Next-generation sequencing methods now allow rapid and cost-effective sequencing of DNA on a scale not previously possible. This offers great opportunities for the research of Mendelian disorders, but also significant challenges. The sequencing of exomes, or whole genomes, has emerged as a powerful clinical research tool, with targeted gene analyses generally being preferred in the clinical diagnostic setting. These methods have been employed here with the aim of identifying novel genetic causes of inherited heart disorders and to gain insights into the utility and limitations of these techniques for clinical diagnosis in these disorders. Data produced from the introduction of a targeted multi-gene next-generation sequencing test into clinical practice has been studied. Variation within the mitochondrial genome has been analysed to assess the importance of mitochondrial DNA variants in patients with hypertrophic cardiomyopathy. The m.4300A>G mutation is identified as an important cause of this disorder, with other previously cardiomyopathy-associated and novel variants also identified. Such multi-gene tests can facilitate interpretable and phenotype-relevant results, but at the expense of limiting more extensive data acquisition. Whole-genome sequencing has been performed in five families with different autosomal dominant inherited heart disease phenotypes of unknown genetic aetiology. In two of these likely pathogenic variants were identified, one in the gene encoding titin (TTN) and the other in the calcium channel subunit gene CACNA1C. In vitro studies were undertaken to support the pathogenicity of the TTN variant and understand the functional effects of this. In the other three families either multiple candidate gene variants were identified or no clear candidate variant was identified. This highlights the difficulties in interpreting these results, even in carefully selected families. Overall, although the research benefits of exome or genome studies are evident, the interpretation and validation of genetic variant data produced remains highly challenging for clinical diagnosis.

In recent years, the genetic landscape of B-cell derived lymphoid malignancies, including chronic lymphocytic leukemia (CLL), has been rapidly unraveled, identifying recurrent genetic mutations with potential clinical impact. Interestingly, ~30% of all CLL patients can be assigned to more homogeneous subsets based on the expression of a similar or “stereotyped” B-cell receptor (BcR). Considering that biased distribution of genetic mutations was recently indicated in specific stereotyped subsets, in paper I, we screened 565 subset cases, preferentially assigned to clinically aggressive subsets, and confirm the SF3B1 mutational bias in subset #2 (45%), but also report on similarly marked enrichment in subset #3 (46%). In contrast, NOTCH1 mutations were predominantly detected in subsets #1, #8, #59 and #99 (22-34%). This data further highlights a subset-biased acquisition of genetic mutations in the pathogenesis of at least certain subsets. Aberrant NF-κB signaling due to a deletion within the NFKBIE gene previously reported in CLL warranted extended investigation in other lymphoid malignancies. Therefore, in paper II, we screened 1460 patients with various lymphoid malignancies for NFKBIE deletions and reported enrichment in classical Hodgkin lymphoma (27%) and primary mediastinal B-cell lymphoma (PMBL) (23%). NFKBIE-deleted PMBL cases had higher rates of chemorefractoriness and inferior overall survival (OS). NFKBIE-deletion status remained an independent prognostic marker in multivariate analysis. EGR2 mutations were recently reported in advanced stage CLL patients; thus, in paper III we screened 2403 CLL patients for mutations in EGR2. An overall mutational frequency of 3.8% was reported and EGR2 mutations were associated with younger age, advanced stage and del(11q). EGR2 mutational status remained an independent marker of poor outcome in multivariate analysis, both in the screening and validation cohorts. Whole-genome sequencing (WGS) of 70 CLL cases, assigned to poor-prognostic subsets #1 and #2 and indolent subset #4, were investigated in Paper IV and revealed a similar skewing of SF3B1 mutations in subset #2 and NOTCH1 mutations in subset #1 to that reported in Paper I. Additionally, an increased frequency of the recently proposed CLL driver gene RPS15 was observed in subset #1. Finally, novel non-coding mutational biases were detected in both subset #1 and #2 that warrant further investigation.

La mucoviscidose est une maladie génétique autosomique causée par une mutation dans le gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Mon travail s’est décomposé en deux parties principales : d’une part j’ai réalisé une revue de la littérature sur l’analyse des génomes bactériens isolés de patients mucoviscidosiques comparativement aux génomes des mêmes espèces isolées dans d’autrescontextes et d’autre part j’ai analysé les génomes de trois espèces bactériennes (Microbacterium yannicii, Chryseobacterium oranimense et Haemophilus parahaemolyticus). L’analyse exhaustive des génomes bactériens issus de patients atteints de mucoviscidose a révélé une extraordinaire évolution de ces génomes en fonction du temps et des traitements reçus par ces patients qui témoigne de la capacité qu’ont ces bactéries à s’adapter à leur écosystème notamment par l’acquisition de nouveaux gènes par transfert latéral de gènes. Ce travail montre l’extraordinaire plasticité des génomes bactériens dans un milieu donné et à ce titre le poumon de patients atteints de mucoviscidose représente un modèle unique pour comprendre l’évolution des génomes bactériens. De plus, notre travail a permis d’identifier leurs mécanismes moléculaires de résistance aux antibiotiques. Les travaux à venir sur l’étude des métagénomes de prélèvements chez ces patients pourrait permettre de répondre à ces questions dans le futur. La découverte de nouvelles espèces et / ou émergentes va nous permettre d’avoir une image plus complète de la mucoviscidose qui pourrait conduire à une meilleure connaissance de la maladie et donc à une meilleure prise en charge thérapeutique. / Cystic fibrosis is an autosomal genetic disorder caused by a mutation in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. Pulmonary infection is the major problem faced by patients with cystic fibrosis. My work is divided into two main parts: first I made a review of the literature on the analysis of bacterial genomes isolated from CF patients compared to the genomes of the same species isolated in autrescontextes and other part I analyzed the genomes of three species of bacteria (Microbacterium yannicii, Chryseobacterium oranimense and Haemophilus parahaemolyticus). The comprehensive analysis of bacterial genomes from cystic fibrosis patients revealed an extraordinary evolution of these genomes with time and treatment received by these patients reflects the ability of these bacteria to adapt to their particular ecosystem the acquisition of new genes by lateral gene transfer. This work shows the extraordinary plasticity of bacterial genomes in a given environment and as the lungs of patients with cystic fibrosis represents a unique model for understanding the evolution of bacterial genomes. In addition, our work has identified their molecular mechanisms of resistance to antibiotics. Future work on the study of metagenomes sampling in these patients could help to answer these questions in the future. The discovery of new species and / or emerging will allow us to have a more complete picture of cystic fibrosis which could lead to a better understanding of the disease and thus a better therapeutic management.

Antimicrobial resistance (AMR) is a worldwide public health concern. While it is evident that the use of antibiotics creates selection pressure for the evolution of antibiotic resistance genes, there are still considerable knowledge gaps relating to the status quo of antibiotic use, emergence of resistant pathogens in different livestock production systems and spread within human and animal communities. This thesis includes a survey of antibiotic use in the dairy sector within a specific area of Zambia and analysis of AMR and virulence factors in E. coli isolated from dairy cattle and diarrhoea human patients with the following objectives. 1. To investigate the usage of antibiotics in the dairy sector and the drivers for use. 2. To determine the prevalence and patterns of antimicrobial resistance in E. coli isolated from faecal samples of dairy cattle. 3. To use whole genome sequencing (WGS) to investigate the molecular epidemiology of resistance determinants in E. coli strains isolated from both dairy cattle and humans. 4. To assess the zoonotic potential of isolated E. coli focusing on Shiga toxin-producing E. coli (STEC) and relationship to STEC associated with clinical disease in the UK. In view of these objectives, the first part of the work was carried out in Zambia and involved a questionnaire, a field survey, isolation of E. coli from dairy cattle faecal samples and phenotypic testing for AMR. In addition, E. coli isolates were obtained from another study that was focused on human patients presenting with diarrhoea at the University Teaching Hospital in Lusaka. The second part involved whole genome sequencing and molecular analyses of E. coli for resistance and virulence genotypes at the Roslin Institute (UK). For the field study, a stratified random sample of 104 farms was studied, representing approximately 20% of all dairy farms in the region. On each farm, faecal samples were collected from a random sample of animals and a standardised questionnaire on the usage of antibiotics was completed. An E. coli isolate was obtained from 98.67% (371/376) of the sampled animals and tested for resistance against the six types of antibiotics (tetracycline, ampicillin, sulfamethoxazole/trimethoprim, cefpodoxime, gentamicin and ciprofloxacin). These E. coli were then analysed together with those from humans for genotypes in the laboratory and from Illumina short read whole genome sequences using bioinformatics tools. Tetracylines and penicillin were the commonly used antibiotics in dairy herds. This finding was in line with the resistance phenotypes detected in E. coli isolated from the dairy cattle. The most prevalent AMR was to tetracycline (10.61; 95%CI: 7.40-13.82), followed by ampicillin (6.02; 95%CI: 3.31-8.73), sulfamethoxazole/ trimethoprim (4.49; 95%CI: 2.42-6.56), cefpodoxime (1.91; 95%CI: 0.46-3.36), gentamicin (0.89; 95%CI: 0.06-1.84) and ciprofloxacin (0%). The risk analysis indicated that AMR was associated with livestock diseases (lumpy skin disease and foot rot), exotic breeds (Jersey and Friesian), location, farm size and certain management practices. Analysis of whole genome sequences showed that isolates from humans had both higher levels and a greater diversity of resistance alleles than the cattle isolates. Common genotypes in both populations were: tetA (16%), tetB (10%), tetC (2%) for cattle isolates with tetA (32%), tetB (22%) and tetD (1%) in human isolates. Other common genotypes were blaTEM (56%), sul1 (29%), sul2 (66%), strA4 (57%) and strB1 (64%) in isolates of human origin while blaTEM (15%), sul1 (3%), sul2 (17%), strA4 (13%) and strB1 (19%) were in the cattle isolates. Whilst the E. coli isolates from cattle encoded resistance to common antibiotics of limited significance to human clinical medicine, isolates from humans had additional extended spectrum beta-lactamases (blaOXA, blaCMY, blaNDM, and blaDHA, blaOKP and blaCTX-M) that encode for resistance to essential antibiotics such as third generation cephalosporins and carbapenems. This was an evidence that AMR is an ongoing public health subject in Zambia but the exclusivity of certain resistances in the human population points to limited or no exchange of genotypes between E. coli of human origin and those from cattle. AMR in humans was probably independently selected by the use of antibiotics of clinical importance such as cephalosporin and fluoroquinolones. The virulence analysis focused on STEC, 11% (41/371) of E. coli isolates from cattle contained Shiga toxin genes (stx) while none (0/73) of the human isolates were positive. Phylogenetic analysis showed a random distribution of bovine STEC, with no indication of clonal spread. Although 89% (16/18) of the STEC tested had a cytotoxic effect on Vero cells, indicative of Shiga toxin production, only three (O45, O111, O157) belonged to one of the seven serogroups (O26, O157, O111, O103, O121, O145 and O45) associated with life-threatening enterohaemorrhagic E. coli (EHEC) infections in humans. In line with this, only the O157 serotype encoded a type 3 secretion system. This shows that, while Stx-encoding strains are common in these dairy herds of Zambia, they are not strain backgrounds known to pose an immediate threat to human health as they lack colonisation factors that are found in typical human EHEC. However, we must remain vigilant as emergence of EHEC strains in these animals remains an ever-present threat.

CCCTC-binding factor (CTCF) binds DNA, thereby helping to partition the mammalian genome into discrete structural and regulatory domains. In doing so, it insulates chromatin and fine-tunes gene activation, repression, and silencing. Complete removal of CTCF from mammalian cells causes catastrophic genomic dysregulation, most likely due to widespread collapse of 3D chromatin looping within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction in CTCF expression are viable but have an increased incidence of spontaneous multi-lineage malignancies. In addition, CTCF is mutated in many human cancers and is thus implicated as a tumour suppressor gene. This study aimed to interrogate the genome-wide consequences of a reduced genomic concentration of Ctcf and its implications for carcinogenesis. In a genetically engineered mouse model, Ctcf hemizygous cells showed modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these were enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, several hundred genes concentrated in cancer-related pathways were dysregulated due to changes in transcriptional regulation. Global chromatin structure was preserved but some loop interactions were destabilised, often around differentially expressed genes and their enhancers. Importantly, these transcriptional alterations were also seen in human cancers. These findings were then examined in a hepatocyte-specific mouse model of Ctcf hemizygosity with diethylnitrosamine-induced liver tumours. Ctcf hemizygous mice had a subtle liver-specific phenotype, although the overall tumour burden in Ctcf hemizygous and wild-type mice was the same. Using whole genome sequencing, the highly reproducible mutational signature caused by DEN exposure was characterised, revealing that Braf(V637E), orthologous to BRAF(V600E) in humans, was the predominant oncogenic driver in these liver tumours. Taken together, while Ctcf loss is partially physiologically compensated, chronic CTCF depletion dysregulates gene expression by subtly altering transcriptional regulation. This study also represents the first comprehensive genome-wide and histopathological characterisation of this commonly used liver cancer model.

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.

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.

<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>

A compound-heterozygous variant occurs when a child inherits a variant from each parent, with these variants occurring at a different position within the same gene and on opposite homologous chromosomes. These inherited variants may result in two nonfunctional versions of the same gene. Compound-heterozygous variants cannot be identified unless a patients' DNA sequence data is phased. Phasing is a computationally demanding process that requires the use of multiple software tools in order to determine which nucleotide was inherited from which parent. First, in Chapter 1, we review the literature to better understand what research has been conducted on the role of compound-heterozygous variants in pediatric cancers and what methods are being used to identify them. In Chapter 2, we develop a pipeline to make it easier for us and other researchers to phase and identify compound-heterozygous variants using VCF files from trios or individuals. We then use this pipeline in Chapter 3 to survey the prevalence of compound-heterozygous variants across 7 pediatric disease types. We show the importance of identifying compound heterozygous and what information would be missed if this variant type was not included in study design. In Chapter 4, we develop a software tool to phase trio data using a combination of Mendelian inheritance logic and an existing phasing software program. We show that our software tool increases the total number of variants that can be phased. Finally, in Chapter 5, we use phased data of three nuclear families, each family having one child with pediatric cancer, to evaluate the potential to use inherited genomic variants to inform diagnostic decisions. The work contained within this dissertation shows the importance of not overlooking compound-heterozygous variants when trying to identify potentially causal genes in pediatric disease. In addition, this work provides software tools that are openly available for other researchers to use; these tools make it easier to phase patient DNA sequence data and to identify compound-heterozygous variants.