La première partie de nos travaux a consisté au séquençage de génomes complets de trois souches pathogènes et de trois souches de portage de Staphylococcus lugdunensis pour les comparer aux 15 génomes complets disponibles sur NCBI. Aucun déterminant génétique associé au contexte de virulence ou de portage de S. lugdunensis n’a été identifié. Cependant, nous avons mis en évidence la présence d’éléments génétiques mobiles et des variations dépendantes des complexes clonaux,définis par MultiLocus Sequence Typing, au sein de loci potentiellement associés à la virulence. Des variations ont été observées dans un locus homologue à celui de Staphylococcus aureus codant le système de sécrétion Ess/type VII (SST7). Nous avons mis en évidence huit organisations génétiques chez cette espèce présentant pourtant une structure de population clonale. La seconde partie de nos travaux a consisté à la caractérisation phénotypique et moléculaire du SST7 chez S. lugdunensis par la formation d’un mutant de délétion du gène essC codant une protéine essentielle à la sécrétion. Nos résultats suggèrent que le SST7 serait impliqué dans la translocation de protéines prédites in silico comme impliquées dans la virulence. Néanmoins, dans des modèles de cytotoxicité cellulaire et d’infection du nématode Caenorhabditis elegans, aucune atténuation de la virulence n’a été observée chez la souche mutante malgré une perte de sa capacité à lyser les erythrocytes, comparativement à la souche sauvage. Nos travaux ont également permis de développer et d’évaluer le pouvoir discriminant de trois nouvelles méthodes de typage constituant des outils très prometteurs pour l’épidémiologie moléculaire des infections à S. lugdunensis. / The first part of this study consisted in whole genome sequencing of three pathogenic and three carriage strains of S. lugdunensis and comparison with the 15 genomes available in the NCBI. No genetic determinant was associated to the pathogenic or carriage context. However, we have highlighted the presence of mobile genetic elements and MultiLocus Sequence Typing clonal complex dependent variations within loci potentially associated with virulence. Variations wereobserved in the ess locus homologous to that of Staphylococcus aureus encoding the type VII secretion system (T7SS). We showed eight genetic organizations in this species with a clonal population structure. The second part of our work consisted in a phenotypic and molecular characterization of T7SS in S. lugdunensis by construction of a deletion essC gene mutant. This gene encodes a protein requiredfor protein secretion. Our results suggest that T7SS could be involved in translocation of proteins predicted as implicated in virulence in silico. Nevertheless, no virulence attenuation was observed in cells cytotoxicity assay and Caenorhabditis elegans virulence assays between wild-type and mutant strains which yet has lost the ability to lyse erythrocytes. We also developed and evaluated discriminating power of three new typing methods, which are very promising tools for the molecular epidemiology of S. lugdunensis infections.

• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. • Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages. • This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. • The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

<p>Kratki uzastopni ponovci predstavljaju klasu mikrosatelitskih segmenata DNK, rasprostranjenih &scaron;irom genoma čoveka. Građeni su od uzastopno ponavljajućih sekvenci dužine 2-6 parova nukleotida. Zahvaljujući različitom broju ponavljanja repetitivne jedinice, većina mikrosatelitskih markera pokazuje visok stepen polimorfizma dužine, koji je moguće ispitati primenom tehnike lančane reakcije polimeraze. Pored utvrđivanja spornih srodničkih odnosa, analiza X hromozom mikrosatelitskih markera može se uspe&scaron;no koristiti i u oblastima kriminalistike, humane identifikacije, populaciono-genetičkim i demografskim istraživanjima i dr. Cilj istraživanja je izrada populacione studije, iz koje će se izračunati broj i frekvencija alela, struktura i frekvencija haplotipova, utvrditi vrednosti relevantnih statističkih parametara, oceniti mogućnost primene analize X-STR markera u slučajevima iz oblasti medicinske kriminalistike, humane identifikacije i ve&scaron;tačenja spornih srodničkih odnosa u populaciji Vojvodine. Istraživanjem je obuhvaćeno 200 odraslih, međusobno nesrodnih osoba. Izolacija DNK materijala iz krvnih mrlja vr&scaron;ena je Chelex metodom, a amplifikacija dobijenih uzoraka DNK metodom PCR, uz kori&scaron;ćenje komercijalnog Mentype&reg; Argus X-12 PCR Amplification Kit &ndash; a. Razdvajanje i detekcija dobijenih fragmenata izvr&scaron;eno je kapilarnom elektroforezom GeneScan i Genotyper programom. Statististička obrada rezultata izvr&scaron;ena je pomoću Arlequin i GENEPOP programa. Za vizuelizaciju interpopulacionih genetičkih odnosa, upotrebljen je program POPTREE2 i koordinatna analiza (Principal Coordinate Analysis - PCoA). Dobijeni rezultati ukazuju da se analiza ispitivanih X-STR markera može uspe&scaron;no primeniti u slučajevima iz oblasti medicinske kriminalistike, humane identifikacije i ve&scaron;tačenja spornih srodničkih odnosa u populaciji Vojvodine, kao i da mogu poslužiti kao osnova za dalja istraživanja u&nbsp; populacionogenetičkim, antropolo&scaron;kim, demografskim i drugim oblastima.</p> / <p>Short tandem repeats (STR) represent a class of microsatellites, widely spread throughout the human genome, consisting of tandemly repeated sequences of 2-6 bp. Related to variation in the number of repeat unit displayed, most of microsatellites show a high degree of length polymorphism, investigated by the PCR techniques. The aim of this research is to create a population study, which will be used to calculate allele and haplotype frequencies, determine the value of relevant statistical parameters and assess the possibility of applying X-STR markers analysis in the fields of forensics, human identification and kinship testing. The study included 200 unrelated adults. DNA isolation was performed by Chelex method and DNA amplification by PCR, using commercial Mentype Argus X-12 PCR Amplification Kit. Separation and detection of fragments was obtained by capillary electrophoresis using Gene Scanand Genotyper program. Statistical analysis of the result was performed using Arlequin and GENEPOP program. For visualization of inter population genetic distances POPTREE2 program and coordinate analysis (PCoA) was used. The results show that the analysis of X-STR markers can be successfully applied in the field of forensics, human identification and kinship testing in the population of Vojvodina, as well as to serve as a basis for further research in population genetic, anthropological, demographic and other scientific areas.</p>

<p>Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. iii The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines.</p>

<p>Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. iii The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines.</p>

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes. / QC 20100819

Introduction: Colonization of the anogenital tract of pregnant women by Group B Streptococcus (GBS) is the main risk factor for early-onset neonatal sepsis. Identification of maternal colonization allows antimicrobial prophylaxis and prevention of vertical transmission. Objectives: To identify the genetic diversity and phenotypic characteristics of GBS using molecular biology techniques and culture in specific medium, and the epidemiological aspects of pregnant women colonized by this bacterium. Methods: It was performed a cross-sectional study, anogenital samples of 316 pregnant women were collected between 35 and 37 weeks and submited to culture in specific medium, antimicrobial susceptibility test, multiplex PCR, multi locus sequence typing (MLST) and multi locus variable number of tandem repeat analysis (MLVA). The epidemiological aspects of colonized and non-colonized pregnant women were also investigated. Results: It was obtained a prevalence of 36.4% by culture and 38.6% by PCR. Multiplex PCR had sensitivity of 100%, specificity of 96.5%, positive predictive value of 94.3% and negative predictive value of 100%. The most common serotypes were Ia, V, II and III. Resistance genes were identified in 34 samples. Sensitivity to penicillin was universal, 24.3% presented resistance to erythromycin and 14.8% to clindamycin. Evaluating the epidemiological variables, there were no differences between the colonized and non-colonized pregnant women. Diversity index and number of genotypes found by MLST was 0.608 and 6, and by MLVA was 0.840 and 15, respectively. Conclusion: We found a high prevalence of maternal GBS colonization, with serotype distribution similar to the Americas region. Multiplex PCR was more accurate than culture, and maternal epidemiological variables showed no difference when evaluating presence or absence of bacterial colonization, its serotypes and antimicrobial resistance. MLVA showed a higher discrimination capacity among the unrelated strains than MLST. / Submitted by Otto Henrique May Feuerschuette (otto.feurschuette@unisul.br) on 2018-04-26T00:54:22Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) OTTO TESE CORRIGIDA PÓS BANCA (1).pdf FINAL.pdf: 3081595 bytes, checksum: afad5beba2687558aafe2f1705d36e01 (MD5) / Approved for entry into archive by Silvane Cauz (silvane.cauz@unisul.br) on 2018-04-26T12:14:53Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) OTTO TESE CORRIGIDA PÓS BANCA (1).pdf FINAL.pdf: 3081595 bytes, checksum: afad5beba2687558aafe2f1705d36e01 (MD5) / Made available in DSpace on 2018-04-26T12:14:53Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) OTTO TESE CORRIGIDA PÓS BANCA (1).pdf FINAL.pdf: 3081595 bytes, checksum: afad5beba2687558aafe2f1705d36e01 (MD5) Previous issue date: 2018 / Introdução: A colonização do trato anogenital de gestantes pelo Estreptococo do grupo B (EGB) é o fator de risco primário para a sepse neonatal precoce. A identificação da colonização materna permite a profilaxia antimicrobiana e prevenção da transmissão vertical. Objetivos: Identificar a diversidade genética e as características fenotípicas do EGB utilizando-se técnicas de biologia molecular e cultura em meio específico, e avaliar os aspectos epidemiológicos de gestantes colonizadas por essa bactéria Métodos: Foi realizado um estudo transversal com 316 amostras anogenitais de gestantes entre 35 e 37 semanas para realização de cultura em meio específico, teste de sensibilidade aos antimicrobianos, PCR multiplex, multi locus sequence typing (MLST) e multi locus variable number of tandem repeat analysis (MLVA). Pesquisou-se também os aspectos epidemiológicos dessas gestantes. Resultados: Foi encontrada prevalência de 36,4% pela cultura e 38,6% pela PCR. A PCR multiplex apresentou sensibilidade de 100%, especificidade de 96,5%, valor preditivo positivo de 94,3% e valor preditivo negativo de 100%. Os sorotipos mais encontrados foram Ia, V, II e III. Os genes de resistência foram identificados em 34 amostras. A sensibilidade à penicilina foi universal, 24,3% apresentaram resistência à eritromicina e 14,8% à clindamicina. Avaliando-se as variáveis epidemiológicas, não se identificaram diferenças entre as gestantes colonizadas e não colonizadas. O índice de diversidade e o número de genogrupos encontrados pelo MLST foi 0,608 e 6, e pela MLVA foi 0,840 e 15, respectivamente Conclusão: Constatou-se uma alta prevalência de colonização materna, com distribuição dos sorotipos semelhante à região das Américas. A PCR multiplex foi mais acurada que a cultura, e as variáveis epidemiológicas maternas não apresentaram diferença significativa ao avaliar-se a presença ou não de colonização bacteriana, seus sorotipos e a resistência aos antimicrobianos. A MLVA apresentou uma capacidade de discriminação entre as cepas não relacionadas maior do que a MLST

Magister Scientiae - MSc / Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines. / South Africa

No description available.

Die Lichtensteinhöhle ist eine Klufthöhle im Berg Lichtenstein in den Harzausläufern. Im anthropogenen Teil der Höhle wurden größere Mengen disoloziert vorliegender Menschenknochen gefunden. Über assoziierte archäologische Artefakte und 14C-Datierungen erfolgte eine Einordnung ins 10.–9. Jh. v. Chr.. Aufgrund eines Überzuges mit Gipssinter und konstant niedriger Temperaturen war ein herausragend guter Erhaltungszustand der Knochen und der enthaltenen aDNA gegeben. Dies ermöglichte umfangreiche anthropologische Forschungsarbeiten an den menschlichen Überresten aus der Lichtensteinhöhle. Eine zentrale Fragestellung zu Beginn der Forschungsarbeiten war, ob es sich um eine Opferstätte oder einen Bestattungsplatz handelt. Es konnte für die zunächst identifizierten 40 Individuen ein ausgewogenes Geschlechterverhältnis und eine Altersverteilung über alle Altersklassen hinweg nachgewiesen werden. Zudem konnten mittels molekulargenetischer Methoden verwandtschaftliche Beziehungen zwischen den Individuen aufgedeckt werden. Die Verwandtschaftsrekonstruktion ergab den Stammbaum eines Familienclans. Damit lagen eindeutige Hinweise für eine Nutzung als Bestattungsplatz vor. Während molekulargenetischer Reihenuntersuchungen verschiedener Skelettelemente und morphologischer Zuordnungen von Skelettelementen zu Individuen wurde deutlich, dass Knochen von mehr Individuen als den 40 bislang identifizierten vorhanden waren. Zudem deutete sich an, dass für nahezu alle Individuen nicht alle Knochen in der Höhle aufgefunden worden waren. Das Fehlen von Skelettelementen warf die Frage auf, ob es sich bei der Lichtensteinhöhle nicht um einen Primär- sondern um einen Sekundärbestattungsplatz handeln könnte. Im aktuell durchgeführten Forschungsprojekt wurden, unter Verwendung morphologischer und molekulargenetischer Methoden, die Zuordnungen der dislozierten Knochen zu Individuen zu Ende geführt. Die rekonstruierten Individuen wurden umfassend morphologisch und molekulargenetisch untersucht, mit dem Ziel, die demografische Struktur der Population zu erschließen und die Verwandtschaftsrekonstruktion auszuweiten. Zudem wurde den Fragen der Nutzungsdauer und der genauen Nutzungsart der Höhle nachgegangen. Es konnten 60 Individuen identifiziert werden. Nur für zwei der Individuen wurden alle bei den Zuordnungen berücksichtigten Skelettelemente vorgefunden. An den Knochen zeigten sich nur wenige Fälle degenerativer Veränderungen. Dies ließ darauf schließen, dass die in der Lichtensteinhöhle bestatteten Menschen nicht übermäßig harter körperlicher Belastung ausgesetzt waren. Spuren massiver Gewalteinwirkung fehlten vollständig. Dies macht es unwahrscheinlich, dass die bestattete Population in kriegerische Auseinandersetzungen involviert war. Einige wenige verheilte Frakturen an Rippen oder Schlüsselbein lassen sich problemlos auf Alltagsunfälle zurückführen. Spuren von Mangel- oder Stressphasen waren nur in Einzelfällen nachweisbar. Dies deutet darauf hin, dass die Bestatteten zu Lebzeiten kontinuierlichen Zugang zu ausreichenden Nahrungsressourcen hatten. Das Geschlechterverhältnis war ausgewogen und die Altersverteilung entsprach in den Grundzügen der für eine historische Population zu erwartenden. Eine fesgestellte Unterrepräsentanz von Individuen der Altersklasse Infans I könnte als Hinweis darauf interpretiert werden, dass tatsächlich Sekundärbestattungen praktiziert wurden und die sehr kleinen, fragilen Knochen der Infans I Individuen zum Zeitpunkt der Umbettungen bereits vergangen waren. In begleitenden Arbeiten durchgeführte statistischen Analysen verschiedener Merkmale, wie z.B. Unterschiede im Grad der DNA-Degradierung, lieferten weitere Hinweise in die Richtung, dass es sich bei der Lichtensteinhöhle um einen Sekundärbestattungsplatz handeln dürfte. Für alle neu identifizierten Inividuen wurden mittels molekulargenetischer Analysen die genetischen Fingerabdrücke sowie die mitochondraialen und Y-chromosomalen Haplotypen bestimmt. Die anschließende Verwandtschaftsrekonstruktion ergab einen erweiterten Stammbaum, in dem für 47 der 60 Individuen entweder direkte Verwandtschaft oder aber Verwandtschaft in mütterlicher oder väterlicher Familienlinie belegt ist. Der Stammbaum umfasst insgesamt sechs Generationen. Dies entspricht – bei einer angenommenen Generationendauer von 20 Jahren – einer Nutzungsdauer von 120 Jahren und passt somit gut zum archäologisch ermittelten Nutzungszeitraum. Die Auswertung der Diversität der mitochondrialen und Y-chromosomalen Haplotypen ergab Hinweise auf eine patrilokale Gesellschaftsform. In begleitenden Arbeiten wurden weitere genetische Marker – z.B. für die Augen- und Haarpigmentierung, die immungenetische Ausstattung oder auch für den Laktosetoleranzstatus – analysiert. Insgesamt zeigte sich, dass sich in vielerlei Hinsicht die genetische Ausstattung heutiger Populationen im Vergleich zu der vor 3.000 Jahren nicht grundlegend unterscheidet. Lediglich für die Frequenz des Laktosetoleranz verursachenden Allels war eine deutliche Zunahme seit der Bronzezeit zu verzeichnen.