Dissertations / Theses on the topic 'Genomic techniques'

Constructing the genomic median of several given genomes is crucial in developing evolutionary trees, since the genomic median provides an estimate for the ordering of the genes in a common ancestor of the given genomes. This is due to the fact that the content of DNA molecules is often similar, but the difference is mainly in the order in which the genes appear in various genomes. The mutations that affect this ordering are called genome rearrangements, and many structural differences between genomes can be studied using genome rearrangements. In this thesis our main focus is on applying combinatorial optimization techniques to genomic median problems, with particular emphasis on the breakpoint distance as a measure of the difference between two genomes. We will study different variations of the breakpoint median problem from signed to unsigned, unichromosomal to multichromosomal, and linear to circular to mixed. We show how these median problems can be formulated in terms of problems in combinatorial optimization, and take advantage of well-known combinatorial optimization techniques and apply these powerful methods to study various median problems. Some of these median problems are polynomial and many are NP-hard. We find efficient algorithms and approximation methods for median problems based on well-known combinatorial optimization structures. The focus is on algorithmic and combinatorial aspects of genomic medians, and how they can be utilized to obtain optimal median solutions.

The PhD research studied two aspects in tilapia, firstly the analysis of sex determination in Nile tilapia (evidence of complex sex-determining systems) and secondly the genetic management of the tilapia species, using different genomic analysis approaches. This research started with the development of two techniques: minimally invasive DNA sampling from fish mucus, which was found to be suitable for standard genotyping and double-digest restriction-site associated DNA sequencing – ddRADseq; and pre-extraction pooling of tissue samples for ddRADseq (BSA-ddRADseq), which was found to be suitable for identifying a locus linked to a trait of interest (sex in this case). The first molecular evidence concerning the sex determination in genetically improved farmed tilapia (GIFT) was described using BSA-ddRADseq. Given the multiple stock origin of GIFT, surprisingly only a single locus (in linkage group 23) was found to be associated with the phenotypic sex across the population. The first evidence of LG23 influence on phenotypic sex in the Stirling population of Nile tilapia was also found. Different combinations of estrogen hormones and high temperature were tested for feminising Nile tilapia: a combined treatment of estrogen hormone and high temperature was found to be more efficient in feminising Nile tilapia than the estrogen alone. A set of species-diagnostic SNP markers were tested which were found to be suitable to distinguish pure species (O. niloticus, O. mossambicus and O. aureus), and these were used to analyse species contribution to GIFT and a selected tilapia hybrid strain. The results of the current research added novel information to our understanding of sex determination in Nile tilapia, which will be helpful in the development of marker-assisted selection in GIFT and other Nile tilapia strains towards the production of all male offspring. The methods developed also have broader applicability in genetic and genomics research.

Tesis por compendio
[ES] La longevidad de las semillas, o el tiempo durante el cual permanecen las semillas viables, es de gran importancia para la conservación de la biodiversidad, la agricultura y la economía. Además, el estudio de este parámetro puede contribuir a conocer mejor los mecanismos moleculares comunes a todos los organismos para prevenir el envejecimiento. Una de las principales estrategias de las semillas para ralentizar su envejecimiento consiste en detener su metabolismo, a través de su deshidratación. Otros mecanismos moleculares para evitar daños son el aislamiento frente al entorno a través de la cubierta de la semilla, y la producción de antioxidantes y otras moléculas para evitar el daño oxidativo, uno de los principales causantes del envejecimiento de las semillas. Los mecanismos de reparación mitigan parte del daño acumulado. El organismo modelo de plantas Arabidopsis thaliana brinda la oportunidad de la realización de estudios genómicos para el estudio de, en este caso, la longevidad de las semillas para descubrir nuevos factores genéticos y mecanismos moleculares determinantes. Este conocimiento servirá para entender mejor los procesos de deterioro de las semillas y que también será clave para aumentar la longevidad de estas. Mediante el uso de variedades naturales genotipadas de Arabidopsis thaliana y un estudio de asociación del genoma conocido como GWAS, seguido de estudios de genética reversa, se han identificado 12 nuevos genes relacionados con la longevidad de las semillas, relacionados con la protección del embrión, el control del daño oxidativo, y la permeabilidad de la cubierta de la semilla. El desarrollo de la cubierta de la semilla está determinado por factores de transcripción. Plantas mutantes en diversos factores de transcripción involucrados en el desarrollo de la cubierta de la semilla presentan una longevidad alterada. La sobreexpresión de los factores de transcripción AtHB25 y COG1 provoca que las semillas presenten una mayor longevidad debido a una incrementada deposición de poliésteres lipídicos. Estas barreras de poliésteres lipídicos son la cutícula, formada por cutina, y la suberina. Ambas participan positivamente en la protección del embrión frente al ambiente exterior. Estudios genómicos de ambos factores de transcripción han demostrado que AtHB25 regula directamente a enzimas biosintéticos de los monómeros de suberina y cutina, y COG1 regula la expresión de enzimas relacionados con la polimerización de poliésteres lipídicos y lignina. La regulación en la que participa AtHB25 es muy importante debido a la alta conservación de las secuencias genómicas y funciones de AtHB25 en angiospermas, y parece involucrado en la respuesta a bajas temperaturas. Por otra parte, COG1, que está involucrado en la percepción de luz, regula parte del desarrollo del tegumento externo a través de la regulación de AP2, un factor clave en el establecimiento de la identidad de tejido de este tegumento de la cubierta de la semilla, donde se localiza la suberina. AtHB25 y COG1 están involucrados en la adaptación de la longevidad de la semilla a través de señales ambientales como la temperatura y la luz, respectivamente, regulando la deposición de poliésteres lipídicos.
[CAT] La longevitat de les llavors, o el temps que romanen les llavors viables, es de gran importància per la conservació de la biodiversitat, l'agricultura i l'economia. A més a més, l'estudi d'aquest paràmetre pot contribuir a conèixer millor els mecanismes moleculars comuns a tots els organismes per prevenir l'envelliment. Una de les principals estratègies de les llavor per retardar el seu envelliment consisteix detenir el seu metabolisme, mitjançant la seua deshidratació. Altres mecanismes moleculars per evitar danys son el seu aïllament de l'entorn per mitjan de la coberta de la llavor, i la producció d'antioxidants i altres molècules per evitar el dany oxidatiu, un dels principal causants del envelliment de les llavors. Els mecanismes de reparació mitiguen part del dany acumulat. L'organisme model Arabidopsis thaliana brinda la oportunitat de la realització d'estudis genòmics per a l'estudi de, en aquest cas, la longevitat de les llavors per descobrir nous factors genètics y mecanismes moleculars determinants. Aquest coneixement servirà per entendre millor els processos de deteriorament de les llavor i serà clau per augmentar la longevitat d'aquestes. Mitjançant l'ús de varietats naturals genotipades d'Arabidopsis thaliana i un estudi d'associació del genoma conegut com GWAS, seguits d'estudis de genètica inversa, s'han identificat 12 nous gens relacionats amb la longevitat de les llavors, relacionats amb la protecció de l'embrió, el control del dany oxidatiu, y la permeabilitat de la coberta de la llavor. El desenvolupament de la coberta de la llavor està determinada per factors de transcripció. Plantes mutants a diversos factors de transcripció involucrats al desenvolupament de la coberta de les llavors presenten una longevitat alterada. La sobreexpressió dels factors de transcripció AtHB25 i COG1 provoca que les llavors presenten una major longevitat degut a una deposició de polièsters lipídics incrementada. Aquestes barreres de polièsters lipídics son la cutícula, formada per cutina, i la suberina. Ambdues participen positivament la protecció de l'embrió enfront de l'entorn exterior. Estudis genòmics d'ambdós factors de transcripció han demostrat que AtHB25 directament regula a enzims biosintètics dels monòmers de suberina i cutina i COG1 regula enzims relacionats amb la polimerització de polièsters lipídics i lignines. La regulació en la que participa AtHB25 es molt important degut a l'alta conservació de les seqüències genòmiques i funcions de AtHB25 en angiospermes, i parteix estar involucrat en la resposta a baixes temperatures. Per altra banda, COG1, que està involucrat en la percepció de la llum, regula part del desenvolupament del integument extern mitjançant la regulació de AP2, un factor clau en l'establiment de la identitat de teixit de aquest integument de la coberta de la llavor, on es localitza la suberina. AtHB25 i COG1 estan involucrats en l'adaptació de la longevitat de la llavor per mitjan de senyals ambientals com la temperatura i la llum, respectivament, regulant la deposició de polièsters lipídics.
[EN] Seed longevity, or period that seeds remain viable, is important for biodiversity conservation, agriculture and economy. In addition, the study of this parameter could ease the knowledge about molecular mechanisms common to all organisms to prevent aging. One of the main strategies of seeds to reduce their aging consists to stop their metabolism, through drying. Other molecular mechanisms to avoid damages are the isolation from the environment with the seed coat, and the production of antioxidants and other molecules to avoid oxidative damage, one of the main seed aging causes. Repair mechanisms relieve part of the accumulated damage. The model plant Arabidopsis thaliana provides the opportunity to carry out genomic studies for the research of, in this case, seed longevity to discover determinant genetic factors and molecular mechanisms. This will serve to better understand seed deterioration processes and it will be key to increase seed longevity. Using natural genotyped varieties of Arabidopsis thaliana and a genome-wide association study (GWAS) followed by reverse genetic studies, 12 new genes related to seed longevity have been identified. They are related to embryo protection, oxidative damage control, and seed coat permeability. Seed coat development is determined by transcription factors. Mutant plants in some transcription factors involved in the seed coat development present altered seed longevity. The over-expression of the transcription factors AtHB25 and COG1 resulted in seeds with increased longevity due to an increased lipid polyester deposition. These lipid polyesters barriers are the cuticle, formed by cutin, and the suberin layer. Both participate positively in the embryo protection from the external environment. Genomic studies of both transcription factors have revealed that AtHB25 directly regulates biosynthetic enzymes of suberin and cutin monomers, and COG1 regulates the expression of enzymes related to the polymerization of lipid polyesters and lignin. The regulation involving AtHB25 is crucial due to the high conservation of genomic sequences and functions of AtHB25 in angiosperms, and it seems to be involved in the response to low temperatures. On the other hand, COG1, which is involved in light perception, regulates part of the development of the external integument through its regulation by AP2, a key factor in establishing the tissue identity of this seed coat integument, where suberin is located. AtHB25 and COG1 are involved in seed longevity adaptation through environmental signals such as temperature and light, respectively, regulating lipid polyesters deposition.
Agradezco a las instituciones públicas la inversión en investigación. Gracias a ella, los laboratorios, el personal y los distintos equipos se han podido financiar. Gaetano fue quien me ayudó enormemente conseguir la beca FPI del Ministerio de Economía y Competitividad BES-2015-072096, asociada al proyecto de investigación nacional BIO2014-52621-R-AR
Renard Meseguer, J. (2021). Identification of genes related to seed longevity in Arabidopsis thaliana using genomic molecular techniques [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/170554
TESIS
Compendio

Butterflies are among the best studied animals, but despite the research efforts carried out during centuries, our knowledge on their diversity and on the mechanisms generating it is still incomplete. In order to understand how butterflies diversify, the speciation continuum of six study cases was examined using morphometrics and several genetic techniques (from sequencing specific markers to genomics). The analysis of phenotypic and genetic variation combined with gene flow evidence allowed to identify the states of the speciation continuum, i.e. to study the relationships between populations. This approach was used as a framework (1) to make grounded taxonomic hypotheses and (2) to extract clues about the mechanisms that drive speciation. As a result, we described and proposed several cases of overlooked and oversplit taxa. We documented the existence of three types of mechanisms producing diversification in butterflies: drift, selection and hybridisation. Selection acted through adaptation to biotic environmental factors, which caused reproductive character displacement, host plant shift and allochrony mediated by adaptation to host plant flowering period. Additionally, the genetic techniques employed were evaluated and their advantages and limitations discussed
Les papallones són un dels animals més ben estudiats però, malgrat els esforços dedicats a la seva recerca, el coneixement que tenim sobre la seva diversitat i sobre els mecanismes que la generen és encara incomplet. Per tal d'entendre com les papallones diversifiquen, s'ha examinat el continu de l'especiació en sis casos mitjançant l'ús de la morfometria i de diverses tècniques genètiques (des de la seqüenciació de marcadors específics fins a la genòmica). L'anàlisi de la variació fenotípica i genètica combinada amb evidències sobre el flux genètic ha permès identificar els estats del continu de l'especiació, i.e. l'estudi de les relacions entre poblacions. Aquesta aproximació s'ha usat com a marc (1) per fer hipòtesis taxonòmiques fonamentades i (2) per extreure pistes sobre els mecanismes que dirigeixen l'especiació. Com a resultat, hem descrit i proposat diversos casos de tàxons que havien passat desapercebuts o que s'havien dividit excessivament. Documentem l'existència de tres tipus de mecanismes productors de diversitat en papallones: deriva, selecció i hibridació. La selecció actuà mitjançant l'adaptació a factors ambientals biòtics, que causaren desplaçament de caràcters reproductius, canvi de planta nutrícia i a\ll ocronia produïda per l'adaptació al període de floració de la planta nutrícia. Addicionalment, les tècniques genètiques emprades són avaluades i els seus avantatges i inconvenients discutits.

Mon projet de doctorat était de créer un pipeline pour taxono-génomique pour la comparaison de plusieurs génomes bactériens. Deuxièmement, je automatisé le processus d'assemblage (NGS) et annotation à l'aide de divers logiciels open source ainsi que la création de scripts de maison pour le laboratoire. Enfin, nous avons intégré le pipeline dans la description de plusieurs espèces bactériennes de laboratoire sur. Cette thèse est divisée principalement en Taxono- génomique et Microbiogenomics. Les avis de la section taxono-génomique, décrit sur les avancées technologiques en génomique et métagénomique pertinentes dans le domaine de la microbiologie médicale et décrit la stratégie taxono-génomique en détail et comment la stratégie polyphasique avec des approches génomiques sont reformatage de la définition de la taxonomie bactérienne. Les articles décrivent les bactéries cliniquement importantes, leur séquençage complet du génome et les études génomiques comparatives, génomiques et taxono-génomique de ces bactéries. Dans cette thèse, j'ai inclus les articles décrivant ces organismes: Megasphaera massiliensis, Corynebacterium ihumii, Collinsella massiliensis, Clostridium dakarense. Bacillus dielmoensis, jeddahense, Occidentia Massiliensis, Necropsobacter rosorum et Pantoea septica. Oceanobacillus
My PhD project was to create a pipeline for taxono-genomics for the comparison of multiple bacterial genomes. Secondly I automated the process of assembly (NGS) and annotation using various open source softwares as well as creating in house scripts for the lab. Finally we incorporated the pipeline in describing several bacterial species from out lab. This thesis is subdivided mainly into Taxono-genomics and Microbiogenomics. The reviews in taxono-genomics section, describes about the technological advances in genomics and metagenomics relevant to the field of medical microbiology and describes the strategy taxono-genomics in detail and how polyphasic strategy along with genomic approaches are reformatting the definition of bacterial taxonomy. The articles describes clinically important bacteria, their whole genome sequencing and the genomic, comparative genomic and taxono-genomic studies of these bacteria

Magister Scientiae - MSc
Viral genomes consist of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The viral RNA molecules are responsible for two functions, firstly, their sequences contain the genetic code, which encodes the viral proteins, and secondly, they may form structural elements important in the regulation of the viral life-cycle. Using a host of computational and bioinformatics techniques we investigated how predicted secondary structure may influence the evolutionary dynamics of a group of single-stranded RNA viruses from the Picornaviridae family. We detected significant and marginally significant correlations between regions predicted to be structured and synonymous substitution constraints in these regions, suggesting that selection may be acting on those sites to maintain the integrity of certain structures. Additionally, coevolution analysis showed that nucleotides predicted to be base paired, tended to co-evolve with one another in a complimentary fashion in four out of the eleven species examined. Our analyses were then focused on individual structural elements within the genome-wide predicted structures. We ranked the predicted secondary structural elements according to their degree of evolutionary conservation, their associated synonymous substitution rates and the degree to which nucleotides predicted to be base paired coevolved with one another. Top ranking structures coincided with well characterized secondary structures that have been previously described in the literature. We also assessed the impact that genomic secondary structures had on the recombinational dynamics of picornavirus genomes, observing a strong tendency for recombination breakpoints to occur in non-coding regions. However, convincing evidence for the association between the distribution of predicted RNA structural elements and breakpoint clustering was not detected.

Thesis (MSc)--University of Stellenbosch, 2008.
ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification.
AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.

L'objectif de notre thèse est d'appliquer la génomique en temps réel pour déchiffrer les caractéristiques génomiques bactériennes et les événements de recombinaison du génome des bactéries atypiques ainsi que leur impact sur les maladies infectieuses. Au cours de ma thèse, nous avons effectué une revue sur les outils bioinformatiques les plus courants utilisés en microbiologie clinique et mis en évidence l’impact de la recombinaison sur le comportement des bacteries. Le deuxième projet de notre thèse est de déchiffrer une epidémis de Staphylococcus saprophyticus causant des infections urinaires en utilisant la technologie MALDI-TOF MS et une analyse comparative du génome de S. saprophyticus pour comprendre leur évolution génomique. Nous avons démontré qu'il existe un groupe de S. saprophyticus géographiquement restreint à Marseille comparé au souches de Nice. De plus, nous avons montré que S. saprophyticus qui était initialement considéré comme une bactérie saprophyte a evolué pour devenir une bactérie pathogène à travers des recombinaisons massives et des « single nucleotide polymorphism », résultant d'une perte significative de gènes. Le troisième projet de notre thèse est une analyse comparative des génomes d'Enterococcus faecalis et d'E.faecium isolé chez l'homme, les animaux et l'environnement pour déchiffrer la différence de propagation et l'acquisition de déterminants antimicrobiens. Nous avons démontré qu'il existe une association directe entre l'absence de système CRISPR, la présence du gène ardA et l'acquisition de gènes de résistance à la vancomycine, qui différencient E. faecalis de E. faecium. Enfin nous avons decrit un nouveau genre bacterien Nissabacter
The objective of our thesis is to applied the Real-time genomic approaches to decipher bacterial genomic features and genome recombination events of atypical bacteria and their impact on infectious diseases. During my thesis, we have reviewed the most common bioinformatics tools applicable in clinical microbiology and highlight how bacterial genome recombination have impacted their behaviour. The second project of our PhD is to decipher a community outbreak of Staphylococcus saprophyticus involved in (UTI) using MALDI-TOF MS technology and a comparative genome analysis of clinical and non-clinical S. saprophyticus to understand their genomic evolution. We demonstrated that there is a geographically restricted cluster of S. saprophyticus circulating in Marseille community as compared to Nice. Moreover, we showed that S. saprophyticus which was initially considered as a saprophytic bacterium has drifted to becoming a pathogenic bacterium through massive genome recombination and single nucleotide polymorphism events, resulting from a significant loss of genes. The third project of our work is a comparative genome evolutionary analysis of Enterococcus faecalis and Enterococcus faecium isolated from human, animals, and environment to decipher the difference in spread and the acquisition of antimicrobial determinants. We demonstrated that there is a direct association between the absence of CRISPR system, the presence of gene ardA and the acquisition of vancomycin resistance genes, which differentiate E. faecalis from E. faecium. Our final project was focused on the discovering of a new genus Nissabacter and its description

Dans le champ de la recherche en génomique, comme dans d'autres domaines très informatisés, les bases de données et les biobanques sont organisées en infrastructures. Ce nouveau modèle organisationnel doit permettre de soutenir l'effort technique et collaboratif requis pour traiter des Big Data, c'est-à-dire des jeux de données trop volumineux et complexes pour être traités en utilisant les méthodes classiques. L'établissement de ces nouveaux environnements constitue un véritable défi technique et philosophique. Il requiert, pour être opérationnel, des cadres réglementaires adaptés, ouverts à la fois à l'internationalisation et à des perspectives de long terme, mais certains de ces changements ne sont pas compatibles avec les procédures éthiques courantes, notamment la procédure de consentement éclairé. L'éthique de la recherche en génomique doit donc être repensée. Faut-il puiser dans la technique les nouvelles solutions de gouvernance de la recherche ? Ou bien est-il plus juste de répondre à ces évolutions en analysant les situations de tension morale suscitées par de nouveaux développements et en décidant de les traiter en fonction de ce à quoi nous tenons collectivement ? L'enjeu de ce travail, qui relève d'une approche pragmatiste, consiste à cultiver une attitude réflexive à propos des changements en cours dans la recherche en génomique. Cette tâche suppose d'expliciter le rôle des biobanques et des bases de données dans la production, la validation et la publication de la recherche génomique. Il est également nécessaire de rendre compte des tensions auxquelles le développement de ces dispositifs donne lieu lorsqu'ils sont incompatibles avec les procédures actuelles. On peut alors examiner si les dispositifs tels qu'ils sont conçus sont désirables dans les contextes où ils sont développés, soulignant ainsi la dimension politique de l'éthique de la recherche. Cette thèse repose sur l'analyse de situations concrètes issues de projets de recherche dans lesquels nous avons été impliquée. Nous utilisons aussi plusieurs disciplines étudiant la science telle qu'elle se fait (philosophie, anthropologie, sociologie et histoire). Au cours de cet examen, l'idée régulatrice de personne-membre est proposée, pour favoriser la prise en compte des appartenances sociales et politiques du sujet de l'éthique de la recherche en génomique
In genomic research, as in other highly computerised scientific fields, databases and biobanks are today (re-)organised into infrastructures. This new organisational model should support the technical and collaborative effort needed to deal with Big Data, that is, data sets that are too large and too complex to be treated with conventional methods. Establishing these new environments is an actual technical challenge that requires, in order to be operational, appropriate regulatory frameworks that are both open to internationalisation and long-term prospects. But some of these changes are not consistent with current ethics procedures, including the informed consent process. The ethics of genomics research must therefore be reconsidered by asking whether it is in technology that we must draw new solutions for the governance of research or whether we must respond to these evolutions by proposing a political treatment to clarify what we value collectively. This work, which is based on a pragmatist approach, intends to cultivate a reflexive attitude on the changes being made in genomic research by describing situations of moral tension. This requires elucidating the role of biobanks and databases in the production, validation and publication of genomic research; accounting for the conflicts of values to which the development of these devices can give rise when they are incompatible with the current procedures and thus to examine whether the devices as conceived are desirable in the contexts where they are developed. This thesis is based on the analysis of concrete situations, resulting from research projects in which we have been involved or from studies of science in practices (philosophy, anthropology, sociology and history). During this examination, the regulatory idea of a person-member is proposed, in order to favor the consideration of the social and political affiliations of the subject of ethics to research in genomics

A neoplasia endócrina múltipla tipo 1 (NEM1) é uma doença genética, de herança autossômica dominante, caracterizada pelo desenvolvimento de tumores endócrinos acometendo, principalmente, hipófise, paratireoide e pâncreas/duodeno endócrinos. É causada, principalmente, por mutação germinativa no gene supressor tumoral MEN1 (11q13). A tumorigênese segue o modelo de Knudson (1971). O diagnóstico genético de famílias com NEM1 reconhece os portadores assintomáticos de mutação MEN1, permite o diagnóstico e tratamento precoce de tumores, promove a redução da morbimortalidade relacionada à NEM1 e exclui familiares não portadores de mutação do rastreamento clínico periódico. O diagnóstico genético de NEM1 tem sido realizado por meio da técnica de sequenciamento Sanger. Entretanto, limitações desta técnica a tornam menos custo efetiva, devido a sua reduzida capacidade de geração de dados, que leva a necessidade de obtenção de produtos de PCR de até 700 pb para adequada leitura do sequenciamento. Além disto, condições específicas do gene MEN1, como a ausência de \"hot spots\" mutacionais, levam a necessidade de sequenciamento de toda sua extensão (7Kb) e contribuem para tornar esta técnica laboriosa e dispendiosa. A subdivisão do gene para sequenciamento Sanger pode ocultar informações, principalmente de regiões intrônicas, que podem ser importantes para o desenvolvimento da doença. Tais dificuldades impedem a incorporação do diagnóstico gênico de NEM1 na prática clínica. Desde 2005, estão disponíveis tecnologias denominadas NGS (Next- Generation Sequencing), que consistem em ferramentas para o sequenciamento genético com capacidade aumentada de geração de dados, tornando-as mais atrativas e de melhor custo-benefício. O NGS confere, ainda, maior velocidade ao processo de obtenção de dados e detém a capacidade de realizar a leitura completa do gene, incluindo regiões promotoras e intrônicas. Por isto, torna a leitura mais ampla e informativa, sem desconsiderar aspectos qualitativos. Dentre várias opções de NGS disponíveis, plataformas leves são consideradas mais adequadas para aplicação clínica, destacando-se as plataformas Ion PGM e Illumina MiSeq. Uma forte tendência tem sido mostrada de migração do sequenciamento Sanger para o NGS, incluindo a aplicação da mesma em diagnóstico genético de doenças complexas e de câncer hereditário. Entretanto, não há estudos prévios envolvendo NGS em NEM1. Diante disto, foi avaliado a qualidade desta técnica como método de diagnóstico genético em NEM1 em comparação ao sequenciamento Sanger. Objetivos: validação da técnica de NGS utilizando como parâmetro o sequenciamento Sanger; avaliação da sensibilidade, especificidade e relação custo-benefício do NGS. Para tal, foram analisados 76 casos-índices com diagnóstico clínico de NEM1 na plataforma Illumina MiSeq. As análises foram subdivididas em duas fases. O enriquecimento da região genômica do gene MEN1 foi realizado por meio de PCR longa. Com base nos dados obtidos foi possível aferir 96% de reprodutibilidade entre as diferentes fases do estudo e aproximadamente 99% de precisão para detecção de variantes. Exatidão, sensibilidade e especificidade resultaram em 100%. Não houve falsos-positivos ou negativos. A técnica de NGS também se mostrou mais custo-efetiva do que o sequenciamento Sanger. Este estudo permitiu validar e introduzir esta técnica como ferramenta de diagnóstico gênico de NEM1 para rastreamento genético de casos-índices
The multiple endocrine neoplasia type 1 (MEN1) is a genetic, autossomic and dominant disease and is correlated with the development of endocrine tumors affecting pituitary gland, parathyroid, endocrine pancreas or duodenum. It is mainly caused by a germinative mutation in tumor suppressor gene MEN1 (11q13). The tumorigenesis follow the Knudson\'s model (1971). Genetic diagnosis of families with MEN1 is essential to recognizes asymptomatic mutation carriers, and allows an earlier detection and treatment of tumors leading to a reduction of mortality and morbidity associated to MEN1. Furthermore, it can exclude family members that do not carry mutations from the periodical screening. The genetic diagnosis for MEN1 is held using Sanger sequencing. However, limitations of this technique make it less cost-effective, mostly, the less capacity of data generation that leads to the need of PCR products up to 700 bp to obtain a suitable read. Moreover, specific conditions of the MEN1 gene contributes to make this process more laborious and expensive, like the need to read all gene sequence (7kb) to make a correct analysis due to the absence of \"hot spots\". This way, the need of \"fragmentation\" to allow the sequencing can hide important information to disease development, mostly in introns. These limitations preclude the clinical application of genetic diagnosis of MEN1. Since 2005, new technologies are available; they are called Next Generation Sequencing (NGS) and consist in a new tool that allow the same sequencing, but with a larger data generation capacity, making them more attractive and costeffective. The NGS also gives a higher speed to the process of data acquiring and allows the complete read of gene, including promoters and introns. Therefore, it makes the results more informative, not forgetting quality aspects. Among lot of options of NGS available, lighter platforms are recommended, for example, Ion PGM and Illumina MiSeq. A strong tendency has been shown in order to change the Sanger sequencing to NGS, including clinical application to genetic diagnosis of complex diseases and inherited cancer. However, there is not previous studies evaluating NGS to MEN1 genetic diagnosis. Thus, present study evaluated NGS as a genetic diagnosis method for MEN1, comparing with Sanger sequencing. This study aimed to validate the NGS method using as model the Sanger sequencing and evaluated sensibility, specificity and costeffectiveness of NGS. For this purpose, 76 index-cases with clinical MEN1 diagnosis were analyzed on Illumina MiSeq. Analyzes were divided in two phases. After analyzes, 96% of reproducibility and 99% of precision were calculated. Accuracy, sensibility and specificity were resulted in 100%. There were not falses negatives or positives. NGS showed more cost-effectiveness with lower costs. This study allowed validation of genetic screening of MEN1 indexcases applying NGS platform

Microarray analysis measures the expression of thousands of mRNAs simultaneously, thereby creating genome-wide transcriptional snapshots of biological states. The novel technique of connectivity mapping is a tool that identifies functional chemical similarities by identifying similarities in the transcriptional signatures they induce. As this is a relatively new technique, the aims of this work were two-fold: Firstly, to optimise the method of connectivity mapping, and secondly to establish its potential uses, by applying the technique in several model systems. Connectivity mapping currently requires query signatures to be in Affymetrix geneidentifier format, thus, gene-expression profiles from other microarray platforms must be converted to Affymetrix identifiers, which can introduce error. Converting the connectivity map database into a universal gene-naming nomenclature reduces the error and allows expansion of the database, making use of existing microarray data resources. The use of gene-expression patterns for disease states, combined with connectivity mapping, to identify potential therapeutic agents, was explored. Small molecules capable of sensitising doxorubicin-resistant cells to doxorubicin and preventing cell death in several Huntington’s disease models were discovered. This provided new insights into therapeutic targets for these conditions and suggests the exciting possibility that transcription patterns can be used to establish and identify novel drugtarget relationships. To discover functional chemical similarities, connectivity mapping was used to investigate the effects of the small molecule apogossypol, which to induces endoplasmic reticulum (ER) aggregation. Connectivity mapping facilitated the discovery of a diverse subset of chemicals that also induced ER aggregation, some of which had been extensively functionally characterised. This provided both mechanistic insight and physiological relevance to the ER aggregation phenomenon. This work clearly demonstrates that, although in its infancy, connectivity mapping is an invaluable resource with potential applications at many stages of the drug discovery process, from cellular pathway dissection and drug target identification, to drug discovery and toxicological assessment.

Although increased genome sequencing e orts have increased our understanding of genomic variability within many bacterial species, there has been limited application of this knowledge towards assessing current molecular typing methods and developing novel molecular typing methods. This thesis reports a novel in silico comparative genomic framework where the performance of typing methods is assessed on the basis of the discriminatory power of the method as well as the concordance of the method with a whole-genome phylogeny. Using this framework, we designed a comparative genomic ngerprinting (CGF) assay for Listeria monocytogenes through optimized molecular marker selection. In silico validation and assessment of the CGF assay against two other molecular typing methods for L. monocytogenes (multilocus sequence typing (MLST) and multiple virulence locus sequence typing (MVLST)) revealed that the CGF assay had better performance than these typing methods. Hence, optimized molecular marker selection can be used to produce highly discriminatory assays with high concordance to whole-genome phylogenies. The framework described in this thesis can be used to assess current molecular typing methods against whole-genome phylogenies and design the next generation of high-performance molecular typing methods from whole-genome sequence data.
xiii, 100 leaves : ill. ; 29 cm

Motivation: A rapid technological development in the biosciences and in computer science in the last decade has enabled the analysis of high-dimensional biological datasets on standard desktop computers. However, in spite of these technical advances, common properties of the new high-throughput experimental data, like small sample sizes in relation to the number of features, high noise levels and outliers, also pose novel challenges. Ensemble and consensus machine learning techniques and data integration methods can alleviate these issues, but often provide overly complex models which lack generalization capability and interpretability. The goal of this thesis was therefore to develop new approaches to combine algorithms and large-scale biological datasets, including novel approaches to integrate analysis types from different domains (e.g. statistics, topological network analysis, machine learning and text mining), to exploit their synergies in a manner that provides compact and interpretable models for inferring new biological knowledge. Main results: The main contributions of the doctoral project are new ensemble, consensus and cross-domain bioinformatics algorithms, and new analysis pipelines combining these techniques within a general framework. This framework is designed to enable the integrative analysis of both large- scale gene and protein expression data (including the tools ArrayMining, Top-scoring pathway pairs and RNAnalyze) and general gene and protein sets (including the tools TopoGSA , EnrichNet and PathExpand), by combining algorithms for different statistical learning tasks (feature selection, classification and clustering) in a modular fashion. Ensemble and consensus analysis techniques employed within the modules are redesigned such that the compactness and interpretability of the resulting models is optimized in addition to the predictive accuracy and robustness. The framework was applied to real-word biomedical problems, with a focus on cancer biology, providing the following main results: (1) The identification of a novel tumour marker gene in collaboration with the Nottingham Queens Medical Centre, facilitating the distinction between two clinically important breast cancer subtypes (framework tool: ArrayMining) (2) The prediction of novel candidate disease genes for Alzheimer’s disease and pancreatic cancer using an integrative analysis of cellular pathway definitions and protein interaction data (framework tool: PathExpand, collaboration with the Spanish National Cancer Centre) (3) The prioritization of associations between disease-related processes and other cellular pathways using a new rule-based classification method integrating gene expression data and pathway definitions (framework tool: Top-scoring pathway pairs) (4) The discovery of topological similarities between differentially expressed genes in cancers and cellular pathway definitions mapped to a molecular interaction network (framework tool: TopoGSA, collaboration with the Spanish National Cancer Centre) In summary, the framework combines the synergies of multiple cross-domain analysis techniques within a single easy-to-use software and has provided new biological insights in a wide variety of practical settings.

Genome sequencing of a large number Firmicute species has recently been completed, including some of the highly oxygen-sensitive butyrate-producing bacteria, belonging to the Lachnospiraceae and Ruminococcaceae families, which have been isolated at the Rowett Institute of Nutrition and Health. However, detailed knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of detailed genomic annotation and pathway analysis, and lack of genetic manipulation techniques. Therefore, the aim of this work was the genomic analysis of the carbohydrate-utilisation and motility genes, and establishment of genetic manipulation techniques for a selected group of these bacteria, specifically the Roseburia/Eubacterium rectale group and Faecalibacterium prausnitzii. This involved the establishment of a Roseburia/E. rectale pan-genome consisting of genome sequences from eleven strains (three of which are first introduced in this work), representing five species. 1840 Carbohydrate-active enzymes (CAZymes), 932 of which were glycoside hydrolases (GHs), were identified in this pan-genome. The GH complement of each strain was used to predict dietary niches of these bacteria in the human colon. The members of the Roseburia/E. rectale group were predicted to have the core capacity to utilise starch, with specific members possessing specialised dietary niches. The motility loci of selected members of the Roseburia/E. rectale group were annotated, and the gene orders of these loci were highly conserved between different members of the group. The motility of these bacteria was shown to be affected by the carbon source utilised for growth. This was followed by the design of methods to allow the transfer of autonomously-replicating plasmids into Roseburia/E. rectale species. The modular plasmids pMTL83151 and pMTL82151 were transferred from an E. coli donor into Roseburia inulinivorans A2-194. pMTL83151 could also be transferred into Eubacterium rectale A1-86 and T1-815. This technique has enabled the heterologous expression of a β-(1,3-1,4)-glucanase enzyme in R. inulinivorans A2-194 and E .rectale T1-815.

Genome Wide Association Studies (GWAS) have uncovered many genetic regions which are associated with autoimmune disease risk. In this thesis, I present methods which I have developed to build upon these studies and enable the analysis of the causal variants of these diseases. Colocalization methods disentangle whether potential causal variants are shared or distinct in related diseases, and enable the discovery of novel associations below the single-trait significance threshold. However, existing approaches require independent datasets to accomplish this. I extended two methods to allow for the shared-control design; one of these extensions also enables fine mapping in the case of shared variants. My analysis of four autoimmune diseases identified 90 regions associated with at least one disease, 33 of which were associated with 2 or more disorders; 14 of these had evidence of distinct causal variants. Once associated variants have been identified, we may wish to test some aggregate property, such as enrichment within an annotation of interest. However, the null distribution of GWAS signals showing association with a trait and preserving expected correlation due to linkage disequilibrium is complicated. I present an algorithm which computes the expected output of a GWAS, given any arbitrary definition of "null", and hence can be used to simulate the null distribution required for such a test. Commonly, GWAS report only summary data, and determining which genetic variants are causal is more difficult; the strongest signal may merely be correlated with the true causal variant. I have developed a statistical method for fine mapping a region, requiring only GWAS p-values and publicly available reference datasets. I sample from the space of potential causal models, rejecting those leading to expected summary data excessively different from that observed. This removes the need for the assumption of a single causal variant. In contrast to other summary statistic methods which allow for multiple causal variants, it does not depend upon availability of effect size estimates, or the allelic direction of effect and it can infer whether the pattern of association is likely caused by a non-genotyped SNP without requiring imputation. I discuss the effect of choice of reference dataset, and the implications for other summary statistics techniques.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2003.
Includes bibliographical references (p. 157-160).
Genome Scanning is a powerful new technique for DNA sequencing. The method presented in this thesis uses an atomic force microscope with a functionalized cantilever tip to sequence single stranded DNA immobilized to a mica surface. The functionalized cantilever tip hybridizes with only one base type (A, C, T, or G) and results in distinct peaks in the AFM-produced image. Genome Scanning has been successful at identifying 40 base strands of synthesized DNA and has been shown to detect a particular base type on 48 kilobase strands of lambda DNA. Currently, Genome Scanning is only accurate to 3-26 bases at a time, however, it can achieve a sequencing speed of 6000 bases/sec. In other words, Genome Scanning can be used to sequence the 3 billion bases of the human genome in 5.78 days.
by Ahmed Elmouelhi.
S.M.

Protein function and dysfunction, and their intimate ties to protein structure, has been a core focus of research for several decades. More recently, research into the lack of structure in proteins has reached fever pitch. Intrinsically disordered proteins (IDPs) are proteins (or protein regions) that exist as collapsed or extended, dynamically mobile conformational ensembles, either at secondary or tertiary level, whilst remaining biologically active. The properties of IDPs can impede their study; they are often inherently unstable, are vastly wide-ranging in molecular weight and often difficult to express in large quantities. Mass spectrometry (MS) has evolved into a tool for the study of dynamic systems such as IDPs due to its large dynamic range, high sensitivity, low sample consumption and its lack of bias towards the folded state of a protein. The addition of ion mobility separation to mass spectrometry analysis (IM-MS) provides insight into the conformations adopted by proteins and their complexes, measuring their rotationally averaged collision cross section which can be compared with coordinates from other biophysical techniques such as X-ray crystallography, NMR and to molecular modelling. The work presented in this thesis uses both MS and IM-MS, along with several other biophysical techniques, to interrogate a number of IDPs which are implicated in cancer. Firstly, variable temperature IM-MS is used to probe several proteins of increasing disorder; structured protein cytochrome c, the tumour suppressor protein p53 and the oncoprotein Murine Double Minute 2 (Mdm2), performing IM-MS measurements at a range of temperature from 200 K to 571 K to elucidate the gas-phase unfolding behaviour of each protein. The interaction between p53 and Mdm2 is a current target for cancer drug therapy. Hence MS and IM-MS, alongside circular dichroism and hydrogen-deuterium exchange are next employed to determine the effect of several known small molecule ligands on the conformations adopted by these disordered domains. The significant structuring of both of these disordered proteins upon binding to their respective ligands can be observed using IM-MS, but is not apparent when using other biophysical techniques, highlighting the ability of IM-MS to capture conformational changes occurring in solution on a short timescale. The regulation of disorder in cells is postulated to be mediated by proline residues. I investigate the impact of proline replacement on the populations of conformers presented by p53 using a range of mutants and then go on to study how these mutations impact upon the binding stoichiometry, affinity and conformational preference of p53 for its interaction partner Mdm2. Finally, the disordered melanoma associated antigen 4 MAGE-A4, and its ability to bind to p53 and block its transcriptional activity is probed using MS and IM-MS.

Gene finding in eukaryotic genomes is an essential part of a comprehensive approach to modern systems biology. Most methods developed in the past rely on a combination of computational prediction and external information about gene structures from transcript sequences and comparative genomics. In the past, external sequence information consisted of a combination of full-length cDNA and expressed sequence tag (EST) sequences. Much improvement in prediction of genes and gene isoforms is promised by availability of RNA-seq data. However, productive use of RNA-seq for gene prediction has been difficult due to challenges associated with mapping RNA-seq reads which span splice junctions to prevalent splicing noise in the cell. This work addresses this difficulty with the development of methods and implementation of two new pipelines: 1/ a novel pipeline for accurate mapping of RNA-seq reads to compact genomes and 2/ a pipeline for prediction of genes using the RNA-seq spliced alignments in eukaryotic genomes. Machine learning methods are employed in order to overcome errors associated with the process of mapping short RNA-seq reads across introns and using them for determining sequence model parameters for gene prediction. In addition to the development of these new methods, genome annotation work was performed on several plant genome projects.

Notre projet a consisté à analyser par aCGH 316 patients présentant une malformation cardiaque congénitale (MCC)à type de transposition des gros vaisseaux, tétralogie de Fallot ou coarctation de l'aorte avec pour objectif la détection de microdélétions et microduplications génomiques et ainsi l'identification de nouveaux gènes contribuant à ces MCC. L'identification de nouveaux gènes permet une meilleure compréhension des mécanismes moléculaires et embryologiques à l'origine de ces malformations et un meilleur conseil génétique. Notre étude a conduit à l'identification de 21 microdélétions et 50 microduplications rares de novo ou héritées dont certaines comprennent des gènes candidats aux MCC. L'analyse bioinformatique des données a permis de montrer que de nombreuses microdélétions/microduplications comprennent des gènes possédant des sites de fixation de FOXC1. Ces microdélétions/microduplications pourraient donc altérer l'expression de certains gènes régulés par FOXC1 au cours du développement cardiaque. Par ailleurs, parmi les remaniements identifiés, nous avons détecté une délétion d'environ 1Mb en amont de SOX9 chez des patients avec malformation cardiaque et syndrome de Pierre Robin. Nous avons montré que cette délétion comprend plusieurs activateurs cardiaques putatifs. Ces résultats suggèrent que la dérégulation de SOX9 peut être responsable de MCC. Enfin, nous avons identifié une duplication partielle de la région 5' du gène SEMA3D donnant lieu à un ARNm tronqué de SEMA3D. Ces résultats suggèrent que la portion tronquée de SEMA3D pourrait perturber la migration des cellules de la crête neurale au cours du développement et ainsi contribuer aux MCC
Our project consists of the analysis by aCGH of a series of 316 patients with Congenital Heart Defects (CHD) such as transposition of the great arteries, tetralogy of Fallot and coarctation of the aorta with the aim to detect genomic microdeletions and microduplications and thus to identify new genes contributing to CHD. The identification of new genes contributes to improve the understanding of the molecular and embryological mechanisms underlying these defects and enable better genetic counseling. Our study led to the identification of rare de novo or inherited 21 microdeletions and 50 microduplications, some of them comprising candidate genes for CHD. Bioinformatic analysis of the data demonstrated that many microdeletions/microduplications include genes with FOXC1 transcription factor binding sites. These microdeletions/microduplications might hamper the expression of genes that are regulated by FOXC1 during heart development. Otherwise, among the rearrangements identified, we detected a ~1 Mb deletion upstream of SOX9 in patients presenting with heart defects and Pierre Robin syndrome. We have shown that this deletion includes several putative cardiac enhancers. These results suggest that deregulation of SOX9 might be responsible for CHD. Finally, we identified a duplication of the 5’ half of SEMA3D, generating a truncated poly-A tailed mRNA of SEMA3D. These results suggest that truncated SEMA3D may have hampered the migration of cardiac neural crest cells during heart development, and thus contributed to CHD

The ability of bacteria to thrive in a variety of host environments depends on their capacity to sense and respond to a wide array of stressors. E. coli encounters many stresses during transit through the gastro-intestinal tract, including acid stress. Acid stress response in E. coli is regulated by a complex network called AR2. The AR2 network comprises several local regulators that collate signals from multiple two-component systems (TCS) including RcsBD, EvgAS and PhoPQ. We combined lab-based evolution and whole genome re-sequencing to generate and identify mutations that confer increased acid resistance in E. coli K-12. All of these mutations map in the gene encoding EvgS, the sensor kinase of the EvgAS TCS. Using a luciferase reporter system and phenotypic assays we characterised the nature of these evgS mutations and their contribution to acid resistance. We also used high-temporal resolution luciferase reporter assays to uncover novel aspects of this network and implicate PhoP in the repression of acid resistance. Finally, we used our evgS mutants to characterise novel interactions within the AR2 network between the two component systems RcsBD and EvgAS. These results are discussed in relation to the role of regulatory networks in bacteria.

During the Human Genome Project the first hundred billion bases were sequenced in four years, however, the second hundred billion bases were sequenced in four months (NHGRI, 2013). As efforts were made to improve every aspect of sequencing in this project, cost became inversely proportional to the speed (NHGRI, 2013). Human Genome Project ended in April 2003 but research in faster and cheaper ways to sequence the DNA is active to date (NHGRI, 2013). On the one hand, these advancements have allowed the convenient and unbiased generation and interrogation of a variety of omics datasets; on the other hand, they have substantially contributed towards the ever-increasing size of biological data. Therefore, informatics techniques are indispensable tools in the field of biology and medicine due to their ability to efficiently store and probe large datasets. Bioinformatics is a specialized domain under informatics that focusses on biological data storage, organization and analysis (NHGRI, 2013). Here, I have applied informatics approaches such as database designing and web development in the context of biological datasets or bioinformatics, to create a novel web-based resource that allows users to explore the comprehensive transcriptome of common aquatic tunicate named Oikopleura dioica (O .dioica), and access their associated annotations across key developmental time points, conveniently. This unique resource will substantially contribute towards studies on development, evolution and genetics of chordates using O. dioica as a model. Mendelian or single-gene disorders such as cystic fibrosis, sickle-cell anemia, Huntington’s disease, and Rett’s syndrome run across generations in families (Chial, 2008). Allelic variations associated with Mendelian disorders primarily reside in the protein-coding regions of the genome, collectively called an exome (Stenson et al., 2009). Therefore, sequencing of exome rather than whole genome is an efficient and practical approach to discover etiologic variants in our genome (Bamshad et al., 2011). Renal agenesis (RA) is a severe form of congenital anomalies of the kidney and urinary tract (CAKUT) where children are born with one (unilateral renal agenesis) or no kidneys (bilateral renal agenesis) (Brophy et al., 2017; Yalavarthy & Parikh, 2003). In this study, we have applied exome-sequencing technique to selective human patients in a renal agenesis (RA) pedigree that followed a Mendelian mode of disease transmission. Exome sequencing and molecular techniques combined with my bioinformatics analysis has led to the discovery of a novel RA gene called GREB1L (Brophy et al., 2017). In this study, we have successfully demonstrated the validation of exome sequencing and bioinformatics techniques to narrow down disease-associated mutations in human genome. Additionally, the results from this study has substantially contributed towards understanding the molecular basis of CAKUT. Discovery of novel etiologic variants will enhance our understanding of human diseases and development. High-throughput sequencing technique called RNA-Seq has revolutionized the field of transcriptome analysis (Z. Wang, Gerstein, & Snyder, 2009). Concisely, a library of cDNA is prepared from a RNA sample using an enzyme called reverse transcriptase (Nottingham et al., 2016). Next, the cDNA is fragmented, sequenced using a sequencing platform of choice and mapped to a reference genome, assembled transcriptome, or assembled de novo to generate a transcriptome (Grabherr et al., 2011; Nottingham et al., 2016). Mapping allows detection of high-resolution transcript boundaries, quantification of transcript expression and identification of novel transcripts in the genome. We have applied RNA-Seq to analyze the gene expression patterns in water flea otherwise known as D. pulex to work out the genetic details underlying heavy metal induced stress (unpublished) and predator induced phenotypic plasticity (PIPP) (Rozenberg et al., 2015), independently. My bioinformatics analysis of the RNA-Seq data has facilitated the discovery of key biological processes participating in metal induced stress response and predator induced defense mechanisms in D. pulex. These studies are great additions to the field of ecotoxicogenomics, phenotypic plasticity and have aided us in gaining mechanistic insight into the impact of toxicant and predator exposure on D. pulex at a bimolecular level.

Uvod: Virusni gastrointestinalni sindrom je aktuelni zdravstveni problem u celom svetu. To važi kako u razvijenim zemljama, tako i u zemljama u razvoju, a posebno u nerazvijenim zemljama, gde je drugi po redu uzrok mortaliteta. Nagli početak bolesti, praćen pojavom velikog broja tečnih stolica, mukom, povraćanjem, bolovima u stomaku, temperaturom, malaksalošću, ima za posledicu dehidrataciju. U svim starosnim grupama obolelih, a naročito kod sasvim male dece, starih i imunodeficitarnih osoba može da dođe do smrtnog ishoda, ukoliko se brzo ne postavi tačna etiološka dijagnoza bolesti i ne pristupi se odmah nadoknadi vode i elektrolita, kao i primeni svih ostalih mera simptomatske terapije. Brzo postavljena tačna dijagnoza, što se najbolje postiže real-time PCR testom, sprečava pojavu komplikacija, pa i fatalnog ishoda bolesti. Istovremeno, omogućava primenu odgovarajućih epidemioloških mera da se spreči nastanak epidemija i njihovo širenje. Cilj ovog istraživanja bio je da se tačno utvrdi incidenca virusnog gastrointestinalnog sindroma u Vojvodini i učestalost pojave epidemijskog i sporadičnog javljanja ove bolesti. Cilj je bio i postavljanje algoritma za primenu real-time PCR testa u dijagnostici virusnog gastrointestinalnog sindroma u budućem radu. Isto tako, cilj je bio da se molekularnom analizom, sekvenciranjem delova genoma pozitivnih uzoraka stolice, izvrši genetska tipizacija i odredi filogenetska pripadnost virusa. Materijal i metode: Tokom petogodišnjeg istraživanja molekularnim real-time PCR testom pregledane su 1003 obolele osobe sa simptomima virusnog dijarealnog sindroma, starosti od mesec dana do preko 90 godina. Pregledani su na rota, noro, astro i enterične adenoviruse. Na osnovu podataka iz anketnih upitnika i istorija bolesti, detaljno su analizirani svi klinički pokazatelji (javljanje bolesti tokom godine, trajanje bolesti, simptomi). Procena težine kliničke slike vršena je prema Vesikari skali. Svi podaci su upoređivani prema vrsti virusnog uzročnika, prema starosti obolelih, godinama trajanja istraživanja i epidemijskom i sporadičnom javljanju oboljenja. Dobijeni podaci su statistički obrađeni, tabelarno i grafički prikazani. Rezultati: U petogodišnjem periodu real-time PCR testom pregledan je uzorak od 1003 obolele osobe različite starosti na 4 virusna uzročnika dijarealnog sindroma (rota, noro, astro i enterične adenoviruse). Virusni dijarealni sindrom dokazan je kod 709 obolelih (70,69%). Najčešće su dokazane rotavirusne infekcije u 28,81%. Statistički značajno najčešće rotavirusi su bili utvrđeni kod dece do 5 godina (38,90%), ali u visokom procentu i kod dece uzrasta 6 do 14 godina (24,83%). Deca mlađa od 5 godina imala su statistički značajno najtežu kliničku sliku, bila su češće hospitalizovana i imala su statistički značajno višu temperaturu. Pored više temperature kod obolelih od rotavirusa, klinička slika je kod ovih bolesnika bila teža i bolest je duže trajala nego kod obolelih od drugih virusa. Norovirusna infekcija je dokazana u 23,03% obolelih i to statistički značajno češće kod odraslih osoba, starijih od 20 godina. Od kliničkih simptoma kod ovih bolesnika statistički značajno češće su dokazani muka, povraćanje i bolovi u stomaku, nego kod obolelih od drugih virusa. Norovirusi su značajno češće bili uzročnici epidemijskog javljanja bolesti. Astrovirus je dokazan kod znatno manjeg broja obolelih (u 2,29%) i to samo kod dece do 5 godina i dece uzrasta 6 do 14 godina. Infekcija izazvana enteričnim adenovirusima dokazana je kod 13,36% bolesnika. Njačešće je utvrđena kod dece uzrsta do 5 godina i 6 do 14 godina. Oboleli od adenovirusa imali su statistički značajno blažu kliničku sliku bolesti. Dva virusna uzročnika u uzorku stolice dokazana su u 3,19% osoba, obično u toku epidemijskog javljanja bolesti. Ovi bolesnici su imali bitno težu kliničku sliku. Najviše obolelih od dijarealnog sindroma bilo je u hladnim mesecima, mada su dijagnostikovani i tokom cele godine. U petogodišnjem periodu utvrđene su 22 epidemije u kolektivima i 9 porodičnih epidemija. Epidemijsko javljanje bolesti bilo je statistički značajno najčešće kod najstarijih bolesnika (starijih od 50 godina), a sporadično javljanje bilo je statističko značajno najčešće kod dece. U cilju potvrde tačnosti dijagnostike virusa u ispitivanim uzorcima real-time PCR testom, genotipizacije, kao i detaljnije molekularne analize, izabrani su reprezentativni uzorci pozitivni na rota, noro, astro ili adenoviruse. Delovi genoma ovih uzoraka su amplifikovani, a zatim sekvencirani. Sekvencirani izolati rotavirusa pripadali su grupi A i tipovima G1P[8], G2P[4], G3P[8] i G9P[8]. Sekvencirani izolati norovirusa pripadali su genogrupi I tipu 2, zatim genogrupi II tipovima 1, 2, 4 i 17. Sekvencirani izolati astrovirusa pripadali su grupi klasičnih astrovirusa i tipovima 1, 4 i 5. Sekvencirani izolati adenovirusa pripadali su grupi F i tipovima 40 i 41, kao i grupi C tipu 2. Pripadnost dobijenih sekvenci u ovom istraživanju, dodatno je potvrđena izradom filogenetskog stabla za sekvence pozitivne na rota, noro, astro ili adenoviruse. Zaključak: Incidenca virusnog dijarealnog sindroma u Vojvodini (70,69%) vrlo je visoka i viša je nego što je bilo pretpostavljeno prilikom prijave teze (u hipotezi). Real-time PCR test treba da bude redovno korišćen u budućem dijagnostičkom radu, jer dovodi do brze dijagnostike, čak i ako su virusi prisutni u malom broju u uzorcima tečnih stolica, što je utvrđeno tokom ovog dijagnostičkog rada. Ispitivani virusi treba da budu redovno dijagnostikovani kod obolelih od dijarealnog sindroma i to u svim starosnim grupama, tokom epidemijskog i sporadičnog javljanja oboljenja.
Introduction: Viral gastrointestinal syndrome is a current ongoing health problem worldwide. This is true of both developed and developing countries, especially underdeveloped ones where it is the second leading cause of mortality. Sudden onset of the disease—accompanied by the occurrence of large numbers of liquid stools, nausea, vomiting, abdominal pain, fever, and exhaustion—leads to dehydration. A fatal outcome can occur in all age groups of patients, especially very young children, the elderly, and the immuno-deficient, unless an accurate etiological diagnosis of the disease is quickly established, followed by a prompt institution of fluid and electrolyte placement, and implementation of other symptomatic therapy measures. Quick establishment of an accurate diagnosis, which is best achieved using the real-time PCR test, prevents the onset of complications, including a potentially fatal outcome of the disease. Simultaneously, it enables the implementation of appropriate epidemiological measures to prevent epidemic outbreaks and their spread. The aim of this study was to accurately determine the incidence of viral gastrointestinal syndrome in Vojvodina and the frequency of epidemic and sporadic occurrence of this disease. The aim was also to set up an algorithm for the application of the real-time PCR test in diagnostics of viral gastrointestinal syndrome in future work. Likewise, the aim was to carry out genetic typing and determine phylogenetic affiliation of the virus using molecular analysis and sequencing of parts of genomes from positive stool samples. Material and Methods: During a five-year study, 1003 patients with symptoms of viral diarrheal syndrome, aged from one month to more than 90 years old, were examined using molecular real-time PCR test. They were screened for rota, noro, astro, and enteric adenoviruses. Based on the data from survey questionnaires and medical case history, all clinical indicators were meticulously analyzed (disease occurrence during the year, disease duration, symptoms). The assessment of the clinical severity was carried out according to the Vesikari Clinical Severity Scoring scale. All data were compared according to the type of the viral causing agent, age of the patients, duration of research in years, and epidemic and sporadic occurrence of the disease. Obtained data were statistically analyzed, tabulated, and graphically displayed. Results: In a five-year period, a sample of 1003 patients of different ages was screened for four different viral causing agents of diarrheal syndrome (rota, noro, astro, and enteric adenoviruses) using the real-time PCR test. Viral diarrheal syndrome was confirmed in 709 patients (70.69%). The most commonly found were rotavirus infections in 28.81% of the cases. Rotaviruses were statistically significantly most common in children younger than 5 years old (38.90%), but were also found in high percentage in children aged 6-14 years old (24.83%). Children under 5 years of age had statistically significantly highest clinical severity and fever, and were more frequently hospitalized. In addition to higher fever in patients with rotavirus, clinical severity in these patients was also higher, and the disease lasted longer than in patients with other viruses. Norovirus infections were reported in 23.03% of the subjects, statistically significantly more frequently in adults over 20 years of age. Regarding the clinical symptoms in these patients, nausea, vomiting, and abdominal pain were statistically significantly more common than in patients with other viruses. Noroviruses were significantly more common as causing agents of epidemic disease outbreaks. Astrovirus was found in a significantly smaller number of patients (in 2.29%), and only in children under 5 years of age and children aged 6-14 years old. Enteric adenovirus infections were reported in 13.36% of the subjects. They were most commonly found in children younger than 5, and those aged 6- 14 years old. Adenovirus sufferers had statistically significantly milder clinical disease. Two viral causing agents in the stool sample were found in 3.19% of the subjects, usually during an epidemic disease outbreak. These patients had a significantly more severe clinical disease. Highest numbers of sufferers from diarrheal syndrome occurred during the cold months, although they were diagnosed throughout the year. In a five-year period, 22epidemics in collective groups and 9 family epidemics were identified. Epidemic outbreaks of the disease were statistically significantly most frequent in the elderly patients (older than 50), while sporadic occurrences were statistically significantly most frequent in children. Representative samples positive for rota, noro, astro, or adenoviruses were selected in order to confirm the accuracy of virus diagnostics in samples tested by the real-time PCR test, and perform genotyping as well as more detailed molecular analyses. Parts of the genomes of these samples were amplified and then sequenced. Sequenced rotavirus isolates belonged to group A and types G1P[8], G2P[4], G3P[8], and G9P[8]. Sequenced norovirus isolates belonged to genogroup I type 2, and genogroup II types 1, 2, 4, and 17. Sequenced astrovirus isolates belonged to the group of classical astroviruses and types 1, 4, and 5. Sequenced adenovirus isolates belonged to group F and types 40 and 41, as well as group C type 2. The affiliation of the obtained sequences in this study was further confirmed by creating a phylogenetic tree for sequences positive for rota, noro, astro, or adenoviruses. Conclusion: The incidence of viral diarrheal syndrome in Vojvodina (70.69%) is very high—higher than what was assumed at the time of the thesis submission (in the hypothesis). The real-time PCR test should be regularly used in future diagnostic work, since it leads to rapid diagnostics even if viruses are present in small numbers in liquid stool samples, as determined in the course of this diagnostic study. The investigated viruses should be regularly tested in patients with diarrheal syndrome belonging to all age groups during both epidemic and sporadic occurrences of the disease.

In this essay I present my creative input and development as a musician since fall 2020 and the compositions that has come out of it. Furthermore, I reflect on preparing these songs for my exam concert with my band Paradise League, as well as adding a trumpet synth called EVI (Electric Valve Instrument) to the instrumentation. Having experienced embouchure fatigue and a lack of stamina to follow through with longer sets, I am also pursuing a more effective technique. Hence, I analyze my practice methods and discuss my trumpet teachers, their tools and the effects thereof. Through the combination of fundamental, soloistic and flexibility exercises I developed a more effortless playing style with a wider artistic expression. There were vast technical differences between the EVI and the trumpet. Despite some minor challengers when switching between the two instruments, the EVI opened up a different set of improvisational ideas, broadening my soloistic framework.

This essay aims to describe and problematize gender roles and master suppression techniques in Stephenie Meyer’s Twilight Saga. This is done in order to enable me, in my future profession as a teacher, to start an emancipatory discussion in class where pupils can become conscious of different ways of reading the love story. I will use the following two research questions to fulfil the purpose: 1) investigate which gender roles that appears in the book’s main characters Bella and Edward and 2) which master suppression techniques that colours their relationship. To answer the questions gender theory and ideology-critics are used. Gender theory is first and foremost used to analyse gender roles whereas ideology-critics is a method of reading that highlights the importance of taking the society and thereto connected values in to account. Applying these two theories on the book it becomes clear that the main characters follows traditional gender roles for what is seen as typical feminine and masculine behaviour; Bella is caring, passive, sexually loyal, and addicted and Edward is aggressive, physically strong and fast, stubborn, dominant and protective. Further more, it is also evident that these roles are accompanied by a number of master suppression techniques used by Edward, such as: make Bella invisible and silly, keep information from her, and use of violence and threats. By using the knowledge in a pedagogical fashion pupils can be energized to start critically reflecting about these stereotypical roles and thereby emancipate from them. They will realize that Stephenie Meyer’s Twilight Saga is a re-production of patriarchal gender structure through an emotional love story.

Titel: Att härska genom internkommunikation – En observationsstudie om kvinnliga ledare Nivå: Examensarbete på grundnivå i ämnet företagsekonomi Författare: Rama Malki och Malin Frisell Handledare: Monika Wallmon och Svante Brunåker Datum: 2020 - juni Syfte: Syftet med denna studie är att öka förståelsen för hur kvinnliga ledare använder och utsätts för härskartekniker, med fokus på internkommunikation. Metod: Denna studie är kvalitativ och utgår från en deduktiv ansats. Den empiriska insamlingen av data har genomförts med hjälp av fem ostrukturerade observationer som sedan har tolkats och analyserats. Analys & slutsats: Kvinnliga ledare använder och utsätts framförallt för härskartekniker som är av snällare art eller spelar på känslor, dessutom finns det indikationer på att både biologiskt kön och kommunikationsstil kan påverka vilka tekniker som används. Flera olika härskartekniker används av kvinnliga ledare, och det finns en risk att teknikerna försämrar effektiviteten i kommunikationen. Vi kan därför dra slutsatsen att de kvinnliga ledare vi har observerat bör fortsätta utveckla sin kommunikation genom ökad medvetenhet om härskartekniker, för att den ska bli mer framgångsrik. Examensarbetets bidrag: Härskarteknikerna som har observerats har synliggjorts genom verbal och icke-verbal kommunikation, men även via skämt och seriösare tonläge. Studien har indikerat att kvinnliga ledare som har en mer relationsorienterad kommunikation tenderar att använda sig av härskartekniker som är av snällare art.Studien har även indikerat att de kvinnor som använder sig av en mer manlig kommunikationsstil kan riskera att misstas för att använda härskartekniker. Förslag till vidare studier: Jämförelser mellan män och kvinnor inom olika branscher, och genom större studier, för att kunna dra slutsatser om huruvida det går att generalisera användningen av härskartekniker till biologiskt kön och kommunikationsstil. Ytterligare ett förslag på vidare studier är att studera vilken påverkan stereotyphot har på härskartekniker. Nyckelord: Härskartekniker, internkommunikation, kvinnliga ledare, ledarskap, stereotyper.
Title: To rule through internal communication – An observational study about female leaders Level: Student thesis, final assignment for Bachelor Degree in Business Administration Authors: Rama Malki and Malin Frisell Supervisor: Monika Wallmon and Svante Brunåker Date: 2020 - June Aim: The aim of this study is to increase the understanding of how female leaders uses and are exposed to master suppression techniques, focusing on internal communication. Method: This study is qualitative with a deductive approach. The empirical material has been conducted and implemented through five unstructured observations and has later been interpreted and analyzed. Analysis & conclusions: Women use and are exposed to master suppression techniques that are more kind or utilizes feelings, and that there are indications that both biological gender and communication styles can affect which techniques that are used. Various master suppression techniques are used by female leaders and risk to impair the effectivity of the communication. Therefore, we can draw the conclusion that the female leaders we have observed should continue to improve their communication by increasing awareness about master suppression techniques, in order to communicate more successfully. Contribution of the thesis: The master suppression techniques have been observed through nonverbal and verbal communication, but also through jokes and a more serious tone. This study has indicated that female leaders have more of a relationship-oriented communication, tend to use master suppression techniques of a kinder nature. The study has also indicated that those women using a masculine communication style, risk to be misunderstood for using master suppression techniques. Suggestions for future research: Comparisons between men and women, in various branch of industries and through larger studies to draw conclusions whether it is possible to generalize master suppression techniques to biological gender and communication style. Furthermore, the effects that stereotypical threats have on master suppression techniques could be researched. Key words: Master suppression techniques, internal communication, female leaders, leadership, stereotypes.

Hintergrund: Karzinoid-Tumoren des embryonalen Mitteldarms sind seltene intestinale neuroendokrine Tumoren, bei denen zum Zeitpunkt der Diagnose häufig Metastasen vorliegen. Im Gegensatz zu Karzinoiden des Vorderdarms und Respirationstraktes sind sie nicht mit der Multiplen Endokrinen Neoplasie Typ 1 (MEN1) vergesellschaftet. Die Mechanismen ihrer Tumorigenesis sind weitgehend unbekannt. Methoden: Tumorgewebe acht sporadischer, maligner Dünndarm-Karzinoide war Objekt dieser Studie über Verlust der Heterozygotie ("Loss Of Heterozygosity" (LOH)) mit 131 fluoreszierenden Mikrosatelliten. DNA Sequenz-Analyse mit Oligonucleotid Primern, die Exon 8-11 des SMAD4/DPC4 Gens flankieren sowie immunhistochemische Färbung mit Smad4/DPC4 antikörpern wurde durchgeführt. Ergebnis: Chromosom 18 wies Deletionen in 88% der Tumoren auf. Alle außer einem Tumor hatten sowohl 18p als auch 18q verloren, in einem der Tumoren war eine kleine Region telomer zu den SMAD4/DPC4/DCC Genen auf 18q21 verloren. Andere Chromosomen waren nur in drei Tumoren betroffen. LOH auf Chromosom 11q13, dem MEN1 Lokus, wurde nicht gefunden.Sequenzierung der DNA und immunhistochemische Färbung für das SMAD4/DPC4 Gen zeigten keine Aberrationen. Diskussion: Die Funde der Chromosom 18 Deletionen weisen eindeutig auf ein entscheidendes Ereignis in der Tumorigenese von Karzinoiden des Mitteldarms hin. An der Entstehung dieser Tumoren könnte ein mutmaßliches Tumor Suppressor Gen beteiligt sein, welches auf Chromosom 18 lokalisiert ist. Dahingegen ist SMAD4/DPC4 wahrscheinlich nicht in die Tumorneogenese von Carcinois Tumoren involviert.
Background: Midgut carcinoid tumors are rare malignant tumors with origin in the neuroendocrine cells of the small intestine. Due to secretion of a variety of peptide hormones and biogenic amines they cause the carcinoid syndrome. Metastases are often present at first diagnosis. Despite this, patients have a realistic chance to survive for a prolonged period (30% (unresectable/metastatic disease) -79% (non-metastatic disease) 5-year survival rate) if treated by a combination of surgery and medication. Unlike their foregut counterparts, midgut carcinoid tumors are not or rarely associated with the multiple endocrine neoplasia type 1 (MEN1) syndrome. The genetic back-ground to tumorigenesis of these neoplasms is unknown. In contrast, the events involved in tumorigenesis of gastroenteropancreatic adenocarcinomas are better characterized with frequent mutations e.g. of the Smad4/DPC4, Smad2/MADR2/JV18-1 and DCC genes on chromosome 18. Methods: Eight metastatic midgut carcinoids were analysed by a genome-wide screening for loss of heterozygosity using 131 PCR-amplified fluorescent-labelled microsatellite markers. DNA sequence analysis using oligonucleotide primers flanking exons 8-11 of the Smad4/DPC4 gene and immunohistochemical staining with Smad4/DPC4 antibodies was performed. Results: Chromosome 18 was deleted in seven out of eight tumors (88%). All but one of these tumors had lost both 18p and 18q, the remaining tumor had lost the long arm but retained the short arm. Several other chromosomal alleles were lost in a subset of the tumors. Loss of heterozygosity (LOH) on chromosome 11q13, the MEN 1 locus, was not found. Smad4/DPC4 wild-type sequence and normal immunohistochemical staining for Smad4/DPC4 protein was found for all analysed tumors. Conclusions: Our finding of a high frequency of chromosome 18 deletions in 88% of the tumors strongly suggests that midgut carcinoid tumorigenesis might involve inactivation of a candidate tumor suppressor gene located in that region while Smad4/DPC4 is unlikely to be involved in that process. A more detailed analysis of the genetic events in midgut carcinoid tumors is warranted to clarify their neogenetic origin.

Efter att ha gått en kurs i fri improvisation där jag fick utforska min violas möjligheter till att producera ljud, väcktes en nyfikenhet hos mig kring utökade speltekniker som kan förekomma i nutida konstmusik, så kallade extended techniques. Jag fick tips om etydsamlingen Viola Spaces av Garth Knox där olika extended techniques presenteras och bestämde mig för att studera in fyra av dem. De etyderna jag valde behandlar olika stråktekniker, nämligen sul ponticello, sul tasto, tremolo och olika sätt att dra stråken på. Under arbetets gång redogör jag för de svårigheter jag stötte på under min inlärningsprocess av etyderna samt hur jag gick till väga för att lösa dem. Arbetet redovisas dels genom videoinspelningar som jag gjorde i samband med mina övningstillfällen och dels genom material från de anteckningar jag förde under processen. Jag tar också upp tolkningsfrågor av notationen och reflekterar kring instruktionerna om hur musiken ska framföras. Arbetet har resulterat i att jag känner mig bättre förberedd på att spela nutida konstmusik än vad jag gjorde tidigare. Jag har också utökat min kunskap om olika stråktekniker.

This study provides a comprehensive genetic overview on the endangered Italian wolf population. In particular, it focuses on two research lines. On one hand, we focalised on melanism in wolf in order to isolate a mutation related with black coat colour in canids. With several reported black individuals (an exception at European level), the Italian wolf population constituted a challenging research field posing many unanswered questions. As found in North American wolf, we reported that melanism in the Italian population is caused by a different melanocortin pathway component, the K locus, in which a beta-defensin protein acts as an alternative ligand for the Mc1r. This research project was conducted in collaboration with Prof. Gregory Barsh, Department of Genetics and Paediatrics, Stanford University. On the other hand, we performed analysis on a high number of SNPs thanks to a customized Canine microarray useful to integrate or substitute the STR markers for genotyping individuals and detecting wolf-dog hybrids. Thanks to DNA microchip technology, we obtained an impressive amount of genetic data which provides a solid base for future functional genomic studies. This study was undertaken in collaboration with Prof. Robert K. Wayne, Department of Ecology and Evolutionary Biology, University of California, Los Angeles (UCLA).

Thesis advisor: Tim van Opijnen
Thesis advisor: Welkin Johnson
Pathogenic bacteria can experience various stress factors during an infection including antibiotics and the host immune system. Whether a pathogen will establish an infection largely depends on its survival-success while enduring these stress factors. We reasoned that the ability to predict whether a pathogen will survive under and/or adapt to a stressful condition will provide great diagnostic and prognostic value. However, it is unknown what information is needed to enable such predictions. We hypothesized that under a stressful condition, a bacterium triggers responses that indicate how the stress is experienced in the genome, thereby correctly identifying a stress response holds the key to enabling such predictions. Bacterial stress responses have long been studied by determining how small groups of individual genes or pathways respond to certain environmental triggers. However, the conservation of these genes and the manner in which they respond to a stress can vary widely across species. Thus, this thesis sought to achieve a genome-wide and systems-level understanding of a bacterial stress response with the goal to identify signatures that enable predictions of survival and adaptation outcomes in a pathogen- and stress-independent manner. Here, we first set up a multi-omics framework that maps out a stress response on a genome-wide level using the human respiratory pathogen Streptococcus pneumoniae as a model organism. Under an environmental stress, gene fitness changes are determined by transposon insertion sequencing (Tn-Seq) which represents the phenotypic response. Differential expression is profiled by RNA-Seq which represents as the transcriptional response. Much to our surprise, the phenotypic response and transcriptional response are separated on different genes, meaning that differentially expressed genes are poor indicators of genes that contribute to the fitness of the bacterium. By devising and performing topological network analysis, we show that phenotypic and transcriptional responses are coordinated under evolutionary familiar stress, such as nutrient depletion and host infection, in both Gram-positive and -negative pathogens. However, such coordination is lost under the relatively unfamiliar stress of antibiotic treatment. We reasoned that this could mean that a generalizable stress response signature might exist that indicates the level to which a bacterium is adapted to a stress. By extending stress response profiling to 9 antibiotics and 3 nutrient depletion conditions, we found that such a signature indeed exists and can be captured by the level of transcriptomic disruption, defined by us as transcriptomic entropy. Centered on entropy, we constructed predictive models that perform with high accuracy for both survival outcomes and antibiotic sensitivity across 7 species. To further develop these models with the goal to eventually enable predictions on disease progression, we developed a dual RNA-Seq technique that maps out the transcriptomic responses of both S. pneumoniae and its murine host during lung infection. Preliminary data show that a high entropy is observed in the pathogen’s transcriptome during clearance (a failed infection) compared to a successful/severe infection, while the host transcriptome exhibits a pro-inflammatory and active immune response under the severe infection. Lastly, we characterized evolutionary trajectories that lead to long-term survival success of S. pneumoniae, for instance this means that the bacterium successfully adapts to the presence of an antibiotic and becomes resistant or can grow successfully in the absence of a formerly critical nutrient. These trajectories show that adaptive mutations tend to occur in genes closely related to the adapted stress. Additionally, independent of the stress, adaptation triggers rewiring of transcriptional responses resulting in a change in entropy from high to low. Most importantly, we demonstrate that by combining multi-omics profiles with additional genomic data including gene conservation and expression plasticity, and feeding this into machine learning models, that adaptive evolution can become (at least partially) predictable. Additionally, the genetic diversity in bacterial genomes across different strains and species can indeed influence a bacterium’s adaptation trajectory. In conclusion, this thesis presents a substantial collection of multi-omics stress response profiles of S. pneumoniae and other pathogenic bacteria under various environmental and clinically-relevant stresses. By demonstrating the feasibility of predictions on bacterial survival and adaptive outcomes, this thesis paves the way towards future improvements on infectious disease prognostics and forecasting the emergence of antibiotic resistance
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

The general aim of this PhD thesis was to study, develop and apply different breeding techniques in melon species in order to produce new commercial cultivars. The three specific objectives of this thesis are: 1. To study and evaluate the commercial value and the parthenogenetic capacity of seven genotypes of C. melo var. Inodorus “Piel de Sapo” type to obtain DH lines which might be further used as parental lines for commercial hybrid F1 seed production. The parthenogenetic generation of DHs from the seven genotypes was evaluated and optimized through the analysis and description of the different steps of the process, assaying: three haploid embryo rescue protocols, previously described in the literature; three chromosome doubling methods; and, a new cytometry flow method for evaluating the ploidy-level. 2. To develop the applicability of CRISPR/Cas9 system in melon by performing a gene knockout of the melon phytoene desaturase gene (CmPDS) in protoplasts and plants. 3. To study and highlight the current methodology in major crops for DH production, the availability of chromosome-doubling methods to obtain DH lines, and the opportunities for HI-mediated genome-editing systems in DH technology. Specifically, the focus was put on haploid inducer-mediated genome-editing systems in cucurbit species to give new insights, opportunities and challenges that may be valuable for developing this technique in cucurbits and other species.

Les études d'association sur un génome complet (GWAS) sont conçues pour découvrir les combinaisons de points de polymorphisme (SNP) associées à des maladies. La découverte de ces associations permet d'élaborer de meilleures stratégies pour détecter, traiter ou prévenir les maladies. Récemment, l'utilisation de techniques d'extraction de patterns discriminatif a été investiguée dans le cadre de problématiques GWAS. Toutefois, la découverte de combinaisons de SNP dans de grands jeux de données GWAS est encore difficile à cause de la complexité des algorithmes utilisés. La thèse se propose donc d'améliorer l'état de l'art des approches d'extraction de motifs discriminants, dans le cadre d'extraction de combinaisons de SNP corrélées à un phénotype d'intérêt. Plusieurs solutions ont été proposées, s'attaquant aux problèmes majeurs en GWAS : évaluation de la force d'association, découverte efficace de combinaisons de SNP et visualisation de ces combinaisons. Les approches proposées sont également prometteuses pour d'autres tâches de bioinformatique comme la découverte d'expressions génique, la détection de motifs de phosphorylation et la détection de motifs de régulation
Discovering high-order SNP combinations associated with diseases is an important task of bioinformatics. Once new genetic associations are identified, they can be used to develop better trategies to detect, treat and prevent the diseases. Recently, this issue has been effectively tackled with discriminative pattern mining algorithms. However, the number of SNPs is often very large, discovering of SNP combinations remains many challenges. To address these challenges this thesis has been advanced the state-of-the-art discriminative pattern mining techniques to discover SNP combinations associated with interesting phenotype. Different solutions have been proposed in this thesis to tackle GWAS analysis. These solutions focus on efficient association strength evaluation, statistically significant discriminative SNP combinations discovery and interesting SNP combinations visualization. The solutions proposed in this thesis are also promising for other tasks of bioinformatics such as differential gene expression discovery, phosphorylation motifs detection and regulatory motif combination mining

Genetic control strategies are a novel method for reducing populations of pest insects such as the yellow fever mosquito Aedes aegypti, a major vector of several important arboviral diseases. This thesis describes efforts to develop new tools to engineer the Ae. aegypti genome and to better understand existing tools, and furthermore to use these to engineer a gene drive system in Ae. aegypti. The piggyBac transposon was found to be extremely stable in the germline of Ae. aegypti, and transposons engineered into the germline could not be remobilized with either an endogenous or exogenous source of piggyBac transposase. Conversely, somatic remobilization of piggyBac transposons was found to be readily detectable in the presence of a source of active transposase, the first report of such remobilization in Ae. aegypti. Toward new tools for genome engineering, the site-specific integrase from the phage φC31 was successfully used to promote exchange between a transgene cassette inserted into the genome of Ae. aegypti and a cassette in a plasmid vector, in the first demonstration of recombinase mediated cassette exchange technology in a pest insect species. The integrases from phages φRV1 and Bxb1 were not found to be active in the germline of the mosquito. Finally, development of a gene drive system in Ae. aegypti using an RNAi-mediated killer-rescue mechanism was attempted. Tissue-specific expression of tTAV-regulated-toxic effectors genes, using the promoter regions of the blood meal induced genes Carboxypeptidase A-1, 30Kb and Vitellogenin A, was possible, but sex-specificity was not achieved. A blood meal inducible lethal phenotype was not possible using the chosen promoters, with expression of the effectors either leading to death in early development or to a sublethal phenotype. RNAi against tTAV fused to the Mnp fragment of the dengue virus’ genome was tissue specific, but was found to be highly effective in the fat body suggesting that the Vitellogenin A was the best candidate for the engineering of killer-rescue systems in the mosquito.

Résumé : La phase haploïde de la spermatogenèse (spermiogenèse) est caractérisée par une modification importante de la structure de la chromatine et un changement de la topologie de l’ADN du spermatide. Les mécanismes par lesquels ce changement se produit ainsi que les protéines impliquées ne sont pas encore complètement élucidés. Mes travaux ont permis d’établir la présence de cassures bicaténaires transitoires pendant ce remodelage par l’essai des comètes et l’électrophorèse en champ pulsé. En procédant à des immunofluorescences sur coupes de tissus et en utilisant un extrait nucléaire hautement actif, la présence de topoisomérases ainsi que de marqueurs de systèmes de réparation a été confirmée. Les protéines de réparation identifiées font partie de systèmes sujets à l’erreur, donc cette refonte structurale de la chromatine pourrait être génétiquement instable et expliquer le biais paternel observé pour les mutations de novo dans de récentes études impliquant des criblages à haut débit. Une technique permettant l’immunocapture spécifique des cassures bicaténaires a été développée et appliquée sur des spermatides murins représentant différentes étapes de différenciation. Les résultats de séquençage à haut débit ont montré que les cassures bicaténaires (hotspots) de la spermiogenèse se produisent en majorité dans l’ADN intergénique, notamment dans les séquences LINE1, l’ADN satellite et les répétions simples. Les hotspots contiennent aussi des motifs de liaisons des protéines des familles FOX et PRDM, dont les fonctions sont entre autres de lier et remodeler localement la chromatine condensée. Aussi, le motif de liaison de la protéine BRCA1 se trouve enrichi dans les hotspots de cassures bicaténaires. Celle-ci agit entre autres dans la réparation de l’ADN par jonction terminale non-homologue (NHEJ) et dans la réparation des adduits ADN-topoisomérase. De façon remarquable, le motif de reconnaissance de la protéine SPO11, impliquée dans la formation des cassures méiotiques, a été enrichi dans les hotspots, ce qui suggère que la machinerie méiotique serait aussi utilisée pendant la spermiogenèse pour la formation des cassures. Enfin, bien que les hotspots se localisent plutôt dans les séquences intergéniques, les gènes ciblés sont impliqués dans le développement du cerveau et des neurones. Ces résultats sont en accord avec l’origine majoritairement paternelle observée des mutations de novo associées aux troubles du spectre de l’autisme et de la schizophrénie et leur augmentation avec l’âge du père. Puisque les processus du remodelage de la chromatine des spermatides sont conservés dans l’évolution, ces résultats suggèrent que le remodelage de la chromatine de la spermiogenèse représente un mécanisme additionnel contribuant à la formation de mutations de novo, expliquant le biais paternel observé pour certains types de mutations.
Abstract : Germline mutations may arise from several endogenous and exogenous mechanisms in both male and female. However, recent next-generation sequencing (NGS) data confirmed that de novo mutations arise primarily in males. This observation suggests that specific spermatogenesis events are involved in the male mutation bias. One potential origin for male-driven mutations is the differentiation of spermatids into spermatozoa, which involves one of the most striking and global chromatin remodeling processes, where histone-bound chromatin is converted into highly condensed protaminated DNA toroid. Using pulse-field gel electrophoresis and comet assay on flow cytometry sorted cells, it was established that chromatin remodeling process is characterized by a transient surge in DNA double strand breaks (DSBs) in the whole population of murine spermatids, which get repaired by the end of spermiogenesis. Using a highly active nuclear extract and immunofluorescences, topoisomerases and markers of DNA repair systems were shown at these steps. Since haploid cells cannot rely on homologous recombination for templated DNA repair, it was hypothesized that this process may be genetically unstable and largely responsible for the observed male de novo mutations bias. Although very challenging, a method allowing the specific genome-wide mapping of DSBs using NGS was developed to establish the genomic distribution of DSBs during chromatin remodeling. It was shown that intergenic regions were enriched in DSBs, particularly LINE1, satellite DNA and simple repeats. Motif finding on potential hotspots showed that proteins from FOX and PRDM families may be implicated. Although homologous recombination cannot take place during spermiogenesis, an enrichment in BRCA1 motif was found, which is also known to be implicated in NHEJ and removal of topoisomerase adducts. Topoisomerase-like SPO11 motif was also enriched suggesting that the meiotic machinery may also be implicated during chromatin remodeling. Moreover, although DSBs tend to accumulate in intergenic regions, gene ontology analysis of hotspot-containing genes showed a marked enrichment in genes related to neurons and brain development. This result hence supports the fact that neurological disease associated mutations are also male biased and associated with advanced paternal age. Since DSB formation during spermiogenesis is conserved through evolution, these results suggest that chromatin remodeling in spermatids represents a significant component in the reported male de novo mutation bias.

Les Spartines jouent un rôle écologique majeur sur les marais salés. Elles représentent un excellent modèle pour appréhender les conséquences écologiques de la spéciation par hybridation et polyploïdie dans le contexte d'invasion biologique. On s'intéresse plus particulièrement, à l'hybridation récente entre une espèce hexaploïde d'origine américaine Spartina alterniflora et une espèce hexaploïde européenne S. maritima ayant donnés deux hybrides F1 (S. x townsendii et S. x neyrautii) et la nouvelle espèce envahissante allododécaploïde (S. anglica). Les nouvelles technologies de séquençage haut-débit facilitent l'exploration de ces génomes peu connus. L'assemblage et l'annotation d'un transcriptome de référence ont permis d'annoter 16 753 gènes chez les spartines hexaploïdes et d'identifier des gènes d'intérêts écologique et évolutif. Une sélection de ces gènes a ensuite été analysée à travers une étude d'expression par PCR quantitative sur les populations naturelles des 5 espèces du complexe. Les résultats ont permis de mettre en évidence une expression homogène intra-populations mais une grande variabilité entre les espèces. L'analyse du génome des Spartines a ciblé prioritairement le développement de ressources génomiques concernant l'espèce S. maritima pour l'analyse des compartiments codant et répété à l'aide de séquençage d'une banque BAC et d'un run de pyroséquençage d'ADN génomique. Les analyses ont permis d'évaluer une proportion d'éléments répétés représentant près de 30% du génome. Les données générées ont alors été comparées avec les génomes séquencés phylogénétiquement proches et ont permis de premières comparaisons entre les spartines et les autres Poaceae.

Syftet med denna studie är att identifiera skillnader och likheter i olika pianolärares undervisningsupplägg, samt undersöka om deras synsätt och metoder går att knyta till de respektive genrer som de undervisar i. Inom forskning och litteratur har motsättningar som kan kopplas till afro och klassiskt undersökts och framställts i kontraster såsom gehör kontra noter eller improvisatoriskt kontra återgivande. Sådana motsättningar ligger till grund för föreliggande studies intresseområde. Studien utgår från ett sociokulturellt perspektiv, vilket innebär att lärande ses som en social aktivitet, där kunskap finns i kommunikation och de redskap som har växt fram i samhället. Datamaterialet består av kvalitativa intervjuer med fyra mer eller mindre aktiva musiker/pianopedagoger (två afrolärare och två klassiska lärare) med olika arbetssituationer. Resultatet visar att individanpassning och musikalisk mening samt glädjefullhet värderas högt i samtliga informanters pianoundervisning. Målet tycks vara att eleverna får uppleva glädje av att kunna musicera, snarare än att de ska bli ”stora” och kända pianister. Motivationen och det främsta ansvaret till att utvecklas anses ligga hos eleven. Fokus på teknik och övning ges efter elevens behov. Musikalitet ska alltid finnas i fokus. Omusikaliska övningar anses vara hämmande för uttrycket och motivationen. Att inkludera improvisation i undervisningen ses som viktigt. Informanterna har dock något olika arbetssätt och mål med improvisation. De klassiska lärarna arbetar efter rytmiska och tonala mallar med syfte att få eleven att utvecklas och/eller våga spela utan noter. Afrolärarna fokuserar mer på att spela till en särskild låt och att hitta personliga uttryck i improvisationen.
The purpose of this study is to identify differences and similarities in various piano teachers’ teaching approach, and investigate whether their approach and methods relate to the respective genres they teach. In research and literature, contradictions that can be linked to ”afro” and ”classical” have been investigated and described in contrasts like ear for music versus sheet music or improvisation versus reproduction. Contractions like that form the basis to this study’s area of interest. The study is based on a socio-cultural perspective, which means that learning is seen as a social activity, where knowledge exists in communication and the tools that have been developed in the community. The data consists of qualitative interviews with four more or less active musicians/piano teachers (two “afro” teachers and two classical teachers) with different work situations. The result shows that personalization, musical meaning and joyfulness are highly valued in all their piano teaching.  The goal seems to be the students´ experiences of joy in making music, rather than that they should become "great" and famous pianists. It is considered that the student has the primary responsibility in evolving and staying motivated. Focus on technique and training is connected to the student's needs. Musicality will always be in focus. “Unmusical” exercises are considered to be inhibiting the expression and motivation. To include improvisation in teaching is seen as important. The informants have somewhat different approaches and goals with improvisation. The classical music teachers work with rhythmic and tonal templates designed to get students to evolve and/or get courage to play without sheet music. “Afro” teachers focus more on playing a particular song and finding personal expressions in improvisation.