<p> The mtDNA control region of the Brown-headed Cowbird (Molothrus ater) was sequenced and comparisons made at the inter- and intraspecific level. Comparison of the control region with that of another Passerine, Darwin's Finch (Geospiza scandens), revealed a high degree of both gross and fine scale structural similarity. At the nucleotide level, this comparison confirmed the presence of a hypervariable domain which evolves at rate approximately 5 times faster than coding mtDNA as well as a relatively conserved central domain which evolves at rate comparable to coding mtDNA. Both species displayed the typical avian mtDNA gene organisation previously described by Desjardins and Morais (1990, 1991) and Quinn and Wilson (in press). However, the most notable structural feature in common was the apparent deletion of the entire left hypervariable domain (CR1). At a finer scale, Conserved Sequence Block (CSB1) was perfectly conserved between cowbird and finch and Conserved Sequence Block 2 (CSB2) was 78% similar. The hypervariable right domain showed the largest degree of sequence divergence between species, 22.7%, while the central domain and phe-tRNA showed much less divergence, 6.47 and 4.41% respectively. At an intraspecific level, in 524 bases of sequence from 31 nestling cowbirds from a population at Delta, Manitoba, only 3 variable sites were detected which defined a total of 4 haplotypes. The average percent sequence divergence for this population was 0.27%. This level of variation within the cowbird population is low compared to other vertebrate populations. This relative lack of variation is largely attributable to the loss of the left hypervariable domain (CR1). The loss of CR1 will limit the control region's usefulness for high resolution population level studies but may make it a useful marker for phylogenetic studies within the class Aves.</p> / Thesis / Master of Science (MSc)

O fator de transcrição mitocondrial A (TFAM) pertence ao grupo das proteínas de alta mobilidade, apresentando um importante papel para a replicação, transcrição e estrutura/organização do DNA mitocondrial (DNAmt). O DNAmt está organizado em um complexo nucleoprotéico, chamado de nucleóide, do qual TFAM é o principal componente protéico, empacotando o DNAmt de forma análoga às histonas no DNA nuclear. Em analogia ao DNA nuclear, foi sugerido que esse empacotamento pode proteger o DNAmt do ataque de espécies oxidantes, enquanto que, por outro lado, poderia também impedir o acesso das enzimas de reparo. Este trabalho visou esclarecer qual o papel de TFAM na proteção do DNAmt e entender como TFAM influencia o reparo do DNAmt. Nossos resultados indicaram que o empacotamento do DNAmt por TFAM pode proteger o DNA da formação de lesões em condições de estresse oxidativo. Células com redução na expressão de TFAM apresentaram taxas alteradas de proliferação e uma menor viabilidade celular após o tratamento com o fotossensibilizador azul de metileno, indicando que TFAM pode contribuir para a manutenção da integridade funcional da mitocondria. A velocidade do reparo do DNAmt, em células Kd-TFAM, foi aparentemente maior, o que indicou a importância da modulação da interação de TFAM com o DNAmt para um reparo rápido e eficiente das lesões oxidadas. Portanto, TFAM desempenha um papel importante para a estabilidade genômica mitocondrial, protegendo o DNAmt dos efeitos deletérios das lesões oxidadas no estresse oxidativo, e também modulando a velocidade do reparo do DNAmt, provavelmente através de modificações/interações que permitam que as enzimas de reparo acessem as lesões no DNAmt. / The mitochondrial transcription factor A (TFAM) belongs to the high mobility group box proteins, and is essencial for replication, transcription and structure/organization of the mitochondrial DNA (mtDNA).The mtDNA is organized in a nucleoproteic complex called the nucleoid, where TFAMis the main protein component,packaging mtDNA in a manner similar to histones in the nuclear DNA. In analogy to the histone role in nuclear DNA, it was suggested that mtDNA packaging by TFAM could protect the mtDNA against oxidized lesions. On the other hand, it could also prevent the access of repair enzymes. This study aimed to understand whether TFAM plays a role in mtDNA stability through these opposing effects of protecting from damage and preventing repair. Our results indicated that TFAM protects the mtDNA against lesion formation upon oxidative stress. Cells with reduced expression of TFAM showed altered proliferation and lower cellular viability after treatment with the photoactivated dye methylene blue, indicating an important role for TFAM in maintaining mitochondrial function and cell survival. MtDNA repair rate was apparently higherin Kd-TFAM cells, which indicated the importance of modulating the interaction of TFAM with mtDNA for a quick and efficient repair of oxidized lesions. Therefore, TFAM plays an important role in maintaining mitochondrial genomic stability by protecting the mtDNA of the deleterious effects of oxidized lesions in oxidative stress, also modulating mtDNA repair, likely through modifications/interactions that modulate its DNA binding activity and access to lesions in mtDNA by DNA repair enzymes.

Sous le terme de maladies mitochondriales, on désigne des maladies multi-systémiques ou à expression tissu-spécifique dues à un déficit de la phosphorylation oxydative qui est assurée par le fonctionnement de 5 complexes protéiques enzymatiques (chaîne respiratoire) parmi lesquelles 13 sous-unités sont codées par le génome mitochondrial, les autres par le génome nucléaire. Ces pathologies recouvrent donc en pratique des maladies génétiques par mutation de l’ADN mitochondrial (ADNmt) mais aussi des maladies génétiques à hérédité mendélienne classique. Dans les cytopathies mitochondriales liées à des mutations de gènes nucléaires, il existe deux sortes de gènes (i) à effet direct correspondant à des gènes codant pour les sous-unités protéiques de la chaîne respiratoire ou leur assemblage, et (ii) à effet indirect correspondant à des gènes codant pour des protéines impliquées dans le maintien et la réplication de l'ADN mitochondrial. Des mutations dans cette dernière classe de gènes peuvent s'accompagner d'anomalies quantitatives ou qualitatives de l'ADNmt : déplétion de l'ADNmt (réduction majeure du nombre de molécules d'ADNmt) et délétions multiples.Après des dosages enzymatiques de l’activité des complexes respiratoires mitochondriaux chez les patients, le ou les types de complexes altérés sont connus. Un grand nombre de gènes mutés responsables de pathologies mitochondriales ont été identifiés, tous codant des constituants des différents complexes de la chaîne respiratoire. Ces dernières années, le groupe d’Agnès Rötig (Hôpital Necker, Paris) a identifié de nouveaux gènes grâce à une approche gènes candidats ou grâce à des tours de génome de familles consanguines de patients qui permettent de délimiter une région chromosomique portant la mutation à l’état homozygote. La validation de l’effet délétère de la mutation identifiée se fait en général en utilisant des organismes modèles d’étude comme les cellules humaines en culture ou bien la levure. Cependant, il reste un grand nombre de cas où la mutation n’a pas pu être identifiée, soit parce que le déficit de tel ou tel complexe ne met pas en jeu un des composants connus de ce complexe ou bien plusieurs complexes de la chaîne respiratoire sont déficitaires mettant en jeu, dans un grand nombre de cas, le maintien de l’ADN mitochondrial pour lequel peu de gènes sont connus.Au laboratoire d’Orsay, nous disposons de deux organismes modèles d’étude, la levure S. cerevisiae et le nématode C. elegans. La levure S. cerevisiae est l’organisme modèle de choix pour étudier les fonctions mitochondriales grâce à ses caractéristiques comme la respiration facultative, mais aussi et surtout par la puissance de sa génétique et le fait que les mitochondries peuvent être transformées. Cependant de par sa respiration facultative et sa division clonale, elle ne se prête pas facilement à des études sur la stabilité de l’ADNmt. En effet, S. cerevisiae perd très facilement son ADNmt après inactivation d’un grand nombre de gènes impliqués dans pratiquement toutes les voies de la biogenèse mitochondriale. Cette levure ne peut donc pas être utilisée de façon simple pour l’étude de la transmission de l’ADN mitochondrial. C’est pourquoi nous nous sommes alors intéressés à l’autre organisme modèle développé au laboratoire, le nématode C. elegans dont ses caractéristiques en font un excellent modèle complémentaire à la levure. / Mitochondrial respiratory chain diseases of nuclear origin represent one of the major causes of metabolic disorders. These diseases are characterized by a huge clinical and genetic heterogeneity which is a major problem in identifying the disease causing gene. Although several gene mutations have already been found in some patients or families, the disease causing gene of the majority is yet to be determined. The overall structure and gene content of the mitochondrial genome and the proteins required for mtDNA transactions are largely conserved from yeast to human offering the opportunity to use animal models to understand the molecular basis of mitochondrial dysfunctions. To expand the number of human candidate genes of mitochondrial diseases involved in mtDNA maintenance, we have developed in this study, the nematode Caenorhabditis elegans as a model organism to identify new proteins involved in mtDNA maintenance by combining RNAi and ethidium bromide exposure. We have developed a large-scale screening method of genes required for mtDNA maintenance in the worm and initially indentified four new C. elegans genes (atad-3, dnj-10, polrmt, phi-37 and immt-1) involved in mtDNA stability. The human homologs of these genes (ATAD3, DNAJA3, POLRMT and ATP5A1) can be now considered as candidate genes for patients with quantitative mtDNA deficiencies. Using our screening design we have begun to screen all the C. elegans genes encoding mitochondrial proteins. Of the 721 estimated C. elegans mitochondrial genes homologous to human genes, we have tested 185 genes and found that 41 genes are required for the maintenance of the mitochondrial genome in post mitotic cells. These genes fall into three main functional categories of metabolism, protein synthesis and oxidative phosphorylation. Finally, in this study, we investigated the reversibility of mtDNA depletion with drugs to counteract POLG dificiency. Three molecules, Chlorhexidine, Resveratrol and Bezafibrate, have been tested to restore normal mtDNA content and worm life cycle. These experiments hold promise for future work using C. elegans as a pharmacological model for mitochondrial diseases.Altogether, the data generated in this work is a starting point for promising advances in the mitochondrial field, showing the relevance of the nematode as a model organism to study fundamental processes as well as human health research.

O fator de transcrição mitocondrial A (TFAM) pertence ao grupo das proteínas de alta mobilidade, apresentando um importante papel para a replicação, transcrição e estrutura/organização do DNA mitocondrial (DNAmt). O DNAmt está organizado em um complexo nucleoprotéico, chamado de nucleóide, do qual TFAM é o principal componente protéico, empacotando o DNAmt de forma análoga às histonas no DNA nuclear. Em analogia ao DNA nuclear, foi sugerido que esse empacotamento pode proteger o DNAmt do ataque de espécies oxidantes, enquanto que, por outro lado, poderia também impedir o acesso das enzimas de reparo. Este trabalho visou esclarecer qual o papel de TFAM na proteção do DNAmt e entender como TFAM influencia o reparo do DNAmt. Nossos resultados indicaram que o empacotamento do DNAmt por TFAM pode proteger o DNA da formação de lesões em condições de estresse oxidativo. Células com redução na expressão de TFAM apresentaram taxas alteradas de proliferação e uma menor viabilidade celular após o tratamento com o fotossensibilizador azul de metileno, indicando que TFAM pode contribuir para a manutenção da integridade funcional da mitocondria. A velocidade do reparo do DNAmt, em células Kd-TFAM, foi aparentemente maior, o que indicou a importância da modulação da interação de TFAM com o DNAmt para um reparo rápido e eficiente das lesões oxidadas. Portanto, TFAM desempenha um papel importante para a estabilidade genômica mitocondrial, protegendo o DNAmt dos efeitos deletérios das lesões oxidadas no estresse oxidativo, e também modulando a velocidade do reparo do DNAmt, provavelmente através de modificações/interações que permitam que as enzimas de reparo acessem as lesões no DNAmt. / The mitochondrial transcription factor A (TFAM) belongs to the high mobility group box proteins, and is essencial for replication, transcription and structure/organization of the mitochondrial DNA (mtDNA).The mtDNA is organized in a nucleoproteic complex called the nucleoid, where TFAMis the main protein component,packaging mtDNA in a manner similar to histones in the nuclear DNA. In analogy to the histone role in nuclear DNA, it was suggested that mtDNA packaging by TFAM could protect the mtDNA against oxidized lesions. On the other hand, it could also prevent the access of repair enzymes. This study aimed to understand whether TFAM plays a role in mtDNA stability through these opposing effects of protecting from damage and preventing repair. Our results indicated that TFAM protects the mtDNA against lesion formation upon oxidative stress. Cells with reduced expression of TFAM showed altered proliferation and lower cellular viability after treatment with the photoactivated dye methylene blue, indicating an important role for TFAM in maintaining mitochondrial function and cell survival. MtDNA repair rate was apparently higherin Kd-TFAM cells, which indicated the importance of modulating the interaction of TFAM with mtDNA for a quick and efficient repair of oxidized lesions. Therefore, TFAM plays an important role in maintaining mitochondrial genomic stability by protecting the mtDNA of the deleterious effects of oxidized lesions in oxidative stress, also modulating mtDNA repair, likely through modifications/interactions that modulate its DNA binding activity and access to lesions in mtDNA by DNA repair enzymes.

Made available in DSpace on 2016-12-08T16:24:09Z (GMT). No. of bitstreams: 1 PGCA11MA078.pdf: 637847 bytes, checksum: a11da742649dfac9bb787886f73c49bf (MD5) Previous issue date: 2011-03-03 / The Brazilian caipira chickens are the result of random mating between different chicken breeds found in Brazil. They are characterized by their hardiness, disease resistance and the adverse conditions of climate and nutrition. In the 80, a change in consumption habits valued natural products, making theses chickens an alternative of great commercial value. However, its low productivity of this prevented the competition with the chicken industry. The output was the development of so-called caipira lines, chickens that blend the hardiness and resistance of native birds with the productivity of Brazilian poultry industry. In Brazil some of these commercial caipira chicken lines were developed, such as Paraíso Pedrês (beef) and Rubro Negra (posture). This study aimed to investigate the genetic variability nuclear and nonnuclear of Brazilian commercial caipira chickens lines through the analysis of ten microsatellite loci and the control region, D-loop, mitochondrial DNA (mtDNA). It was collected blood samples from 92 birds of Paraíso Pedrês lines (42) and Rubro Negrea (50). It was used the polymerase chain reaction technique (PCR) to amplify the samples and the amplified products were subjected to electrophoresis on an automated sequencer ABI 3130 DNA Genetic Analyzer. The number of alleles ranged from 3 (LEI0254) to 32 (LEI0212) for Paraíso Pedrês (PP) lines and 4 (LEI0254) to 31 (LEI0212) for Rubro Negra (RN) lines. The number of alleles per locus was 13,40 and 13,10 for PP and RN lines, respectively. Average expected heterozygosity was 0,824 for PP and 0,604 for RN. In the analysis of mtDNA, 100% of birds had PP haplotype 4, from Europe. In the RN line, haplotype 4 was found in 60% of the samples, the haplotype 3c in 10% and 30% in 3d haplotype, the haplotype 3c e 3d, from Asia. The results indicate that Brazilian lines of chickens examined, have a higher genetic variability observed in the typical commercial lines of chickens and the same was found for non-commercial poultry. For the formation of RN lines was used mostly birds of European origin and some of Asian origin. And for the composition of the PP lines were used only birds of European origin, at least as regards the maternal line / As galinhas caipiras brasileiras são o resultado de cruzamentos aleatórios entre diversas raças de galinhas encontradas no Brasil. Caracterizam-se pela sua rusticidade, resistência a doenças e as condições adversas de clima e alimentação. Nos anos 80, uma mudança nos hábitos de consumo valorizou os produtos naturais, tornando as galinhas caipiras alternativas de grande valor comercial. Porém, sua baixa produtividade inviabilizou a competição desta com a galinha industrial. A saída foi o desenvolvimento das chamadas linhagens caipiras, galinhas que mesclam a rusticidade e resistência das aves caipiras brasileiras com a produtividade das aves industriais. No Brasil foram desenvolvidas algumas destas linhagens, como a Paraíso Pedrês (corte) e a Rubro Negra (postura). Este trabalho teve como objetivo investigar a variabilidade genética nuclear e não nuclear de linhagens de galinhas caipiras brasileiras através da análise de dez lócus de microssatélites e da região controladora, alça-D, do DNA mitocondrial (mtDNA). Foram coletadas amostras de sangue de 92 aves das linhagens Paraíso Pedrês (42) e Rubro Negra (50). Foi utilizada a técnica de reação em cadeia pela polimerase (PCR) para a amplificação das amostras e os produtos amplificados foram submetidos à eletroforese em um sequenciador automático ABI 3130 DNA Genetic Analyser. O número de alelos variou de 3 (LEI0254) a 32 (LEI0212) para a linhagem Paraíso Pedrês (PP), e 4 (LEI0254) a 31 (LEI0212) para a linhagem Rubro Negra (RN). A média de alelos por loco foi de 13,40 e 13,10 para a linhagem PP e RN, respectivamente. A heterozigosidade média esperada foi de 0,824 para PP e 0,604 para RN. Na análise do mtDNA, 100% das aves PP possuíam o haplótipo 4, de origem Européia. Na linhagem RN, o haplótipo 4 foi encontrado em 60% das amostras, o haplótipo 3c em 10% e o haplótipo 3d em 30%, os haplótipos 3c e 3d são de origem Asiática. Os resultados obtidos indicam que as linhagens de galinhas caipiras brasileiras analisadas, apresentam uma variabilidade genética superior às observadas em linhagens comerciais típicas de galinhas e semelhante à observada em aves não comerciais. Para a formação da linhagem RN foi utilizado na sua maioria aves de origem Européia e algumas de origem Asiática. E, para a composição da linhagem PP, foi utilizado somente aves de origem Européia, pelo menos no que se refere à linhagem materna

Made available in DSpace on 2016-12-08T16:24:15Z (GMT). No. of bitstreams: 1 PGCA13MA117.pdf: 974615 bytes, checksum: 1adc6a24dc175e42ba61d10eb2f142ea (MD5) Previous issue date: 2013-02-28 / The domestic chicken (Gallus gallus domesticus) was originated by the jungle red flow chicken (Gallus gallus) and the bankiva chicken (Gallus gallus bankiva). Since the domestication, around 7000 years ago in the Asian Southeast, the chicken came with the human migration, and then scattered to the entire world and arrived to Brazil around 1500 BC. In Brazil has came breeds from Europe and Asia, and they were let in freedom and they crossed at random breeding generating the chicken as know as Brazilian caipira chicken. With development of the national chicken producing the caipira s chickens were forgotten in countryside in third world countries, to be considered less productive. However, with the growing consumption of product considered healthier, this chicken was recovered. In additional, to be more rustic and disease resistance, because their high genetic variability, these chickens are considered an important source for alleles which can be use in the animal genetic improvement. The mitochondrial DNA (mtDNA) is characterized by had an exclusively maternal inheritance and the most used method amplify the mtDNA control region (D-loop), which has a high genetic variability. This study aimed to investigate the origin and composition of the maternal lines on these lineages. . We collected blood samples from 105 Brazilian blue-egg caipira s chickens. The fragments were amplified by PCR, sequenced, and analyzed by the MEGA 4.0 software. As result, 89,52% of the chicken belong to the clade A, 7,62% to C e 2,86% to E, which means that participated animals most from Asian and few from European origin, in the maternal lineages. The results indicate that these strains analyzed of Brazilian chickens, have a higher genetic variability so that observed in non-commercial poultry and conserve a strong founder effect / A galinha doméstica (Gallus gallus domesticus) é originária da galinha vermelha do mato (Gallus gallus) e galinha bankiva (Gallus gallus bankiva). Desde a sua domesticação, que ocorreu por volta de 7000 anos atrás no Sudeste asiático, a galinha acompanhou a migração humana, e assim se espalhou pelo mundo inteiro, chegando ao Brasil por volta do ano de 1500. No Brasil chegaram aves oriundas da Europa e da Ásia, e aqui foram deixadas em liberdade e cruzaram de forma aleatória gerando o que hoje se conhece como galinha caipira. Com o desenvolvimento da avicultura, estas aves foram esquecidas nos quintais de casa, por serem consideradas menos produtivas. No entanto, com o crescente aumento no consumo de produtos considerados naturais, esta ave voltou ao mercado. Por ser mais rústica e resistente a doenças, em virtude de sua variabilidade genética alta, estas aves também são consideradas uma importante fonte de alelos que podem ser utilizados no melhoramento animal. O DNA mitocondrial (mtDNA) caracteriza-se por ser de exclusiva herança materna, sendo o método mais utilizado consiste em amplificar a região controle (alça-D), que possui grande variabilidade genética. Desta forma, este trabalho tem como objetivo determinar a origem e composição das linhas maternas nestas linhagens. Foram coletadas amostras de sangue de 105 galinhas caipiras de ovos azuis oriundas do município de Dois Lajeados-RS. Os fragmentos foram amplificados pela PCR, sequenciados, e analisados pelo software MEGA 4.0. Como resultado, 89,52% das aves pertencem ao haplogrupo A, 7,62% ao C e 2,86% ao E. Para a composição desta ave foram utilizados em sua maioria aves de origem asiática e um pouco de origem europeia, em sua linhagem materna. Os resultados obtidos indicam que estas aves analisadas, apresentam uma variabilidade genética semelhante a aves não comerciais e conservam ainda um efeito fundador muito forte

The genus Rousettus has distributional pattern unique among fruitbats comprising both Asia and Africa and reaching northern distributional limits of the family in Persia, Arabia and Mediterranean basin. This could be ascribed to the ability of echolocation, consequent cave dwelling, and presumably other site-specific adaptations, which enabled dispersal independent of forest block and surviving in Mediterranean type of climate. Using fastly evolving mitochondrial marker, we aimed to assess genetic variability, its geographic distribution and demography of northern populations of the Egyptian fruitbat (Rousettus aegyptiacus). Mitochondrial network indicates deep genetic divergence between disjunct Mediterranean and eastern African parts of the range. Basal position of Sinaic and Jordanian haplotypes within northern clade indicate important role of these regions in colonization of eastern Mediterranean. Generally, the northern haplogroup is moderately diversified with partial geographic localization of particular haplotypes. Significant isolation by distance pattern suggests relatively pronounced site fidelity of particular colonies, at least in terms of maternal gene flow. Landscape genetics analyses indicate discontinuities in distribution of mitochondrial genetic variability, in some cases correlating with...

Der Nachweis von Fäkalindikatorbakterien (wie z. B. E. coli oder intestinale Enterokokken) gibt zwar einen Hinweis auf eine fäkale Belastung im Gewässer, erlaubt jedoch keinen Rückschluss auf den Ursprung. Im Rahmen dieser Dissertation wurden verschiedene Methoden zur Identifizierung von fäkalen Eintragsquellen (Microbial Soruce Tracking, MST) geprüft. Hierzu zählen Kultur-basierte Verfahren, wie der Nachweis von Toxingenen in E. coli-Isolaten, und Kultur-unabhängige Methoden wie die Denaturierende-Gradienten-Gelelektrophorese oder der Nachweis von DNA-Abschnitten, die spezifisch für menschlichen oder tierischen Kot sind. Die Untersuchung von E. coli-Isolaten aus der aquatischen Umwelt auf Toxingene erwies sich in Hinblick auf die Identifizierung von fäkalen Eintragsquellen als nicht zielführend. Problematisch war neben dem hohen Zeitaufwand vor allem das seltene Vorkommen dieser Virulenzfaktoren im untersuchten Gewässer. Enteropathogene E. coli-Bakterien werden nur von infizierten Wirten abgegeben, daher ist der Nachweis der Virulenzgene in Gewässern stark eingeschränkt. Im nächsten Schritt wurden Datenbank-unabhängige Verfahren für die Untersuchungen im Einzugsgebietsmaßstab etabliert. Methoden zur spezifischen Detektion von fäkalen Einträgen durch Menschen, Rinder (Wiederkäuer), Schweine, Hunde, Schafe, Hühner und Pferde standen anschließend für die Identifizierung von fäkalen Einträgen in verschiedenen Einzugsgebieten zur Verfügung. Die Ergebnisse der Untersuchungen belegen, dass mit den etablierten MST-Verfahren sehr empfindliche und spezifische Werkzeuge für die Identifizierung von fäkalen Einträgen im Einzugsgebietsmaßstab zur Verfügung stehen. Durch die Untersuchung von Wasserproben auf das Vorkommen von MST-Markern konnten Aussagen über Auftreten und Stärke fäkaler Belastungen in städtisch und ländlich geprägten Einzugsgebieten gemacht werden. Kontaminationsquellen wurden identifiziert und Vorschläge für gezielte Management¬maßnahmen im Einzugsgebiet abgeleitet. Des Weiteren wurde die MST-Verfahren erfolgreich dazu genutzt Erkenntnisse über den Ursprung von Antibiotikaresistenzgenen im Tai-See (China) zu gewinnen. Die Ergebnisse dieser Arbeit zeigen, dass die MST-Methoden basierend auf wirtsspezifischen Markern für den Einsatz in der Praxis geeignet sind. Zusammen mit der Betrachtung der örtlichen Gegebenheiten ist MST ein hilfreiches und kostengünstiges Werkzeug bei der Ursachen-forschung und der Ermittlung der Herkunft fäkaler Belastungen in der aquatischen Umwelt. Damit trägt es wesentlich zu einer zielorientierten Bewirtschaftung des Gewässers bei.:ABKÜRZUNGSVERZEICHNIS ABBILDUNGSVERZEICHNIS TABELLENVERZEICHNIS FORMELVERZEICHNIS 1. HINTERGRUND 1.1 INDIKATORORGANISMEN 1.2 SOURCE TRACKING-METHODEN 1.2.1 Datenbank-abhängige Methoden 1.2.2 Datenbank-unabhängige Methoden 1.2.3 Source Tracking mit chemischen Substanzen 1.3 ANTIBIOTIKARESISTENZEN IN DER UMWELT 2. ZIELSETZUNG DER ARBEIT 3. MATERIAL UND METHODEN 3.1 MONITORING MIKROBIOLOGISCHER PARAMETER – KULTURVERFAHREN 3.1.1 Coliforme Bakterien und Escherichia coli 3.1.2 Enterokokken 3.1.3 Clostridium perfringens-Sporen 3.2 MOLEKULARBIOLOGISCHE ANALYTIK 3.2.1 PCR-Untersuchung von E. coli-Isolaten auf Virulenzgene 3.2.2 Molekularbiologische Untersuchung von Wasserproben 3.3 UNTERSUCHUNGEN ZUR ERFASSUNG DER CHARAKTERISTIKA DER KULTUR-UNABHÄNGIGEN MICROBIAL SOURCE TRACKING-VERFAHREN 3.3.1 Nachweisempfindlichkeit 3.3.2 Spezifität und Sensitivität 3.3.3 Charakterisierung möglicher Eintragsquellen 3.3.4 Untersuchungen zur Stabilität von Microbial Source Tracking-Markern 3.4 STATISTISCHE AUSWERTUNG 4. BESCHREIBUNG DER UNTERSUCHUNGSGEBIETE 4.1 RHEIN 4.2 GALLUSQUELLE 4.3 EINZUGSGEBIETE DER BERLINER WASSERBETRIEBE 4.3.1 Wasserwerk Tiefwerder 4.3.2 Wasserwerk Kaulsdorf 4.4 EINZUGSGEBIETE DER WSW ENERGIE & WASSER AG 4.4.1 Talsperre Herbringhausen 4.4.2 Talsperre Kerspe 4.5 TAI-SEE 4.6 VERGLEICH DER UNTERSUCHUNGSGEBIETE 61 5. ERGEBNISSE UND DISKUSSION 63 5.1 NACHWEIS VON VIRULENZGENEN ALS MICROBIAL SOURCE TRACKING-WERKZEUG 5.2 DATENBANK-UNABHÄNGIGE UND KULTUR-UNABHÄNGIGE MICROBIAL SOURCE TRACKING-VERFAHREN 5.2.1 Etablierung Datenbank-unabhängiger und Kultur-unabhängiger Microbial Source Tracking-Verfahren 5.2.2 Nachweisempfindlichkeit 5.2.3 Spezifität und Sensitivität der Marker 5.2.4 Charakterisierung möglicher Eintragsquellen 5.2.5 Stabilität von Microbial Source Tracking-Markern 5.3 UNTERSUCHUNG IM EINZUGSGEBIETSMAßSTAB 5.3.1 Gallusquelle 5.3.2 Wasserwerke Tiefwerder und Kaulsdorf 5.3.3 Talsperren Herbringhausen und Kerspe 5.4 EINSATZ VON MICROBIAL SOURCE TRACKING-METHODEN ZUR IDENTIFIZIERUNG DER HERKUNFT VON ANTIBIOTIKARESISTENZEN 6. ZUSAMMENFASSUNG UND AUSBLICK 7. EIGENE VERÖFFENTLICHUNGEN 8. FINANZIELLE FÖRDERUNG 9. LITERATURVERZEICHNIS 10. ANHANG / Although evidence of fecal indicator bacteria (such as E. coli or intestinal enterococci) indicates fecal contamination in the aquatic environment, it does not allow any conclusion regarding the origin of the pollution. With the use of so-called microbial source tracking methods it is possible to trace the origin of such contaminations. In this dissertation, various microbial source tracking methods were tested. These include culture-based methods such as the detection of toxin genes in E. coli isolates, and culture-independent methods such as denaturing gradient gel electrophoresis or the detection of defined DNA sections specific for human or animal feces using quantitative real-time PCR. Analysis of E. coli isolates from the aquatic environment on toxin genes proved to be ineffective in identifying fecal sources of input. In addition to the high effort of time, the problem was the rare occurrence of these virulence factors in the examined water. Enteropathogenic E. coli bacteria carry virulence genes which are only released from infected hosts. Therefore, the detection of virulence genes in weakly to moderately contaminated waters is severely limited. In the next step, culture-independent PCR procedures were established. Afterwards, methods for the specific detection of fecal entries by humans, cattle (ruminants), pigs, dogs, sheep, chickens and horses were available for the identification of fecal entries in various catchment areas. The results of these investigations show that established MST methods provide very sensitive and specific tools for the identification of fecal entries at the catchment scale. Investigation using culture-independent methods provided information on the origin and severity of fecal pollutions in urban and rural catchment areas. Origins of contaminations were identified and concrete recommendations for management action plans in the catchment area were derived. Overall, the results show the potential and the suitability for practical application of molecular biological microbial source tracking methods. Furthermore, the MST methods have been successfully used to gain knowledge about the origin of antibiotic resistance genes in Tai Lake (China). The results of this thesis show that MST methods based on host-specific markers are suitable for practical use. Together with the consideration of local conditions, MST is a helpful and cost-effective tool for causal research and the determination of the origin of fecal contamination in the aquatic environment. Thus it contributes significantly to a goal-oriented management of the water body.:ABKÜRZUNGSVERZEICHNIS ABBILDUNGSVERZEICHNIS TABELLENVERZEICHNIS FORMELVERZEICHNIS 1. HINTERGRUND 1.1 INDIKATORORGANISMEN 1.2 SOURCE TRACKING-METHODEN 1.2.1 Datenbank-abhängige Methoden 1.2.2 Datenbank-unabhängige Methoden 1.2.3 Source Tracking mit chemischen Substanzen 1.3 ANTIBIOTIKARESISTENZEN IN DER UMWELT 2. ZIELSETZUNG DER ARBEIT 3. MATERIAL UND METHODEN 3.1 MONITORING MIKROBIOLOGISCHER PARAMETER – KULTURVERFAHREN 3.1.1 Coliforme Bakterien und Escherichia coli 3.1.2 Enterokokken 3.1.3 Clostridium perfringens-Sporen 3.2 MOLEKULARBIOLOGISCHE ANALYTIK 3.2.1 PCR-Untersuchung von E. coli-Isolaten auf Virulenzgene 3.2.2 Molekularbiologische Untersuchung von Wasserproben 3.3 UNTERSUCHUNGEN ZUR ERFASSUNG DER CHARAKTERISTIKA DER KULTUR-UNABHÄNGIGEN MICROBIAL SOURCE TRACKING-VERFAHREN 3.3.1 Nachweisempfindlichkeit 3.3.2 Spezifität und Sensitivität 3.3.3 Charakterisierung möglicher Eintragsquellen 3.3.4 Untersuchungen zur Stabilität von Microbial Source Tracking-Markern 3.4 STATISTISCHE AUSWERTUNG 4. BESCHREIBUNG DER UNTERSUCHUNGSGEBIETE 4.1 RHEIN 4.2 GALLUSQUELLE 4.3 EINZUGSGEBIETE DER BERLINER WASSERBETRIEBE 4.3.1 Wasserwerk Tiefwerder 4.3.2 Wasserwerk Kaulsdorf 4.4 EINZUGSGEBIETE DER WSW ENERGIE & WASSER AG 4.4.1 Talsperre Herbringhausen 4.4.2 Talsperre Kerspe 4.5 TAI-SEE 4.6 VERGLEICH DER UNTERSUCHUNGSGEBIETE 61 5. ERGEBNISSE UND DISKUSSION 63 5.1 NACHWEIS VON VIRULENZGENEN ALS MICROBIAL SOURCE TRACKING-WERKZEUG 5.2 DATENBANK-UNABHÄNGIGE UND KULTUR-UNABHÄNGIGE MICROBIAL SOURCE TRACKING-VERFAHREN 5.2.1 Etablierung Datenbank-unabhängiger und Kultur-unabhängiger Microbial Source Tracking-Verfahren 5.2.2 Nachweisempfindlichkeit 5.2.3 Spezifität und Sensitivität der Marker 5.2.4 Charakterisierung möglicher Eintragsquellen 5.2.5 Stabilität von Microbial Source Tracking-Markern 5.3 UNTERSUCHUNG IM EINZUGSGEBIETSMAßSTAB 5.3.1 Gallusquelle 5.3.2 Wasserwerke Tiefwerder und Kaulsdorf 5.3.3 Talsperren Herbringhausen und Kerspe 5.4 EINSATZ VON MICROBIAL SOURCE TRACKING-METHODEN ZUR IDENTIFIZIERUNG DER HERKUNFT VON ANTIBIOTIKARESISTENZEN 6. ZUSAMMENFASSUNG UND AUSBLICK 7. EIGENE VERÖFFENTLICHUNGEN 8. FINANZIELLE FÖRDERUNG 9. LITERATURVERZEICHNIS 10. ANHANG

This doctoral thesis is submitted in the form of science publications with impact factor and presents human biological variation in two different ways. The first part is the study of phenotype variation focused on the degree of limb bones bilateral asymmetry. Besides genetic and hormonal factors asymmetry may develop as a response to biomechanical and to environmental factors influencing the individual/population. Therefore this trait can be considered as a measure of person's living conditions, health or environmental stress of different origin or exa- mine the effects of behavioral distinctions as sexual division of labor and diffe- rences in subsistence strategy. The submitted publication (Kujanová et al., 2008) is based on samples of two diachronic populations (medieval and recent) from Bohe- mia with different expected levels of health/environmental stress. The study is fo- cused especially on these aspects that may be indicative of various stresses, such as malnutrition or other nonspecific factors influencing health of persons living in the studied populations. According to the results we propose that bilateral asymmetry observed in the diachronic populations showed some differences supporting the theory that the medieval population was not subjected to as highly stressful condi- tions as the...

O talha-mar Rynchops niger (Rynchopinae) e o trinta-réis-grande Phaetusa simplex (Sterninae), são aves aquáticas migratórias que se reproduzem simultaneamente em muitas praias fluviais da América do Sul. A taxonomia e a sistemática destas subfamílias foram objetos de poucos estudos. Além disso, possuem subespécies cuja delimitação e a caracterização ainda são confusas, além do que, uma revisão rigorosa da validade destes táxons nunca foi feita. E, como consequência dos poucos estudos, existe uma enorme imprecisão sobre a real área de distribuição de cada táxon, não havendo muita informação sobre os locais para onde se movimentam após o período reprodutivo. Assim sendo, este trabalho teve como objetivo caracterizar geneticamente e revisar a taxonomia destas duas espécies polítipas, definindo os seus táxons válidos e distribuição. Para tal, foram utilizados caracteres morfológicos (de plumagem e morfométricos), sequências do gene mitocondrial ND2 e de marcadores associados a sítios de restrição (ddRADseq), a fim de estimar a variação intra e interpopulacional, o fluxo gênico e a estrutura genética; visando também entender o padrão de migração destes táxons na América do Sul e a influência dos rios na taxonomia e na história evolutiva destas aves. De acordo com os resultados conclui-se: P. simplex passa a ser considerado um táxon monotípico, pois não é possível separar as subespécies entre si, nem morfologicamente e nem geneticamente. Apesar das variações genéticas entre as três subespécies de R. niger não serem significativas, estas continuam a ser consideradas como subespécies válidas, pois puderam ser plenamente diagnosticáveis quanto aos caracteres de plumagem e de distribuição. Não existem variações genéticas significativas entre as populações. As populações podem estar passando por um processo de expansão recente ou seleção positiva; ou podem estar se comportando como uma metapopulação. Os grandes rios sul-americanos, juntamente com o ciclo sazonal de precipitação da América do Sul (que altera a dinâmica destes rios), influenciam diretamente na distribuição, e, consequentemente, na evolução das aves aqui analisadas. Neste caso, os rios funcionam como vias de contato (e não como barreiras) entre os indivíduos, contribuindo para o intenso fluxo gênico dos táxons aqui apresentados / The Black Skimmer (Rynchops niger) and the Large-billed Tern (Phaetusa simplex) are migratory waterbirds that breed simultaneously on river beaches throughout South America. Few studies have been conducted on the taxonomy and systematics of these polytypic species and the delimitation and validity of each taxa described has never been studied in detail. As a result, the geographical distribution of both species is poorly understood and there is little information about the whereabouts of their non-breeding grounds. Therefore, the purpose of this study was to characterize genetically and revise the taxonomy and distribution of these two polytypic species. For this it was integrated morphological characters (plumage and morphometrics), mitochondrial sequences (ND2), and single nucleotide polymorphisms (SNPs) inferred from double digestion restriction associated DNA sequencing (ddRADseq) to estimate: 1) intra- and inter-populational variation, 2) gene flow, 3) population genetic structure, 4) migration patterns of these taxa within South America, and 5) assess the influence of rivers on the taxonomy and evolutionary history of these birds. The results lead to the following conclusions: P. simplex should now be considered a monotypic taxon because currently recognized subspecies are neither morphologically nor genetically diagnosable. Although genetic variation between the three subspecies currently recognized in R. niger is not significant, these taxa continue to be considered as valid subspecies because they are fully diagnosable in plumage characters and distributional patterns. There are no significant genetic variation between the populations of both species (R. niger and P. simplex). Populations may be undergoing a process of recent expansion or positive selection or they may be behaving like a metapopulation. Main South American rivers, together with the seasonal precipitation cycles of South America (which changes the dynamics of these rivers), have direct influence on the distribution, and, consequently, on the evolution of these birds. In this case, the rivers function as pathways of contact (and not as barriers) between individuals, contributing to the intense gene flow between these taxa