ABSTRACT Spiny softshell turtles (Apalone spinifera) occur at the northwest limit of their range in Lake Champlain. This species, although widespread across North America, is listed as threatened in Vermont due to habitat destruction and disturbances of anthropogenic origin. The population of spiny softshell turtles in Lake Champlain is isolated from other North American populations and is considered as an independent management unit. Efforts to obtain information on the biology of spiny softshell turtles in Lake Champlain precede 1936 with conservation measures being initiated in 1987. Methods of studying spiny softshell turtles in Lake Champlain have included direct observation, mark-recapture, nest beach monitoring, winter diving, and radio telemetry. Each of these approaches has provided some information to the sum of what is known about A. spinifera in Lake Champlain. For example major nesting beaches, hibernacula, and home range size have been determined. Currently spiny softshell turtles primarily inhabit two areas within Lake Champlain, Missisquoi Bay and the mouth of the Lamoille River. However, the population structure and gene flow between spiny softshell turtles inhabiting the Lamoille and Missisquoi regions remained unknown. A GIS model was created and tested in order to identify additional nesting beaches used by spiny softshell turtles along the Vermont shores of Lake Champlain. Although some additional small potential nesting beaches were found, no additional major nesting sites were found. The GIS model identified the mouth of the Winooski River (the site of a historical population) as potentially suitable nesting habitat; however, no evidence of spiny softshell turtle nesting was found at this site. A series of methods developed for collecting molecular and population genetic data about spiny softshell turtles in Lake Champlain are described, including techniques for DNA extraction of various tissue types and the design of new primers for PCR amplification and sequencing of the mitochondrial control region (mtD-loop). Techniques for circumventing problems associated with DNA sequence alignment in regions of a variable numbers of tandem repeats (VNTRs) and the presence of heteroplasmy within some individuals are also described. The mtD-loop was found to be a suitable marker to assess the genetic structure of the Lake Champlain population of spiny softshell turtles. No significant genetic sub-structuring was found (FST=0.082, p=0.223) and an indirect estimate of the migration rate between Lamoille and Missisquoi regions of Lake Champlain was high (Nm>5.576). In addition to consideration of A. spinifera in Lake Champlain, the mtD-loop was modeled across 46 species in 14 families of extant turtles. The primary structure was obtained from DNA sequences accessed from GenBank and secondary structures of the mtD-loop were inferred, (from thermal stabilities) using the program Mfold, for each superfamiliy of turtles. Both primary and secondary structures were found to be highly variable across the order of turtles; however, the inclusion of an AT-rich fold (secondary structure) near the 3' terminus of the mtD-loop was common across all turtle families considered. The Cryptodira showed conservation in the primary structure at regular conserved sequence blocks (CSBs), but the Pluerodira displayed little conservation in the primary structure of the mtD-loop. Overall, greater conservation in secondary structure than primary structure was observed in turtle mtD-loop. The AT-rich secondary structural element near the 3' terminus of the mtD-loop may be conserved across turtles due to it serving a functional role during mtDNA transcription.

Summarized the results of previous studies, the body size of Niviventer coxingi seemed to be correlated with altitude. For more exact evidence, geographic variations of body lengths and appendage sizes among areas were examined. Moreover, Freckleton et al. (2003) indicated that phylogenetic relationships may affect the results of one¡¦s study while variation of body size is discussed. Therefore, the phylogeographic variation among different areas was also examined in this study. The body lengths of N. coxingi in higher altitude (Fong-gang, 1700 m) were shorter than in lower altitude (Shan-ping 700 m). Body lengths of N. coxingi in Shan-ping were longer than other areas but Wu-shih-kang; and N. coxingi in Fong-gang were longer than those in Wu-tou Mountain; but no significant differences were found among other areas. No significant differences were found in appendage sizes but hind-foot length. Significant differences of the hind-foot length were only found between Shan-ping which had the longest hind-foot lengths in average and those in Wu-tou Mountain which had the shortest. No correlations were found between altitudes and the body length or appendage sizes of N. coxingi. The phylogenetic relationships based on D-loop region of N. coxingi were reconstructed by neighbor-joining, maximum parsimony and maximum-likelihood methods. An N. culturatus was used to be an outgroup. All three trees represented similar patterns. Although some individuals from neighborhood grouped together, some individuals from the same area represented distantly. Moreover, many branches represented in the root of the consensus trees because of the low bootstrap value. The results revealed the geographic variations did not correlated with their phylogenetic relationships and the heat conservation/ dissipation mechanism, which was the traditional explanation of Bergmann¡¦s rule, was not appropriate for N. coxingi, either.

Abstract MtDNA D-loop sequences and cranial morphometrics were used to analyze the phylogenetic relationships of bharal (Pseudois nayaur szechuanensis) populations. The habitat preferences of bharal were also investigated in Helanshan, Ningxia. The results from the analysis of mtDNA D-loop sequences and cranial morphometrics were in accordance. Among all populations analyzed, the phylogenetic relationships between populations in Lixian, Sichuan and Helanshan, Ningxia were most distantly related, while populations in Dulan, Qinghai and Subei, Gansu were close to one another. No matter in summer or winter, bharal generally prefer to stay in meadows, but they would appear in forests and shrubs as well.

An almost unbroken fringing reef runs along the east coast of Africa, the lagoon inside the reef is the foundation of almost all artisanal fisheries. It is a low-tech fishery conducted by many people. Some areas can have up to 19 fishermen per square kilometer. High fishing pressures, coupled with declining fish stocks has led to changes in mean size and reproductive age of many exploited species. There is a vital and urgent need for scientifically based management systems, including the utilization of genetic information to guide management practices. This thesis aims to investigate the presence of genetic structures in the western Indian Ocean. In order to do that we first investigated the historical patterns of connectivity throughout the region (paper I). In papers II and III we focused on local scale connectivity in Kenya and Tanzania and finally in paper IV we investigate the large-scale contemporary gene flow throughout the Western Indian Ocean. In paper III we also investigate the temporal genetic variation at one site and compare it to the small-scale genetic variation along a stretch of the Kenyan coastline. Some overall conclusions that can be drawn from my body of work are: there are genetic structures present in the western Indian Ocean even though the apparent lack of physical barriers. Major oceanic currents aid evolutionary dispersal patterns. A single geographic site need not be genetically homogenous or temporally stable. Island sites are genetically more homogenous than mainland sites. In conclusion, there are clear and distinct genetic structures present especially in Siganus sutor, the most targeted fish for the artisanal fishery in East Africa. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

O câncer de mama é uma das neoplasias mais freqüentes em mulheres, conseqüentemente muitos esforços têm sido feitos na tentativa de se chegar a um diagnóstico mais precoce ou até mesmo na obtenção de maiores índices de cura. Neste sentido, o presente trabalho avaliou três tipos de alterações em nível de DNA mitocondrial com a intenção de verificar relações entre a apresentação clínico-patológica do tumor, na ocasião do ato cirúrgico, a ocorrência de mutações no DNA mitocondrial do tecido neoplásico primário e linfonodos axilares metastásicos. Para tanto, 82 amostras foram analisadas quanto à presença de alterações na alça D (D310 e microsatélite (CA).) e a ocorrência de grandes deleções na região 8295-13738. Os resultados mostraram que variações na região correspondente a alça D do mtDNA, tanto na região D310 quanto no microsatélite (CA)., são freqüentes, confirmado esta região como um "hot-spot" mutacional. Por outro lado, a deleção de 4977pb foi rara (4%) nas amostras analisadas, diferindo de resultados obtidos em outros trabalhos. Uma nova deleção de 5247pb na região 8295¬13738 foram constatadas numa amostra de linfonodos metastásico. Comparação direta entre tumores primários e linfonodos metastásicos mostrou que em 53% dos linfonodos comprometidos apresentam alterações em nível de mtDNA. Estas alterações correspondem a deleções no microsatélite (CA). na região D310 e um caso de deleção de 4977pb. Considerando que as alterações em nível de mtDNA refletem a instabilidade do genoma, tal constatação pode ser tomada como indicativo do maior grau de malignidade das células presentes nos linfonodos metastásicos, e a ocorrência seleção de grupos celulares durante o processo de metastização. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-11T12:50:15Z No. of bitstreams: 1 Dissertacao Leandro Brust.pdf: 1590408 bytes, checksum: 18029852219e400f87c980617a20da0d (MD5) / Made available in DSpace on 2014-06-11T12:50:15Z (GMT). No. of bitstreams: 1 Dissertacao Leandro Brust.pdf: 1590408 bytes, checksum: 18029852219e400f87c980617a20da0d (MD5) / Breast cancer is one ofthe most ftequent diseases among women, consequently much effort has been done in order to have earlier diagnosis or even to achieve higher cure rates. In this way, the present work had assessed three kinds of alterations in mitochondrial DNA aiming to verify possible relations between clinical and pathological data at the time of surgery and the occurrence of mitochondrial DNA mutations in the primary neoplasic tissue and metastasic axillary lymphonodes. In this sense, 82 samples were analyzed with respect to alterations at the D¬loop region (D310 and microsatelite (CA)n) and occurrence of large deletions at the 8295-3738 region. The results showed that variations at the D-Ioop region of the mtDNA, both at the 0310 location and the microsatellite (CA)n are frequent, confirming this region as a mutational hot-spot. Conversely, the deletion of 4977bp was rare (4%) in the analyzed samples, differing from results obtained in other works. A new deletion of 5247 pb within the region 8295-\3738 was found in a metastatic lymphonode sample. Direct comparison between primary tumor and metastatic lymphonodes showed that 53% of the compromised lymphonodes exhibit alterations at the mtDNA level. This alterations correspond to deletions at the microsatellite (CA)n, the D31O region, and one case of 4977bp delection. Considering that alterations at mtDNA level reflect the genomic instability, the present results can be considered as indicative of the higher degree of malignance of the cells present at the metastatic lynphonodes, and the occurrence of clonal selection during the metastatic process.

Goal of this Thesis is the comparison of the Grayling˘s genetic diversity. The complete mitochondrial DNA (mtDNA) control region (1083 base pairs) and gene for cytochrom B (1076 base pairs) were sequenced in 147 European grayling (Thymallus thymallus), from 21 populations in Czech Republic, Slovakia and Bosnia. In samples, 17 haplotypes were detected with average haplotype diversity Hd = 0,5480 and genic differentiation (FST) from 0,001 to the 0.9341. The most common haplotype TH1 was detected in 92 samples. An inferred intraspecific phylogenetic tree revealed three well-separated clades. One clade was represented by Danubian haplotypes from Slovakia. Second highly divergent clade contained individuals from river Vrbas in Bosnia, and the last biggest clade included haplotypes of all three sea basins from Czech Republic. We suppose that the gained results will be later utilized to the choice of suitable conservation measures that will lead to stabilization of the state of ancestral population of this ever more rare species in our region.

O câncer de mama é uma das neoplasias mais freqüentes em mulheres, conseqüentemente muitos esforços têm sido feitos na tentativa de se chegar a um diagnóstico mais precoce ou até mesmo na obtenção de maiores índices de cura. Neste sentido, o presente trabalho avaliou três tipos de alterações em nível de DNA mitocondrial com a intenção de verificar relações entre a apresentação clínico-patológica do tumor, na ocasião do ato cirúrgico, a ocorrência de mutações no DNA mitocondrial do tecido neoplásico primário e linfonodos axilares metastásicos. Para tanto, 82 amostras foram analisadas quanto à presença de alterações na alça D (D310 e microsatélite (CA).) e a ocorrência de grandes deleções na região 8295-13738. Os resultados mostraram que variações na região correspondente a alça D do mtDNA, tanto na região D310 quanto no microsatélite (CA)., são freqüentes, confirmado esta região como um "hot-spot" mutacional. Por outro lado, a deleção de 4977pb foi rara (4%) nas amostras analisadas, diferindo de resultados obtidos em outros trabalhos. Uma nova deleção de 5247pb na região 8295¬13738 foram constatadas numa amostra de linfonodos metastásico. Comparação direta entre tumores primários e linfonodos metastásicos mostrou que em 53% dos linfonodos comprometidos apresentam alterações em nível de mtDNA. Estas alterações correspondem a deleções no microsatélite (CA). na região D310 e um caso de deleção de 4977pb. Considerando que as alterações em nível de mtDNA refletem a instabilidade do genoma, tal constatação pode ser tomada como indicativo do maior grau de malignidade das células presentes nos linfonodos metastásicos, e a ocorrência seleção de grupos celulares durante o processo de metastização. / Breast cancer is one ofthe most ftequent diseases among women, consequently much effort has been done in order to have earlier diagnosis or even to achieve higher cure rates. In this way, the present work had assessed three kinds of alterations in mitochondrial DNA aiming to verify possible relations between clinical and pathological data at the time of surgery and the occurrence of mitochondrial DNA mutations in the primary neoplasic tissue and metastasic axillary lymphonodes. In this sense, 82 samples were analyzed with respect to alterations at the D¬loop region (D310 and microsatelite (CA)n) and occurrence of large deletions at the 8295-3738 region. The results showed that variations at the D-Ioop region of the mtDNA, both at the 0310 location and the microsatellite (CA)n are frequent, confirming this region as a mutational hot-spot. Conversely, the deletion of 4977bp was rare (4%) in the analyzed samples, differing from results obtained in other works. A new deletion of 5247 pb within the region 8295-\3738 was found in a metastatic lymphonode sample. Direct comparison between primary tumor and metastatic lymphonodes showed that 53% of the compromised lymphonodes exhibit alterations at the mtDNA level. This alterations correspond to deletions at the microsatellite (CA)n, the D31O region, and one case of 4977bp delection. Considering that alterations at mtDNA level reflect the genomic instability, the present results can be considered as indicative of the higher degree of malignance of the cells present at the metastatic lynphonodes, and the occurrence of clonal selection during the metastatic process.

Ein großer Bereich der Bioinformatik ist die automatische Annotation von Genen. Hier gibt es einige Systeme, die speziell für die Annotation in mitochondrialen Genomen ausgelegt sind. So sind MOSAS1[16], MITOS2[6] und DOGMA3[20] zu erwähnen. Alle können mehr oder weniger automatisch die Proteine, tRNAs und rRNAs annotieren. Keins von ihnen kann aber die Kontrollregionen bzw. den D- Loop vorhersagen. Diese Arbeit beschäftigt sich mit dieser Lücke und versucht Möglichkeiten zu finden, die eine Automatisierung der Annotation des D-Loop zu erlauben. Als Grundlage für die verschiedenen Annäherungen und die Bewertung der Ergebnisse werden die Annotationen des D-Loops in der NCBI RefSeq 464[13] benutzt. Hier wird eine besondere Aufmerksamkeit auf die Säugetiere gelegt. Dies ist damit begründet, dass es sich dabei um eine übersichtliche Gruppe mit vielen annotierten D-Loop Regionen handelt. So werden mit den Programmen Fragrep [12] und Blast [1] versucht einzelne Merkmale aus dem D-Loop zu annotieren. Diese werden dann zu einer gesamt Annotation des D-Loops im D-Loop-Finder zusammengesetzt.

La réplication des chromosomes est altérée par les facteurs endogènes et/ou exogènes qui perturbent la progression des fourches de réplication. Les cellules doivent donc coordonner la synthèse d’ADN avec des mécanismes assurant la stabilité et le rétablissement des fourches bloquées. La recombinaison homologue (RH) est un mécanisme universel qui permet la réparation de l’ADN et participe au maintien de la réplication des chromosomes. Néanmoins, les mécanismes qui régulent la RH, notamment la RH ectopique versus la RH allélique, restent mal compris. Un autre mécanisme essentiel assurant la stabilité des génomes est l’assemblage de l’ADN néo-synthétisé autour de nucléosomes, conduisant à la constitution de fibres chromatiniennes nécessaires à l’organisation structurale du matériel génétique. Chez Saccharomyces cerevisiae, des défauts d’assemblage de la chromatine conduisent à une instabilité des fourches de réplication et augmentent le taux de RH. Sachant que les chaperonnes d’histones jouent un rôle crucial durant l’assemblage de la chromatine, j'ai décidé de me concentrer sur le rôle de la chaperonne d’histones H3-H4 appelé Chromatin Assembly Factor 1 (CAF-1) dans les mécanismes de RH, chez Schizosaccharomyces pombe. En effet, la RH est associée à une étape de synthèse de l’ADN, et peu de choses sont connues sur l’assemblage de la chromatine au cours de cette synthèse. Mes résultats ont exclu un rôle de CAF-1 dans la recombinaison allelique et le maintien de la stabilité des fourches de réplication. Par contre, CAF-1 joue un rôle important dans les mécanismes de recombinaisons ectopique et dans la formation de réarrangements chromosomiques induits par des blocages de fourches. Mes données suggèrent un modèle selon lequel CAF-1 permet la stabilisation d’intermédiaires de recombinaison précoces (D-loop), via le dépôt de nucleosomes au cours de l’extension par polymérisation de ces intermédiaires. Ainsi CAF-1 neutralise la dissociation des intermédiaires de recombinaison précoces par l’ADN helicase Rqh1. CAF-1 ferait partie d'un équilibre qui règle la stabilité/dissociation des intermédiaires de recombinaison précoces. J'ai montré que le rôle de CAF-1 dans cet équilibre a une importance toute particulière pendant la recombinaison non-allelique, révélant ainsi un nouveau niveau de régulation des mécanismes de RH par l'assemblage de la chromatine. / The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.

No description available.