Venous thromboembolism (VTE) is a common cardiovascular disease that frequently recurs and is associated with significant numbers of death annually. The influence of the hereditary risk factors is not yet firmly established but twin and family studies suggest that heritability is about 50%. Several genetic risk factors have been identified by genomeHwide association studies (GWAS) but they do not explain all of the missing heritability of VTE. NextHgeneration sequencing (NGS) has revolutionized the genetic analysis of disease and has been used to discover the genes underlying unsolved Mendelian disorders. It has also been used to identify rare alleles which may help explain the missing heritability for complex diseases. The study population of this study consisted of 32 randomly chosen VTE patients from the MATSHstudy (Malmö Thrombophilia Study). The seventeen genes that in earlier studies have been shown to be associated with VTE were examined and the identified VTEHrelated mutations were compared to the general population. The results showed that Ion TorrentHsequencing effectively provided good coverage and read depth in all of the sequenced genes. Optimization of the primer panels resulted in higher and more balanced coverage and the quality of the results in this study was on an overall high level. A total of 215 variants were detected – 62 in exons, 8 in splice and 145 in introns. One Mendelian mutation was detected in PROC and rare variants were found in F2 and FGG. The most common risk factor (F5 Leiden) was highly enriched with 25% in this study compared to 3% in a background population. / Venös tromboembolism (VTE) är en vanlig, ofta återkommande, kardiovaskulär sjukdom som associeras med åtskilliga dödsfall årligen. De ärftliga riskfaktorernas påverkan är inte fullständigt kartlagda ännu men tvillingH och familjestudier antyder att ärftligheten kan vara runt 50%. Ett flertal genetiska riskfaktorer har identifierats genom genome$wide association studies (GWAS) men de förklarar inte hela den saknade ärftlighetskomponenten för VTE. NästaHgenerationsHsekvensering (NGS) har revolutionerat den genetiska sjukdomsanalysen och har använts för att upptäcka de gener som ligger bakom tidigare olösta Mendelska sjukdomstillstånd. Man har även använt NGS för att identifiera rara alleler som kan hjälpa till att förklara de saknade ärftlighetskomponenterna för nedärvning av komplexa sjukdomar. Studiepopulationen I den här undersökningen utgjordes av 32 slumpmässigt utvalda VTEHpatienter från Malmö Thrombophilia Study (MATS). De sjutton gener som I tidigare studier har visat sig vara associerade med VTE undersöktes och de identifierade VTEHrelaterade mutationerna jämfördes med en normalpopulation. Resultaten visade att Ion TorrentHsekvensering ger bra täckningsgrad och läsdjup i alla de sekvenserade generna. Optimering av primerHpanelerna resulterade i en mer balanserad täckningsgrad och resultatkvaliteten i den här studien var på en generellt hög nivå. Totalt 215 varianter detekterades – 62 i exon, 8 i splice och 145 i introner. En Mendelsk mutation detekterades I PROC och rara varianter hittades i F2 och FGG. Den starkaste och vanligaste riskfaktorn (F5 Leiden) var högt anrikad i den här studien med 25% jämfört med 3% i en bakgrundspopulation.

A mastite bovina é considerada a doença de maior impacto nos rebanhos leiteiros, exercendo efeito econômico negativo sobre a produtividade e perdas significativas à indústria de laticínios. Tendo em vista os impactos na sanidade animal e os prejuízos econômicos acarretados, o objetivo deste estudo foi caracterizar de forma mais abrangente a comunidade microbiana presente em rebanhos leiteiros com mastite subclínica utilizado o sequenciamento parcial do gene 16S ribossomal RNA (rRNA). Especificamente, foram caracterizadas as comunidades bacterianas presentes em amostras de leite vindas de três fazendas comerciais, sendo que cada fazenda contribuiu com amostras com alta contagem de células somáticas (CCS > 200.000 cel./mL) e com baixa contagem (CCS < 200.000 cel./mL) perfazendo um total de 57 animais. O DNA total foi extraído e amplificado com os oligonucleotídeos iniciadores da região V3 e V4 do gene 16S rRNA. O sequenciamento foi realizado utilizando a tecnologia de sequenciamento de nova geração através do equipamento MiSeq (Illumina - San Diego, EUA). Para efeito de comparação, alíquotas de todas as amostras foram destinadas ao cultivo microbiológico para identificação de bactérias causadoras da mastite. Os fragmentos amplicons de todas as amostras foram submetidos a uma série de análises computacionais utilizando o programa QIIME. Após a avaliação adicional das sequências em nível de espécie, verificou-se que em geral as bactérias diagnosticadas por cultura geralmente não corresponderam com as sequências mais abundantes detectadas pelo sequenciamento. A análise da composição da microbiota de amostras de leite provenientes de animais saudáveis revelou a presença de uma grande diversidade de espécies bacterianas, mesmo que nenhuma bactéria tenha sido detectada por técnica de cultura. A espécie bacteriana mais abundante em todas as amostras foi Staphylococcus chromogenes. Staphylococcus aureus também foi detectada na grande maioria das amostras As diferenças na composição microbiana foram observadas entre as amostras quando a comparação foi feita de forma individual. Estas diferenças foram notórias em composição taxonômica e foram refletidas por intermédio das estimativas de alfa e beta diversidade. Quando a comparação foi realizada por separação de grupos com alta e baixa CCS, essa diferença não foi tão evidente. Com este estudo será possível compreender a diversidade dos microrganismos presentes na glândula mamária de animais saudáveis e com mastite subclínica. Essas informações podem ser úteis podendo contribuir no planejamento de medidas terapêuticas e preventivas mais eficazes da doença. / Bovine mastitis is considered the most impact disease in dairy herds, exerting negative economic effect on productivity and significant losses to the dairy industry. In view of the impact on animal health and carted economic losses, the objective of this study was to characterize more comprehensively the microbial community present in dairy herds with subclinical mastitis using the partial sequencing of 16S ribosomal RNA gene (rRNA). Specifically, the bacterial communities present in samples of milk coming from three commercial farms were identified, and each farm contributed samples with high somatic cell count (SCC> 200,000 cel./mL) and low count (SCC <200,000 cel./ml) for a total of 57 animals. Total DNA was extracted and amplified with primers of the V3 and V4 region of the 16S rRNA gene. Sequencing was performed using the new generation of sequencing technology through MiSeq equipment (Illumina - San Diego, USA). For comparison, aliquots of all samples were intended for microbiological culture for identification of bacteria which cause mastitis. The amplicon fragments of all samples were subjected to a series of computer analyzes using the QIIME program. After further evaluation of the sequences at the species level, it was found that in general the bacteria do not generally diagnosed by culture corresponded to the most abundant sequences identified by sequencing. The analysis of milk samples from microbial composition from healthy animals revealed the presence of a diversity of bacterial species, even though no bacteria have been detected by culture technique. The most abundant bacterial species in all samples was Staphylococcus chromogenes. Staphylococcus aureus was also detected in most samples differences in microbial composition were found between the samples when a comparison was made individually. These differences were noticeable in taxonomic composition and were reflected by means of the estimates of alpha and beta diversity. When comparison was performed by separation of high and low groups with CCS, this difference was not so evident. This study will be possible to understand the diversity of microorganisms present in the mammary gland of healthy animals and with subclinical mastitis. This information can be useful and can contribute in the planning of more effective therapeutic and preventive measures of the disease.

La capacidad de Mycobacterium tuberculosis para sobrevivir dentro del macrófago contribuye grandemente a su patogenicidad, latencia y persistencia durante la infección. Este bacilo induce alteraciones en el ambiente intrafagosomal e inhibe la maduración del fagosoma, favoreciendo su supervivencia intracelular. M. tuberculosis PknG secuestra al macrófago precisamente al evitar la fusión fagosoma-lisosoma. En este sentido, PknG representa una familia de dianas novedosas para enfrentar la necesidad de nuevos antimicrobianos para la tuberculosis latente. Aquí, apuntamos a: (i) elucidar la base estructural-molecular del ATP y Mg2+ como cofactores de PknG; (ii) caracterizar los parámetros cinéticos que gobiernan la formación del complejo PknG:ATP; e, (iii) identificar péptidos capaces de unirse a PknG para investigar experimentalmente su interactoma usando enfoques combinatorios como “Phage Display”. Nuestros resultados confirman que PknG se une exclusivamente al ATP con una constante de disociación (KD) de 108.8  22.9 µM. El Mg2+ estabiliza térmicamente a PknG de forma ATP-dependiente. Análisis de estado pre-estacionario muestran que la unión y disociación del ATP es rápida en el complejo PknG:ATP. Usando PknGN-Ext, TPR resolvimos la estructura cristalina en el estado unido al ADP mientras que demostramos que el ATP imposibilita la cristalización. Los análisis bioinformáticos de las librerías enriquecidas por Phage Display identificaron 57 potenciales peptidos que interactuarían con PknG. Una comparación cercana con el proteoma de M. tuberculosis proporcionó un subconjunto de 20 proteínas que podrían interactuar con PknG. Nuestros resultados confirmaron cinco proteínas asociadas a PknG previamente reportadas: PknG, DnaK chaperona, transportador ABC Rv1747, Proteína Ribosomal L23 y Factor de Elongación Tu, resaltando la validez de nuestra plataforma para descubrir el interactoma de PknG. Así, nuestros resultados revelan interacciones proteína-proteína putativas que podrían participar en la supervivencia micobacteriana, mientras que también proporcionan bases sólidas para desarrollar drogas antituberculosas al interrumpir estas interacciones o explotar estos peptidos tipo compuesto líder. / The ability of Mycobacterium tuberculosis to survive inside the macrophage greatly contributes to its pathogenicity, latency and persistence during infection. This bacillus induces alterations in the intraphagosomal environment and inhibits phagosome maturation, thus promoting mycobacterial survival. M. tuberculosis PknG hijacks the macrophage precisely by avoiding phagosome-lysosome fusion. In this sense, PknG represents a family of novel targets to cope with the need for new antimicrobials for latent tuberculosis. Here, we aimed to: (i) elucidate the structural-molecular basis of ATP and Mg2+ as PknG cofactors; (ii) characterize the kinetic parameters governing PknG:ATP complex formation; and, (iii) identify PknG-binding peptides to experimentally query PknG’s interactome using combinatorial approach such as Phage Display. Our results confirm that PknG exclusively binds to ATP with a dissociation constant (KD) of 108.8  22.9 µM. Mg2+ thermally stabilizes PknG in an ATP-dependent manner. Pre-steady-state analyses show that ATP binding and dissociation are rapid in the PknG:ATP complex. Using PknGN-Ext, TPR we solved the ADP-state crystal structure while showing that ATP precludes crystallization. Phage Display and bioinformatic analyses identified 57 potential PknG binders. A close comparison to the M. tuberculosis proteome provided a subset of 20 proteins that may interact with PknG. Our results confirmed five previously reported PknG-associated proteins: PknG, DnaK chaperone, ABC transporter Rv1747, Ribosomal Protein L23 and Elongation Factor Tu, highlighting our platform’s validity to uncover the PknG interactome. Altogether, our results reveal putative protein-protein interactions that may play a role in mycobacterial survival, while also providing solid bases for the development of anti-tuberculosis drugs by disrupting these interactions or exploiting these lead-like peptide molecules. / Tesis

Les maladies à transmission vectorielle sont dues à des agents pathogènes transmis par des arthropodes hématophages. Ces vecteurs assurent une transmission active (mécanique ou biologique) d’un agent infectieux d’un vertébré vers un autre vertébré. A l’échelle mondiale, les tiques sont responsables de la transmission de la plus grande variété d’agents pathogènes, elles transmettent des microorganismes responsables de maladies bactériennes (borréliose de Lyme, rickettsioses) ou parasitaires (babésioses, theilérioses), ou même virales (encéphalite à tiques).Les Antilles se situent au cœur de la zone Néotropicale des Caraïbes, et constituent une zone à risque pour l’émergence de maladies vectorielles en raison des conditions climatiques favorables aux vecteurs et des échanges intercontinentaux importants (flux illégal d’animaux, oiseaux migrateurs,…). La situation épidémiologique de la zone Caraïbe vis-à-vis des maladies transmises par les tiques est très peu documentée. Les études menées sur le terrain portent essentiellement sur des agents pathogènes affectant les animaux comme Ehrlichia ruminantium, Babesia (bovis et bigemina) et Anaplasma marginale et sont donc loin de pouvoir répondre aux questions concernant le risque d’émergence ou de réémergence de maladies à tique. Ainsi, il est nécessaire et urgent de développer des outils efficaces de surveillance épidémiologique qui permettraient la détection des agents pathogènes, nouveaux, connus ou non suspectés présents dans les tiques. C’est dans ce contexte d’amélioration des performances de veille sanitaire des maladies à tiques dans les Caraïbes que prend place le projet de thèse. La visée de la thèse était de faire un état des lieux des agents pathogènes d’intérêt médical et vétérinaire présents dans les tiques caribéennes à l’aide de techniques de détection à haut débit. Pour cela nous avons d’abord réalisé un séquençage à haut débit d’ARN extraits de tiques collectées en Guadeloupe et en Martinique afin de réaliser un inventaire sans a priori des agents pathogènes (bactéries, parasites, et virus) présents. Cette analyse a permis de mettre en évidence une grande diversité en microorganismes pathogènes au sein de nos échantillons, révélant également la présence de quatre virus appartenant à de nouveaux genres viraux récemment décrits et associés aux arthropodes. Les informations obtenues via le séquençage, additionnées aux données disponibles dans la littérature ont permis de constituer ainsi une liste des agents pathogènes transmis par les tiques nécessitant une surveillance sanitaire dans les caraïbes. A partir de ce répertoire nous avons développé un système de dépistage à haut-débit d’agents infectieux applicable à toute la zone des caraïbes. L’outil de détection est un support microfluidique de type puce à ADN, basé sur la technologie BioMarkTM dynamic arrays (Fluidigm Corporation) qui permet de réaliser de la PCR en temps réel à haut débit afin de détecter simultanément 48 à 96 cibles au sein de 48 à 96 échantillons. Deux puces ont été développées, une première pour le suivi des bactéries et parasites, et une deuxième pour le suivi des virus. Leur performance a été testée sur des échantillons de tiques collectées en Guadeloupe et en Martinique. Ce dépistage à grande échelle a donné un aperçu complet de la situation épidémiologique de 45 bactéries, 17 parasites and 31 virus potentiellement transmis par les tiques dans les Antilles Françaises. La méthode de surveillance développée durant cette thèse représente une amélioration majeure des techniques de veille épidémiologique, permettant la détection rapide et concomitante d’un large panel d’agent pathogène. Elle sera prochainement appliquée au criblage à haut débit des agents infectieux présent dans des tiques collectées à travers la Caraïbe, provenant notamment de Trinité-et-Tobago, Saint-Kitts, la Barbade, et Sainte-Lucie, grâce à la collaboration du réseau CaribVet, et de vétérinaires locaux / Vector-borne diseases are illnesses caused by pathogens transmitted by haematophagous arthropods which provide active transmission (mechanical or biological) of infectious agents from one vertebrate to another. Among these vectors, ticks are known to carry and transmit the greatest variety of pathogens of public health and veterinary importance. They transmit microorganisms responsible for bacterial (Lyme borreliosis, rickettsioses), parasitic (babesiosis, theileriosis), or viral diseases (tick-borne encephalitis).The Antilles are located in the heart of the Caribbean Neotropical Zone. This area can be considered at risk for the emergence of vector-borne diseases mainly due to favorable environmental conditions and intercontinental exchanges (e.g. legal and illegal animal trade, migratory birds). However, the epidemiological situation of the Caribbean area, with regard to tick-borne diseases, is still poorly documented. Indeed, most of field studies only focused on animal pathogens such as Ehrlichia ruminantium, Babesia (bovis and bigemina) and Anaplasma marginale and questions about the risk of emergence or re-emergence of tick-borne diseases remain unanswered. Thus, it is crucial to develop efficient epidemiological surveillance tools that would enable the detection of new, known or unexpected pathogens present in ticks. In this context, the main objective of my thesis was to obtain an overview of pathogens of medical and veterinary interest present in Caribbean ticks using new high-throughput technologies. We first used a high-throughput sequencing approach to determine pathogens present in ticks (bacteria, parasites, and viruses) collected in Guadeloupe and Martinique. This analysis revealed a great diversity of pathogenic agents in our samples and highlighted the presence of four viruses belonging to new viral families recently described and associated with arthropods. Results of sequencing combined with data available in the literature allowed us to make the most exhaustive list of pathogens potentially transmitted by ticks and requiring health surveillance in the Caribbean area. From this pathogen inventory, we developed a system of high-throughput screening of infectious agents applicable to the whole Caribbean area. This molecular tool is a microfluidic system based on the BiomarkTM dynamic arrays technology (Fluidigm Corporation), which enables high-throughput real-time PCR to simultaneously detect 48-96 targets within 48 to 96 samples. Two different chips have been developed, one for bacteria and parasites monitoring, and one for viruses. Their efficiency was tested on tick samples collected in both Guadeloupe and Martinique. This large-scale screening provided a comprehensive overview of the epidemiological situation of 45 bacteria, 17 parasites and 31 viruses potentially transmitted by ticks in the French West Indies. The high-throughput detection tool developed during my thesis represents a major improvement in epidemiological surveillance technology, enabling the rapid and concomitant monitoring of a wide range of pathogens. It will soon be applied to high-throughput screening of infectious agents found in ticks collected throughout the Caribbean, including Trinidad and Tobago, St. Kitts, Barbados, and St. Lucia, thanks to the collaboration with the CaribVet network, and local veterinarians

This thesis presents short DNA alignment tools optimization. These short DNA reads are products of next\nobreakdash-generation sequencing technologies. The results produced by existing align\-ment tools can be influenced by various parameters. For this purpose, an optimization framework to find the optimal values of selected parameters was developed. This framework is based on differencial evolution algorithm and its main goal is to maximize the alignment accuracy. The functionality of the framework was tested on both real and generated data sets of short DNA reads. An accurate alignment is crucial for correct prediction of various genetic characteristics.

Dans cette thèse, nous nous intéressons au problème de recherche incrémentale de motifs dans des séquences fortement similaires (On-line Pattern Matching on Highly Similar Sequences), issues de technologies de séquençage à haut débit (SHD). Ces séquences ne diffèrent que par de très petites quantités de variations et présentent un niveau de similarité très élevé. Il y a donc un fort besoin d'algorithmes efficaces pour effectuer la recherche rapide de motifs dans de tels ensembles de séquences spécifiques. Nous développons de nouveaux algorithmes pour traiter ce problème. Cette thèse est répartie en cinq parties. Dans la première partie, nous présentons un état de l'art sur les algorithmes les plus connus du problème de recherche de motifs et les index associés. Puis, dans les trois parties suivantes, nous développons trois algorithmes directement dédiés à la recherche incrémentale de motifs dans un ensemble de séquences fortement similaires. Enfin, dans la cinquième partie, nous effectuons une étude expérimentale sur ces algorithmes. Cette étude a montré que nos algorithmes sont efficaces en pratique en terme de temps de calcul / In this thesis, we are interested in the problem of on-line pattern matching in highly similar sequences, On-line Pattern Matching on Highly Similar Sequences, outcoming from Next Generation Sequencing technologies (NGS). These sequences only differ by a very small amount. There is thus a strong need for efficient algorithms for performing fast pattern matching in such specific sets of sequences. We develop new algorithms to process this problem. This thesis is partitioned into five parts. In the first part, we present a state of the art on the most popular algorithms of finding problem and the related indexes. Then, in the three following parts, we develop three algorithms directly dedicated to the on-line search for patterns in a set of highly similar sequences. Finally, in the fifth part, we conduct an experimental study on these algorithms. This study shows that our algorithms are efficient in practice in terms of computation time.

MSc (Zoology) / Department of Zoology / See the attached abstract below

Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Methods: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Case report and results: Cherry red macular spotswere first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations aswell as cerebralMRIwere unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to b1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699CNA, p.S233R in exon 4 and c.803ANG; p.Y268C in Exon 5 in NEU1 transcriptNM_000434.3), leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

TITLE: Mutation and substitution rates in sexual and asexual forms: a clue to the persistence of sex in a model group of Cobitis? AUTOR: Jan Röslein DEPARTMENT: Ústav živočišné fyziologie a genetiky AVČR, v.v.i. SUPERVISOR: Mgr. Karel Janko, Ph.D. ABSTRACT: Subject of this thesis is to test several hypotheses about the evolution of asexual reproduction in model group of fish family Cobitis and its mutual competition among sexual and asexual forms, which touches one of the oldest unresolved issues of biology. Specifically, the work deals with the accumulation of non-synonymous mutations, which accelerated accumulation in the genome of clonal lineages theoretically leads to increased extinction compared with sexually reproducing populations (so-called. The theory of Muller's ratchet and Kondrashov's hatchet). This thesis is based on a normalized cDNA sequencing data from oocytes and liver tissue, which has served as a base matrix (generated based on non-normalized cDNA data) for transcriptome sequencing (RNAseq). Consequently, the RNAseq data have served as validation for acquired polymorphisms, detection of differential expression of allele- specific expression (ASE) hybrid biotypes. This diploma thesis balances among the edges of vast spectrum of hypotheses regarding the evolution of the genus hybrid...

Next generation sequencing technologies are changing the way scientific experiments and diseases diagnostics are performed and thus will allow what is called personalized medicine. The sense of presented thesis is to make survey of new approaches to DNA sequencing and demonstrate usage and constraints of bioinformatic analytical tools available to day. Discussed techniques are then applied to the case study of finding molecular basis for rare hereditary disease. Introductory part deals with overview of commercially available sequencing techniques (454 Life Science, Applied Biosystems, Illumina, Helicos). Fundamentals of each method are described and possible further development is outlined. Post sequencing data analysis is than discussed in details. In practical section we demonstrate genome analysis techniques successfully used to reveal causal mutation in the gene responsible for adult form of autozomal neuronal ceroid lipofuscinosis (ANCL). Combination of linkage analysis (Merlin), copy number variant analysis (Genome-Wide Human SNP Array 6.0), analysis of expression profiles (HumanRef-8 v2 Expression BeadChips) and exome sequencing (SOLiD™ 4 System) has been applied to members of one ANCL family. We also paid attention to comparison, evaluation and selection of available mapping algorithms used in...