Η Πλάγια Αμυοτροφική Σκλήρυνση είναι μια ετερογενής νόσος, που οφείλεται στην εκφύλιση των ανώτερων και κατώτερων κινητικών νευρώνων. Αρχικά η νόσος εκδηλώνεται είτε με μυϊκή αδυναμία και σπαστικότητα των άνω ή κάτω άκρων, είτε με δυσχέρεια στην ομιλία. Η εξέλιξη της νόσου είναι μοιραία και ο αναμενόμενος χρόνος επιβίωσης είναι 3 – 5 χρόνια. Με την εμφάνιση της νόσου έχουν συσχετιστεί πολλοί γενετικοί και περιβαλλοντικοί παράγοντες. Ταυτοχρόνως, έχουν προταθεί και πολλοί μηχανισμοί παθογένειας. Παρόλα αυτά, η ALS παραμένει ανίατη και οι υπάρχουσες θεραπείες αποσκοπούν στην βελτίωση της κλινικής εικόνας ή / και στην παράταση της επιβίωσης. Ιδιαίτερα στον ελληνικό πληθυσμό, η γενετική αιτία της νόσου δεν έχει διερευνηθεί επαρκώς. Μεταλλάξεις στα γονίδια SOD1, TARDBP και FUS στις οποίες οφείλεται το 30% των οικογενών περιπτώσεων ALS σύμφωνα με τη βιβλιογραφία, δεν απαντώνται στους ασθενείς με ελληνική καταγωγή. Για αυτό το λόγο, με τη βοήθεια της αλληλούχησης ολόκληρου του γονιδιώματος και χρησιμοποιώντας διάφορες προσεγγίσεις πάνω στα δεδομένα που εξήχθησαν, εστιάσαμε την έρευνά μας σε συγκεκριμένες παραλλαγές. Στόχος μας ήταν ο έλεγχος για πιθανές συσχετίσεις μεταξύ των παραλλαγών αυτών και της εμφάνισης της νόσου. Η επιλογή των παραλλαγών στηρίχθηκε στους κοινούς μεταξύ των ασθενών πολυμορφισμούς που δεν υπήρχαν στο δείγμα αναφοράς. Από αυτή την προσέγγιση, επιλέχθηκαν οι κοινοί πολυμορφισμοί rs6850200 (TBC1D1), rs1861869 (FTO), rs2892469 (FTO), rs4773203 και rs10581954 (A2LD1), rs34030508 (FAM181B) και rs6676539 (KAZN). Η δεύτερη προσέγγιση, περιλάμβανε τις νέες παραλλαγές που εντοπίστηκαν στο γονιδίωμα τουλάχιστον ενός εκ των ασθενών. Μεταξύ αυτών, επιλέχθηκαν 2 παραλλαγές, στην 5’ αμετάφραστη περιοχή των γονιδίων FGF13 και SLC36A1. Σε δεύτερο χρόνο, αντιπαρατέθηκαν οι παραλλαγές των γονιδιωμάτων, με τη βάση δεδομένων Human Gene Mutation Database (HGMD) και συγκεκριμένα, με τις μεταλλάξεις που έχουν συσχετιστεί με την ALS. Ωστόσο, δεν κρίθηκε σκόπιμη η περαιτέρω διερεύνηση των αποτελεσμάτων αυτών. Το δείγμα των ασθενών επεκτάθηκε για τις επιλεγμένες παραλλαγές και τα αποτελέσματα της γονοτύπησης των ασθενών συγκρίθηκαν με τα αποτελέσματα της γονοτύπησης που πραγματοποιήθηκε σε δείγμα αναφοράς. Οι παραλλαγές που έδωσαν στατιστικά σημαντικό αποτέλεσμα, αναλύθηκαν και σε πληθυσμό Σαρδηνίων ασθενών ALS έναντι υγιών ατόμων. Εξετάσθηκε επίσης και η περίπτωση μιας οικογένειας στην οποία υπήρχαν τέσσερα άτομα τα οποία εμφάνιζαν ιδιαίτερο φαινότυπο και είχαν διαγνωστεί με πιθανή ALS. Συλλέχθηκε το πλήρες ιατρικό ιστορικό των ατόμων της οικογένειας που νοσούσαν, ενώ με τη χρήση της μεθόδου νέας γενιάς αλληλούχησης εντοπίστηκε πιθανή μετάλλαξη. Μεταξύ των συνολικά οχτώ παραλλαγών που μελετήθηκαν (από την πρώτη και δεύτερη προσέγγιση), από την ανάλυση των δύο προέκυψε στατιστικά σημαντική διαφορά μεταξύ υγιών και ασθενών σε επίπεδο γονοτύπων, αλλά όχι σε επίπεδο αλληλομόρφων. Οι συσχετίσεις με την ALS αφορούν τους πολυμορφισμούς, rs6850200 (TBC1D1), rs1861869 και rs2892469 (FTO), στον ελληνικό πληθυσμό. Επιπλέον, οι rs1861869 και rs2892469 συνδέθηκαν με απλότυπο. Οι διαφορές αυτές δεν υπήρχαν στο δείγμα των Σαρδηνίων. Αναφορικά με την οικογένεια, επιβεβαιώθηκε η ύπαρξη μετάλλαξης μεγέθους 25 βάσεων στο γονίδιο SPG7 σε ομόζυγη κατάσταση, στα μέλη της οικογένειας που φέρουν τον φαινότυπο της ALS / HSP (Οικογενούς Σπαστικής Παραπληγίας) και σε ετερόζυγη κατάσταση στα υγιή άτομα της οικογένειας που ελέγχθηκαν. / Amyotrophic Lateral Sclerosis is a heterogeneous disorder, caused by upper and lower motor neuron degeneration. At the first stages of the disease, muscle weakness as well as spasticity of upper or lower limbs or alternatively, impaired speech become obvious. The patients deteriorate rapidly and finally die 3 – 5 years after the initiation of the symptoms. Many genetic and environmental factors have been correlated with ALS. At the same time, a wide range of pathogenesis’ mechanisms have been proposed. Nevertheless, ALS remains a fatal disease and the existing therapies, aim to symptom relief and/ or a slight prolongation of life time expectancy. In particular, the genetic causes of ALS in Greek population, have not been thoroughly investigated. Mutations in SOD1, TARDBP and FUS exons to which 30% of FALS cases are attributed according to the bibliography, are under-represented in patients with a Hellenic ancestry. Herein, we obtained whole genome sequencing data from 10 Greek ALS patients. To interpret our data we used several approaches, which then helped us focus on certain variations. The aim of the project was to investigate those variations and their possible association with the disease. The variations of interest were chosen according to the following criteria; polymorphisms (i) being common to all patients, (ii) not present in healthy individuals and (iii) located near genes. From this approach, we chose the common polymorphisms rs6850200 (TBC1D1), rs2892469 (FTO), rs1861869 (FTO), rs4773203 and rs10581954 (A2LD1), rs34030508 (FAM181B) and rs6676539 (KAZN). The second approach, includes the annotation of all the novel variants found in at least one patient. Among these variants, two were chosen, both located in the 5’ untranslated regions of the genes FGF13 and SLC36A1. Subsequently, the patients’ genomes variants were juxtaposed with the Human Gene Mutation Database (HGMD) and only those associated with ALS were selected. In this case, however, no further investigation has been performed. Our patients’ population was expanded for the overall chosen variations and their genotypes were compared to the genotypes of healthy individuals. Whenever the outcome had a statistical significance, a replication study was attempted in Sardinian ALS patients compared to healthy individuals. In parallel, we came across a case of a family with interesting phenotype features; four individuals in this family were diagnosed with possible ALS / HSP (Hereditary spastic paraparesis) and their full medical history was acquired. Through a next generation sequencing method a new possible mutation was identified. Overall, eight variations were studied (from the first and second approach). The polymorphisms rs6850200 (TBC1D1), rs1861869 and rs2892469 (FTO) showed a statistically significant association with ALS at a genotype level in the Greek population. At the same time, rs1861869 and rs2892469 were correlated with the existence of a haplotype. On the contrary, rs1861869 and rs2892469 showed no statistical significant differences, when Sardinian ALS patients were compared to healthy Sardinian individuals. As far as the family mentioned above is concerned, the existence of a homozygous 25bp deletion in the SPG7 gene was confirmed. All the members of the family, who manifested symptoms, bear the deletion.

Gene-fusions are a prevalent class of genetic variants that are often employed as cancer biomarkers and therapeutic targets. In recent years, high-throughput sequencing of the cellular genome and transcriptome have emerged as a promising approach for the investigation of gene-fusions at the DNA and RNA level. Although, large volumes of sequencing data and complexity of gene-fusion structures presents unique computational challenges. This dissertation describes research that first addresses the bioinformatics challenges associated with the analysis of the massive volumes of sequencing data by developing bioinformatics pipeline and more applied integrated computational workflows. Application of high-throughput sequencing and the proposed bioinformatics approaches for the breast and ovarian cancer study reveals unexpected complex structures of gene-fusions and their functional significance in the onset and progression of cancer. Integrative analysis of gene-fusions at DNA and RNA level shows the key importance of the regulation of gene-fusion at the transcription level in cancer.

A tuberculose (TB) embora passível de tratamento efetivo, ainda é um grave problema de saúde pública em diversos países, inclusive no Brasil. Nas últimas décadas houve progressos consistentes no controle da doença, porém o avanço da resistência bacilar ainda é um desafio a ser superado, já que os mecanismos da resistência são bastante complexos e não totalmente conhecidos, o que dificulta o desenvolvimento de testes de sensibilidade com elevada acurácia. O objetivo deste trabalho foi caracterizar as mutações gênicas de cepas de Mycobacterium tuberculosis de pacientes do Brasil e de Moçambique com doença resistente a drogas através do sequenciamento genômico total, além de descrever padrões de mutações obtidos por testes moleculares comerciais e comparar estes dados com resultados de testes fenotípicos. Estudo descritivo e transversal que incluiu 30 isolados (17 do Brasil e 13 de Moçambique), submetidos aos testes moleculares comerciais Genotype MTBDRplus®, Genotype MTBDRsl®, Xpert MTB/RIF® e teste fenotípico BACTEC MGIT 960 SIRE®. Todos os isolados também foram avaliados pelo sequenciamento genômico realizado pelo Illumina MiSeq Sequencing System® e submetidos a análise de mutações que conferem resistência às drogas contra TB utilizando o TB profiler online tool. A sensibilidade e especificidade do sequenciamento genômico para detecção de resistência a rifampicina foi de 87,5% e 92,3%, respectivamente. Além disso, o sequenciamento detectou a mutação (Val170Phe) no gene rpoB em dois isolados de M. tuberculosis de Moçambique. Esta mutação não é detectada pelos testes genotípicos comerciais. A sensibilidade do sequenciamento para a isoniazida foi de 95,6% e a especificidade de 100%. Para a estreptomicina, a sensibilidade foi de 85,7% e a especificidade de 93,3%. Para o etambutol, observamos sensibilidade de 100% e especificidade de 77,2%. As mutações mais frequentes associadas à resistência à rifampicina foram a Ser450Leu e a His445Tyr no gene rpoB. Em relação à isoniazida, predominou a mutação Ser315Thr no gene katG. O sequenciamento genômico, dado seu alto poder discriminatório, tem grande potencial de fornecer informações mais acuradas sobre mecanismo gênicos da resistência bacilar, possibilitando futuramente o aprimoramento de testes diagnósticos mais precisos. / Although there is an effective treatment for tuberculosis (TB), it is still a serious public health problem in several countries, including Brazil. In the last decades, there has been consistent progress in disease control, but the increasing number of disease caused by resistant strains is still a challenge to be overcome, since the mechanisms of resistance are quite complex and not fully known, which difficult the development of susceptibility tests with high accuracy. The aim of this work was to characterize gene mutations of Mycobacterium tuberculosis strains from Brazilian and Mozambican patients with drug-resistant disease through whole genome sequencing, as well as to describe patterns of mutations obtained by commercial molecular tests and to compare these data with results of phenotypic susceptibility tests. It was a cross-sectional study that included 30 isolates (17 from Brazil and 13 from Mozambique). Commercial molecular tests Genotype MTBDRplus(TM), Genotype MTBDRsl(TM), Xpert MTB / RIF(TM) and BACTEC MGIT 960 SIRE(TM) phenotypic test were performed for all isolates. All of them were also evaluated by whole genome sequencing performed by the Illumina MiSeq Sequencing System(TM) and submitted to analysis of mutations that confer drug resistance against TB using the TB profiler online tool. The sensitivity and specificity of whole genome sequencing for detection rifampicin resistance was 87.5% and 92.3%, respectively. Also, whole genome sequencing detected the mutation (Val170Phe) in the rpoB gene in two isolates of M. tuberculosis from Mozambique. This mutation is not detected by commercial genotypic tests. The sensitivity of the whole genome sequencing for isoniazid was 95.6%, and the specificity was 100%. For streptomycin, the sensitivity was 85.7%, and the specificity was 93.3%. For ethambutol, we observed a sensitivity of 100% and specificity of 77.2%. The most frequent mutations associated with rifampicin resistance were rpoB Ser450Leu and His445Tyr. About isoniazid, the katG Ser315Thr mutation was the most frequent. Whole genome sequencing, given its high discriminatory power, has great potential to provide more accurate information about the gene mechanisms of bacilli resistance, making possible the improvement of more accurate diagnostic tests in the future.

La publication du génome de la paramécie (Aury, 2006) a révélé une séquence atypique particulièrement intéressante pour les études de génomique évolutive. Au cours de cette thèse, j’ai mené une analyse bioinformatique détaillée de ce génome en me concentrant particulièrement sur les trois points suivants : 1) Le rôle de deux classes distinctes de petits ARN fonctionnels non codants, l’une intervenant dans les processus de régulation de l’expression des gènes tandis que l’autre participe aux réarrangements génomiques (élimination de fragments d’ADN) associés au cycle sexuel de la paramécie. 2) L’évolution des paires de gènes après une duplication globale de génome (WGD). En effet, avec une WGD relativement récente précédée de deux autres WGDs plus anciennes encore bien visibles, la paramécie est un modèle de choix pour cette étude. Nous avons pu montrer que la rétention des deux copies d’un gène après une WGD est fortement corrélée au niveau d’expression des gènes. Nous proposons un modèle basé sur les coûts et bénéfices de l’expression des gènes pour expliquer cette observation. 3) L’analyse de contraintes sélectives sur les introns pour produire des messagers détectables par le Nonsense-Mediated mRNA Decay (NMD). Ces contraintes sélectives, mises en évidence initialement chez la paramécie, se sont avérées être présentes chez tous les eucaryotes que nous avons pu analyser, ce qui nous amène à questionner l’efficacité des mécanismes d’épissage chez les eucaryotes et le rôle du NMD dans la prévention des erreurs d’épissage. L’ensemble de ces analyses a permis de mieux comprendre un certain nombre de mécanismes évolutifs universels / This work presents a detailed analysis of the paramecium genome, with focusing more precisely on the 3 following topics : 1) The role of two distinct classes of small non-coding RNAs. The first one (siRNAs) being involved in post-transcriptional gene silencing while the other (scanRNAs) plays a crucial role during the massive genomic rearrangements that occur in ciliates after sexual reproduction (Lepère et al. 2009). 2) The evolution of duplicated genes following Whole-Genome Duplications (WGDs). Indeed, the paramecium genome contains evidences for 3 successive WGDs (Aury et al. 2006), which explains why this organisms is perfectly well suited for such an analysis. We show that retention of duplicated genes is strongly correlated to their expression level and we propose a model based on cost and benefit of gene expression to explain this pattern. 3) The analysis of the extremely tiny introns in paramecium (99% of introns are less than 20-33nt in length) revealed the presence of a translational control of splicing in eukaryotes. This work suggests that splicing errors are frequent and that eukaryotic cells rely on the Nonsense-mediated mRNA Decay to detect aberrant transcripts produced by splicing errors (Jaillon et al. 2008). These analyses provide new insights on several evolutionary mechanisms that shape the genomes of eukaryotes

The primary objectives of this project were: 1. to develop improved methods for extraction of DNA from human skeletal remains, 2. to improve STR profiling success of low-copy DNA samples by employing whole genome amplification to amplify the total pool of DNA prior to STR analysis, and 3. to improve STR profiling success of damaged DNA templates by using DNA repair enzymes to reduce the number/severity of lesions that interfere with STR profiling. The data from this study support the following conclusions. Inhibitory compounds must be removed prior to enzymatic amplification; either during bone section pretreatment or by the DNA extraction method. Overall, bleach outperformed UV as a pretreatment and DNA extraction using silica outperformed microconcentration and organic extraction. DNA repair with PreCR™ A outperformed both whole genome amplification and repair with PreCR™ T6. Superior DNA extraction results were achieved using the A6 PMB columns (20 ml capacity column with 6 layers of type A glass fiber filter), and DNA repair with PreCR™ A led to an overall improvement in profile quality in most cases, although whole genome amplification was unsuccessful. Rapid, robust DNA isolation, successful amplification of loci from the sample-derived DNA pool, and an elimination of DNA damage and inhibitors may assist in providing sufficient genetic information from cases that might otherwise lie on the fringe of what is possible to obtain today.

Campylobacter est responsable de la zoonose bactérienne d’origine alimentaire la plus fréquemment reportée en Europe. Cette bactérie étant ubiquitaire, les sources et voies d’infection de l’Homme sont nombreuses. Cependant, afin de diminuer l’incidence de la maladie, il est nécessaire d’identifier les principaux réservoirs impliqués dans les infections humaines. Pour cela, nous avons dans un premier temps investigué la présence de Campylobacter dans trois réservoirs animaux (volaille, bovin, animaux de compagnie), ainsi que la diversité génétique des isolats de C. jejuni, en comparaison à celle d’isolats cliniques, à l’aide des techniques MLST (Multilocus sequence typing) et CGF (Comparative Genomic Fingerprinting). Afin d’identifier l’origine des campylobactérioses avec précision et de compenser notamment les limites techniques de la MLST, 15 marqueurs génétiques ont été sélectionnés comme marqueurs potentiellement indicateurs de l’hôte, après analyse de plus de 800 génomes de C. jejuni. Par la suite, la capacité de la MLST, la CGF40 et des 15 marqueurs à identifier l’origine des campylobactérioses a été étudiée. Ainsi, les 15 marqueurs se sont révélés être particulièrement performants pour l’attribution de sources des campylobactérioses, suivis ensuite par la MLST, tandis que la CGF40 est apparue comme étant peu adaptée. A partir des données MLST et des 15 marqueurs génétiques, une implication majoritaire des volailles et des bovins a été mis en évidence en France, tandis que les animaux de compagnie et l’environnement (comprenant eau et oiseaux sauvages) étaient faiblement impliqués. Ceci permet ainsi de renforcer les efforts de recherche relatifs aux moyens de lutte contre Campylobacter menés dans ces réservoirs. Ce travail a également permis de mettre en évidence de potentielles spécificités nationales dans la dynamique de transmission de C. jejuni à l’Homme. / Campylobacter is the causal agent of the main bacterial foodborne gastroenteritis in Europe. Since Campylobacter is frequently found in animal reservoirs, sources of human infection and transmission routes are various. However, to decrease the human burden of campylobacteriosis, it is essential to quantify the relative importance of the several reservoirs in human infections. For this purpose, we assessed the contamination of chicken, cattle and pets by Campylobacter spp., and further characterized C. jejuni isolates using MLST (Multilocus Sequence Typing) and CGF (Comparative Genomic Fingerprinting) in comparison with French clinical isolates. Then, in order to identify the most likely origin of campylobacteriosis cases in France and overcome MLST limitations in source attribution, about 800 C. jejuni genomes were analyzed which resulted in the identification of 15 genes as promising host segregating markers for source attribution. Subsequently, we assessed the ability of MLST, CGF40 and the 15 host-segregating markers to identify the most likely origin of campylobacteriosis. The 15 host-segregating markers were the most powerful in source attribution, followed by MLST, while CGF40 appeared to be not suitable for source attribution in our study. Based on MLST and the 15 markers, assignments of clinical cases emphasize the significant implication of chicken and ruminant in human infection by Campylobacter, while pets and the environment (including water and wild birds) were slightly involved, reinforcing the interest to focus control strategies on livestock. Finally this work highlights potential national variations in the transmission dynamics of C. jejuni to human.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21614号 / 理博第4521号 / 新制||理||1649(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 平田 聡 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The aim of this thesis is to create an algorithm that allows the alignment of the genome of two organisms by means of suffix structures and to implement it into the programming language environment R. The thesis deals with the description of the construction of the suffix structures and the methods of whole genome alignment. The result of the thesis is a functional algorithm for whole genome alignment by means of suffix structures implemented in the software environment R and its comparison with similar programs for the whole genome alignment.

<p>Humans have altered natural landscape since the agricultural revolution, but it has been most destructive since human globalization and rampant industrialization in the last two centuries. These activities deteriorate and fragments natural habitat of many wild species that creates small isolated populations that lose genetic diversity over time. Loss of genetic diversity reduces the adaptive capacity of a population to respond to future environmental change and increases their extinction risks. Implementing strategies for wildlife conservation is a challenge primarily because of our lack of understanding of the biology of many wild species, the risks they are currently facing, and their evolutionary histories. With the advent of genomic and computational techniques, it is now possible to address these concerns. In my research, I used genomics to study the evolutionary history of the Montezuma Quail (<i>Cyrtonyx montezumae</i>) and created monitoring tools that can be readily applied by wildlife managers for its conservation. Montezuma Quail is a small gamebird found mostly in Mexico with peripheral populations existing in Arizona, New Mexico, and Texas. Montezuma Quail are going through species wide decline in the United States and are listed as vulnerable in the state of Texas due to their small population sizes and geographic isolation from rest of the range. My results show that Texas quail are genetically distinct and significantly less diverse than Arizona quail. Analysis of whole genome sequences from multiple individuals show that due to small population sizes and isolation, Texas quail are significantly more inbred and genetic drift is the major contributor for loss of genetic diversity we see today. Inbreeding is negatively impacting Texas quail as they carry more deleterious alleles within their genome that reduce fitness of the individuals. Demographic models predict that both Arizona and Texas populations were formed via founding bottlenecks around 20,000 years ago. Texas populations have maintained small population sizes since its split from the ancestral populations and are less efficient in purging new deleterious mutations that arise post-bottleneck. The inferences from my research not only carries direct implications for Montezuma Quail conservationists, but also illustrate the power of evolutionary genomics in implementing targeted management strategies for any species that face existential threats in today’s waning world. </p>

Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...