Yes / Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting. / ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Yes / BACKGROUND: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis allows rapid, accurate inferences about the sources, location and timing of transmission. However, in an era of heightened concern for personal privacy and science distrust, such inferences could result in unintended harm and undermine the public´s trust. METHODS: We held interdisciplinary stakeholder discussions and performed ethical analyses of real-world illustrative cases to identify principles that optimise benefit and mitigate harm of M. tuberculosis WGS-driven TB source investigations.RESULTS: The speed and precision with which real-time WGS can be used to associate M. tuberculosis strains with sensitive information has raised important concerns. While detailed understanding of transmission events could mitigate harm to vulnerable patients and communities when otherwise unfairly blamed for TB outbreaks, the precision of WGS can also identify transmission events resulting in social blame, fear, discrimination, individual or location stigma, and the use of defaming language by the public, politicians and scientists. Public health programmes should balance the need to safeguard privacy with public health goals, transparency and individual rights, including the right to know who infects whom or where.CONCLUSIONS: Ethical challenges raised by real-time WGS-driven TB source investigation requires public health authorities to move beyond their current legal mandate and embrace transparency, privacy and community engagement.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT>MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV<15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.

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.