability of cells pre- and post-freezing. Skin fragments were obtained from 6 adult horses (3 females and 3 males) and divided in pieces of about 1 mm3. For culture, the bottoms of two culture bottles were moistened with 1 mL of FCS, 10 fragments of skin were deposited, and 5 ml of DMEM high glucose + 10% FCS, 100 IU/mL penicillin, 100 g/mL streptomycin and 3 g/ml amphotericin B were added with the bottles raised vertically. After 30 min incubation in 5% CO2 in air, the bottles were moved to a horizontal position allowing the culture media to make contact with the tissue fragments. All bottles were cultured for 7 days in 5% CO2 in air until the beginning of confluence. The complete culture media was replaced weekly and, at 70% of confluence, trypsinization (ATV Vernese, Adolfo Lutz Institute) and the first passage were performed. Two passages were performed before freezing. For the second passage, the culture and trypsinization were repeated. For cells subjected to serum starvation (0.5% FCS), culture bottles were produced in duplicate for viability and cellular cycle analysis at 24, 48, 72, 96, 120, 144 and 168 hours of culture. For the confluence groups bottles were produced for the analysis of viability and cellular cycle at the moment cells achieved confluence...(Complete abstract, click electronic address below)

Um componente considerado crítico para o manejo bem sucedido de populações silvestres é a manutenção da variação genética. No intuito de avaliar a magnitude e a distribuição da variabilidade genética existente em populações de Caiman latirostris, nove populações distribuídas ao longo do eixo latitudinal da distribuição da espécie no Brasil e uma população em cativeiro foram estudadas com auxílio de onze locos microssatélites. A diversidade gênica média (He=h) e a heterozigosidade média observada (Ho) apresentaram valores elevados, 0,628 e 0,567, respectivamente, denotando existência de elevada variabilidade genética para esta espécie nas regiões de estudo. A população paulista mesmo estando na zona intermediária de distribuição geográfica no Brasil não possui a maior variabilidade genética. O valor FST estimado foi 0,270 e o RST foi 0,342. Ambas as medidas de diferenciação entre as populações foram significativas (P<0,05). As altas estimativas de FST e RST sugerem a ausência ou fluxo gênico restrito entre essas populações, exceção feita entre as populações de Natal (RN) e João Pessoa (PB), onde não houve diferenciação significativa entre as populações, sugerindo assim que há fluxo gênico entre elas, fato confirmado pelo coeficiente de parentesco. Pôde-se concluir neste trabalho que a distância genética entre a população do litoral é afetada pela existência da Serra do Mar como barreira geográfica pelo fato desta população apresentar as maiores diferenciações genéticas e não se agrupar a nenhuma população pelo método de agrupamento UPGMA, mesmo estando próxima das populações paulista. A distância genética entre as populações parece não acompanhar a distância geográfica, em termos de gradiente latitudinal (r=0,206). Entretanto quando retiramos a população da Ilha do Cardoso esta correlação aumenta significativamente (r=0,540), indicando haver um certo padrão espacial da variabilidade genética entre as populações. O coeficiente médio de parentesco foi baixo entre e dentro das populações estudadas. Com estes resultados podemos começar a entender a dinâmica e estrutura social de populações de Caiman latirostris, e quanto mais se compreende sobre a biologia destes animais mais precisa serão decisões visando condições que permitam a existência continua da espécie. / A component considered critical for the managment well succeed of wild populations it is the maintenance of genetic variation. In the intention of evaluating the extend and the distribution of the existent genetic variability in populations of Caiman latirostris, nine populations distributed along the latitudinal axis of the distribution of the species in Brazil and a population in captivity they were studied with aid of eleven locos microsatellite. The genic mean diversity (He=h) and the observed mean heterozygosity (Ho) across all loci for all populations ranged from 0,628 and 0,567, respectively, denoting existence high genetic variability. The population from São Paulo being in the intermediate zone of geographical distribution in Brazil doesn't possess the largest genetic variability. The value dear FST was 0,270 and RST it was 0,342. Both differentiation measures among the populations were significant (P < 0,05). The higher estimates of FST and RST suggested a absence or low gene flow among those populations, exception done between the populations of Natal (RN) and João Pessoa (PB), where there was not significant differentiation among the populations, suggesting a gene flow pattern among them, fact confirmed by the related coefficient. It could be concluded in this work that the genetic distance among the population of the coast is affected by the existence of the Mountain of the Sea as geographical barrier for the fact of this population to present the largest genetic differentiations and not to group the any population for the grouping method UPGMA, same being close of the populations from São Paulo. The genetic distance among the populations seems not to accompany the geographical distance, in terms of latitudinal gradient (r=0,206). However when we removed the population of Cardoso's Island this correlation it increases significantly (r= 0,540), indicating there to be a certain space pattern of the genetic variability among the populations. The medium coefficient of related was low among and inside of the studied populations. With these results we can begin to understand the dynamics and it structures social of populations of Caiman latirostris.

This study aimed the study of ovarian follicular dynamics in a interovulatory period in nulliparous Santa Ines sheep carrying the polymorphism FecGE. Were used 21 sheep that previously genotyped for FecGE polymorphism by PCR-RFLP. The animals were divided into three groups (n = 7 / group) according to the genotype mutation: WW - non-mutant wild; EW . heterozygote and EE - homozygous. Follicular dynamics were monitored by ultrasound within a period interovulatory. The capture and measurement of the ovaries images were made every 24 hours until detection of estrus, being adjusted to every 12 hours until ovulation. It was found that 15 animals (71.42%) showed the emergence of three waves and six (28.58%) had four follicular waves. Don ft were influence of genotype on the selection, follicles . 2 and . 4 mm, (2,8 } 0,7) and follicular dominance, follicles > 5 mm, (0,9 } 0,1), the interovulatory period (17,5 } 0,2) and the duration of estrus (38,11 } 0,9). The largest number of ovulations was observed in homozygous sheep (2.4 } 0.2) compared to the wild type (1.7 } 0.2), however, heterozygous presenting intermediate value (1.9 } 0.1) but not differ from the others. Despite genetic groups did as not differ to the interval between the onset of estrus and ovulation (25,34 } 1,8), high individual variation was observed among sheep within each genotype. Santa Ines sheep that carriers the polymorphism FecGE express some differences in ovarian follicular dynamics in relation to non-mutants, being represented mainly by the largest number of ovulations in homozygous genetic group. / Este trabalho teve por objetivo o estudo da dinamica folicular ovariana do periodo interovulatorio em ovelhas nuliparas da raca Santa Ines portadoras do polimorfismo FecGE. Foram utilizadas 21 ovelhas previamente genotipadas para o polimorfismo FecGE por PCR-RFLP. Os animais foram divididos em tres grupos (n=7/grupo) de acordo com o genotipo relacionado a mutacao como segue: WW - selvagem nao mutante; EW - heterozigoto e; EE - homozigoto. A dinamica folicular foi acompanhada por ultrassom dentro de um periodo interovulatorio. A captura e mensuracao das imagens dos ovarios foram feitas a cada 24 horas ate a deteccao do estro, sendo ajustadas para cada 12 horas ate a ovulacao. Foi verificado que 15 animais (71,42%) apresentaram a emergencia de tres ondas e seis (28,58%) apresentaram quatro ondas de crescimento folicular. Nao houve influencia do genotipo sobre o numero de foliculos selecionados, . 2 e . 4 mm, (2,8 } 0,7) e dominantes, >5 mm, (0,9 } 0,1), o periodo interovulatorio (17,5 } 0,2) e, a duracao do estro (38,11 } 0,9). O maior numero de ovulacoes foi observado nas ovelhas homozigotas (2,4 } 0,2) em relacao as do tipo selvagem (1,7 } 0,2) sendo as heterozigotas apresentando valor intermediario (1,9 } 0,1), porem nao diferindo dos demais. Apesar dos grupos geneticos nao diferirem quanto ao intervalo entre o inicio do estro e a ovulacao (25,34 } 1,8 h), foi verificada alta variacao individual entre as ovelhas dentro de cada genotipo. Ovelhas nuliparas Santa Ines portadoras do polimorfismo FecGE expressam diferencas no tempo de sobrevivencia folicular e no diametro do foliculo ovulatorio em relacao as nao mutantes, sendo que o grupo genetico homozigoto apresenta um maior numero de ovulacoes.

Indiana University-Purdue University Indianapolis (IUPUI) / Abuse of alcohol during adolescence continues to be a problem, and it has been shown that earlier onset of drinking predicts increased alcohol abuse problems later in life. High levels of impulsivity have been demonstrated to be characteristic of alcoholics, and impulsivity has also been shown to predict later alcohol use in teenage subjects, showing that impulsivity may be an inherent underlying biological process that precedes the development of alcohol use disorders. These experiments examined adolescent drinking in a high-drinking, relatively impulsive mouse population, and assessed its effects on adult drinking and adult impulsivity. Experiment 1: Selectively bred High-Alcohol Preferring (HAP II) mice, which are shown to be highly impulsive, were given either alcohol (free choice access) or water only for two weeks during middle adolescence or adulthood. All mice were given free choice access to alcohol following 30 days without access, in adulthood. Experiment 2: Adolescent HAP II mice drank alcohol and water, or water alone, for two weeks, and were then trained to perform a delay discounting task as adults to measure impulsivity. In each experiment, effects of volitional ethanol consumption on later behavior were assessed. We expected adolescent alcohol exposure to increase subsequent drinking and impulsivity. Adolescent mice consumed significant quantities of ethanol, reaching average blood ethanol concentrations (BECs) of 142 mg/dl in Experiment 1 and 108 mg/dl in Experiment 2. Adult mice reached average BECs of 154 mg/dl in Experiment 2. Mice pre-exposed to alcohol in either adolescence or adulthood showed a transient increase in ethanol consumption, but we observed no differences in impulsivity in adult mice as a function of whether mice drank alcohol during adolescence. These findings indicate that HAP II mice drink intoxicating levels of alcohol during both adolescence and adulthood, and that this volitional intake has long-term effects on subsequent drinking behavior. Nonetheless, this profound exposure to alcohol during adolescence does not increase impulsivity in adulthood, indicating that long-term changes in drinking are mediated by mechanisms other than impulsivity. Importantly, this research demonstrates that the HAP II mouse is a good candidate for a model of heavy adolescent alcohol consumption.

Made available in DSpace on 2015-03-26T13:42:34Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1695521 bytes, checksum: 9a0915b224b9fc02033cedd8cfc42515 (MD5) Previous issue date: 2014-07-30 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The species of the genus Astyanax are widely distributed in the Neotropical region and have few morphological, ecological and behavioral differences, which complicates the taxonomic classification of this taxon. The nominal species Astyanax bimaculatus was described by Linnaeus in 1758 and currently species with two oval spots that occur in several Neotropical basins are considered as belonging to bimaculatus complex. The objective of this study was to determine the cytogenetic and molecular characteristics (mitochondrial DNA) of 19 populations currently assigned to Astyanax bimaculatus from complex coastal basins of southeastern Minas Gerais using conventional staining, nucleolar organizer regions Ag-NORs, C-band, DAPI, FISH and the gene cytochrome oxidase I - COI. Data were compared with other species of the complex. The karyotype 6m +20 sm +18 st +6 t occurred in all populations, with Ag-NORs ranging from two to eight stained chromosomes. The C-banding showed centromeric and pericentromeric markings, evidenced by the fluorochrome DAPI. Fluorescent 18S and 5S rDNA probes marked one chromosome pair respectively. The phylogeny obtained with the COI gene indicated the presence of two haplogroups with large molecular distance between them: haplogroup I was more closely related to the populations of the São Francisco River Basin, while haplogroup II was subdivided into a group which was more related to populations from the upper Paraná (IIA) and another group formed exclusively by coastal haplotypes (IIB). The karyotype uniformity of coastal populations is greater than the recognized Astyanax altiparanae, suggesting the persistence of large populations. Polymorphisms involving C-band patterns indicate that the addition and reduction of heterochromatin blocks are compatible with intraspecific variation. The molecular data suggest that coastal basins received migrants from continental basins, with more recent geodispersion of a coastal haplogroup, probably related to the dynamic confluence of coastal basins during glacial periods. The genetic similarity between São Francisco River Basin and coastal basins indicates that the nominal species Astyanax lacustris does not represent a valid taxon. While the Upper Paraná Basin maintained a molecular identity consistent with the existence of the nominal taxon Astyanax altiparanae that, agreeing with the cytogenetic data, also occurs in the Paraíba do Sul River Basin. It is concluded that the bimaculatus complex in southeastern Brazil is composed by three lineages, including only two species Astyanax aff. bimaculatus present in São Francisco and coastal basins and A. altiparanae present in the Upper Paraná. / As espécies do gênero Astyanax apresentam ampla distribuição na região neotropical e apresentam poucas diferenças morfológicas, ecológicas e comportamentais, o que dificulta a classificação taxonômica desse táxon. A espécie nominal Astyanax bimaculatus foi descrita em 1758 por Linnaeus e, atualmente, espécies com duas manchas ovaladas que ocorrem em diversas bacias neotropicais são consideradas como pertencentes ao complexo bimaculatus. O objetivo deste trabalho foi determinar as características citogenéticas e moleculares (DNA mitocondrial) de 19 populações consideradas como pertencentes ao complexo Astyanax bimaculatus das bacias costeiras do sudeste de Minas Gerais utilizando coloração convencional, regiões organizadoras de nucléolos Ag-NOrs, banda C, DAPI, FISH e o gene citocromo oxidase I - COI. Os dados foram comparados com outras espécies do complexo. O cariótipo 6m+20sm+18st+6t ocorreu em todas as populações, com Ag-NORs variando de dois a oito cromossomos marcados. A banda C mostrou marcações centroméricas e pericentroméricas, evidenciadas pelo fluorocromo DAPI. As sondas 18S e 5S marcaram apenas um par cromossômico respectivamente. A filogenia obtida com o gene COI indicou a presença de dois haplogrupos com grande distância molecular: o haplogrupo I mais aparentado com populações da bacia do rio São Francisco, enquanto o haplogrupo II subdividiu-se em um grupo mais aparentado com populações do alto Paraná (IIA) e outro grupo formado exclusivamente por haplótipos costeiros (IIB). A uniformidade cariotípica das populações costeiras é maior que a reconhecida em Astyanax altiparanae, sugerindo a persistência de grandes populações. Polimorfismos envolvendo padrões de banda C indicam que a adição e redução de blocos de heterocromatina são consideradas variações intraespecíficas. Os dados moleculares sugerem que as bacias costeiras receberam aporte de bacias continentais, com geodispersão mais recente de um haplogrupo costeiro, provavelmente relacionado com a dinâmica de confluência de bacias costeiras durante os períodos glaciais. A similaridade genética entre a bacia do rio São Francisco e as bacias costeiras indica que a espécie nominal Astyanax lacustris não representa um táxon válido. Enquanto a bacia do alto Paraná manteve uma identidade molecular condizente com a existência do táxon nominal Astyanax altiparanae que, condizente com os dados citogenéticos, também ocorre na bacia do rio Paraíba do Sul. Conclui-se que o complexo bimaculatus no sudeste brasileiro é composto de três linhagens, englobando apenas duas espécies: Astyanax aff. bimaculatus presente no São Francisco e nas bacias costeiras e A. altiparanae presente no alto Paraná.

Commercialisées depuis 1996, les plantes génétiquement modifiées produisant une toxine insecticide (toxine Cry) dérivée de Bacillus thuringiensis et appelées plantes Bt ciblent certains Lépidoptères ou Coléoptères ravageurs. Au fil des ans, les surfaces cultivées en plantes Bt sont de plus en plus importantes et contrôlent de larges populations d'insectes. Pour limiter le risque de développement de populations résistantes, une stratégie agricole appelée 'Haute Dose / Zone Refuge' est actuellement recommandée aux Etats-Unis par l'Environmental Protection Agency. Cette stratégie préventive nécessite la plantation d'une 'zone refuge' composée de plantes non-Bt utilisables par le ravageur ciblé et plantée à proximité de la 'zone Bt' qui synthétise une haute dose de toxine Cry. <p><p>Mon projet de recherche s’inscrit dans le cadre de l’évaluation de l'efficacité de cette stratégie et s’articule en deux phases :une phase expérimentale et une phase théorique. La première se concentre sur la caractérisation en laboratoire de l'impact des toxines Cry sur la biologie d'un ravageur. Cette phase constitue un support au volet théorique :la mise au point d’un modèle mathématique évaluant l'efficacité de la stratégie HD/R. L'originalité de ce projet repose entre autre sur l'interactivité entre ces deux volets.<p><p>Volet expérimental. Impact des toxines Cry sur la biologie de Plodia interpunctella. Nous évaluons séparément l'impact d'une gamme de concentrations de deux toxines Cry (CryXX et CryYY) sur une série de paramètres comportementaux et biologiques d'un insecte commun des denrées stockées: Plodia interpunctella (Hübner) (Lepidoptera :Pyralidae). Ces paramètres sont sélectionnés car leur variation pourrait avoir un impact sur l'efficacité de la stratégie HD/R dans le contrôle de la résistance. Il est donc pertinent de les quantifier pour intégrer dans le modèle des ordres de grandeur réalistes et générer des résultats qui ne sont pas uniquement basés sur des spéculations théoriques.<p><p>Volet théorique A. Efficacité de la stratégie HD/R pour des plantes Bt synthétisant une ou deux toxines simultanément. La stratégie 'HD/R' a été développée pour prévenir la résistance envers les plantes Bt synthétisant une seule toxine. Or, depuis 2003, de nouvelles variétés de coton Bt synthétisant simultanément deux toxines Cry sont commercialisées (BollgardII® et WidestrikeTM). Nous évaluons, grâce au modèle que nous avons développé, l'efficacité de cette stratégie lors d'une utilisation exclusive de plantes Bt synthétisant une ou deux toxines.<p><p>Volet théorique B. Impact du ralentissement du développement des insectes sur les plantes Bt sur l'efficacité de la stratégie HD/R. Le volet expérimental met en évidence un allongement de la durée du développement des larves se nourrissant sur une diète contaminée en toxine Cry. Ce ralentissement induit une séparation temporelle entre l'émergence des adultes de la zone Bt et de la zone refuge et perturbe une hypothèse principale de la stratégie HD/R: le croisement aléatoire entre adultes, indépendamment du génotype et de la zone d'origine. Dans ce troisième chapitre, nous étudions l'impact de la perturbation du croisement aléatoire sur l'efficacité de la stratégie HD/R. Nous testons également deux options pour optimiser la stratégie en cas d'asynchronie: l'utilisation de plantes Bt synthétisant une faible concentration en toxine (atténuant le décalage entre l'émergence des adultes) ou l'augmentation de la taille de la zone refuge (favorisant la survie des individus porteurs d'allèle de sensibilité et donc optimisant la dilution de la résistance à la génération suivante). <p><p>Ce travail s'intègre dans une problématique actuelle et utilise des outils de biologie théorique (théories de la dynamique et de la génétique des populations) ainsi que le développement d'un modèle mathématique. Il apporte des éléments de réponse et de réflexion sur l'optimisation de la gestion de la résistance des insectes mais c'est aussi une illustration de la complémentarité entre la biologie expérimentale et théorique.<p><p><p>/<p><p>On the market since 1996, genetically modified plants synthesizing an insecticidal toxin (Cry toxin) stemmed from Bacillus thuringiensis, called Bt plants, target several insect pests (Lepidoptera or Coleoptera). Bt crops cover increasingly larger areas and control important pest populations The Insect Resistance Management Strategy (IRM) strategy currently recommended in the U.S.A. to limit the development of resistant populations is the High Dose / Refuge zone (HD/R) strategy. This pre-emptive strategy requires a refuge zone composed by non-Bt plants, usable by the target insect and in close proximity of the Bt zone synthesizing a high toxin concentration.<p><p>My research project contributes to the effectiveness assessment of this HD/R strategy. It is structured on two main parts: an experimental, and a theoretical section. The first part characterizes the impact of Cry toxins on the biology of an insect pest. It is the basis of the theoretical part: the implementation of a mathematical model, which evaluates the effectiveness of the HD/R strategy.<p>The originality of this project is based on the interactivity of these two components.<p><p>Experimental section. Impact of the Cry toxins on the biology of Plodia interpunctella. We assess the impact of a range of concentrations of two Cry toxins (CryXX et CryYY) on several behavioural and biological parameters of a common pest of stored products: Plodia interpunctella (Hübner) (Lepidoptera :Pyralidae). These parameters are selected because their variation could influence the effectiveness of a HD/R strategy. So, it is important to quantify these parameters so that realistic values can be integrated in our model. The results of the model are thus not based on theoretical assumptions alone.<p> <p>Theoretical section A. Effectiveness of a HD/R strategy with Bt plants synthesizing one or two toxins. Initially, the HD/R strategy has been developed to limit the resistance towards Bt plants synthesizing one toxin. However, since 2003, new Bt cotton varieties synthesize two toxins simultaneously (BollgardII® et WidestrikeTM). We assess, with our model, the effectiveness of this strategy for Bt plants synthesizing one or two toxins.<p><p>Theoretical section B. Impact of the slowing down of the insect development reared on Bt plants on the effectiveness of the HD/R strategy. The experimental part demonstrates that larvae reared on a Bt diet have a protracted development duration. The consequence of this is a temporal separation between adult emergence in the two zones (Bt zone and refuge zone). This could affect the main assumption of the HD/R strategy, i. e. random mating independently of the genotype and of the native zone. In this third chapter, we study the impact of random mating disruption on the effectiveness of a HD/R strategy. We test two options to optimise the strategy in case of asynchrony: the use of Bt plants synthesizing a lower toxin concentration (limiting emergence asynchrony) or increasing the refuge zone size (favouring the survival of insect carrying one or two susceptible allele and thus optimising the dilution of resistance at the next generation). <p><p>This work is applied to a current issue. It uses some of the tools of theoretical biology (theories of population dynamics and population genetics) and develops a mathematical model. It provides some responses and some elements of thought about insect resistance management. It is also an illustration of the complementarity between experimental and theoretical biology.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished