Thèses sur le sujet « Transcriptomic analysi »

Le combat contre les infections bactériennes reste un enjeu majeur de santé publique notamment avec la dissémination des Entérobactéries productrices de carbapénèmases, capables d’hydrolyser l’ensemble des β-lactamines. On assiste à l’émergence de certains clones épidémiques, se distinguant par leur distribution mondiale, leur forte transmissibilité et leur capacité à persister chez les patients. L’exemple le plus parlant est le cas du clone de Klebsiella pneumoniae appartenant au « sequence type » (ST) 258 et responsable de la diffusion mondiale de la carbapénèmase KPC (Klebsiella Pneumoniae Carbapenemase) portée majoritairement par des plasmides de la famille InFIIk. Les raisons du succès de ce clone et de l’association KPC/IncFIIk/ST258 ne sont pas encore totalement élucidées. Par ailleurs, il n’existe pas de corrélation entre le niveau d’expression d’une carbapénèmase, la sensibilité in vitro de la souche vis à vis des carbapénèmes et l’efficacité clinique d’un traitement par ces molécules. Les phénomènes d’héterorésistance aux carbapénèmes sont fréquents chez les souches produisant KPC, mais l’impact clinique est inconnu. Les mécanismes de régulation de l’expression des carbapénèmases ne sont pas élucidés.Les objectifs de cette thèse résident dans l’analyse des facteurs génétiques associés à la diffusion et la persistance de clones multi-résistants ainsi que l’analyse de l’expression des β-lactamases associées.La première partie de ce travail porte sur l’analyse de l’évolution in vivo d’une souche de K. pneumoniae ST258 produisant KPC ayant persisté chez un patient pendant près de 5 ans. L’analyse comparative des génomes provenant de 17 isolats a permis de mettre en évidence une diversification génétique importante ainsi que la sélection de mutations modifiant la virulence de la souche et sa sensibilité aux antibiotiques. Afin de caractériser les raisons du succès de certains plasmides portant KPC, une analyse transcriptomique d’une souche de Escherichia coli TOP10 transformée par un plasmide multi-réplicon IncFIIk-IncFIB exprimant KPC-2, a été réalisée en présence ou non d’imipénème. Les gènes les plus exprimés dans ces conditions sont les gènes de résistance aux antibiotiques et certains gènes essentiels à la réplication du plasmide. La présence d’imipénème affecte peu la transcription des gènes plasmidiques mais induit un stress oxydatif important dans l’ensemble de la souche. Par ailleurs, l’analyse de l’expression du gène blaKPC-2 dans différentes espèces par 5’-RACE a permis de révéler que ce gène de résistance est sous la dépendance de plusieurs promoteurs, dont la force diffère selon le fond génétique. Cette caractéristique pourrait expliquer le succès de certains isoformes du transposon Tn4401 permettant une meilleure expression du gène blaKPC-2, dans certaines espèces. Les outils développés dans cette thèse ont également été appliqués à l’analyse d’un clone d’Enterobacter kobei ST125 dont la céphalosporinase naturelle ACT-28 possède une activité d’hydrolyse accrue vis à vis de l’imipénème. Enfin, l’analyse du génome de la première souche Shewanella sp. produisant une BLSE de type CTX-M-15 a permis de révéler la présence d’un nouveau variant oxacillinase chromosomique avec activité carbapénèmase, appelé OXA-535. OXA-535 est proche d’OXA-436, un autre variant carbapénèmase porté par un plasmide ayant déjà disséminé chez les Entérobactéries. L’analyse de l’environnement génétique des gènes blaOXA-535 et blaOXA-436 confirme le rôle du genre Shewanella comme progéniteur des carbapénèmases de classe D. Ce travail contribue à une meilleure compréhension de la diffusion de certains clones multi-résistants et des mécanismes contrôlant l’expression des gènes de résistance aux β-lactamines
Multidrug resistant bacteria and in particular carbapenemase-producing Enterobacteriaceae remain a major health public challenge. Some successful clones are emerging globally, due to their high transmissibility and their ability to colonize and persist in patients over time. Genomic analyses revealed that the dissemination of KPC carbapenemase is closely related to the spread of Klebsiella pneumoniae of the sequence-type (ST) 258 and to few successful plasmids linked to IncFIIk family. However, the reasons of the association between K. pneumoniae ST258, IncFIIk plasmids and KPC that led to the rapid spread of this clone are currently unknown.Furthermore, there is no correlation between expression level of a carbapenemase-encoding gene, in vitro susceptibility to carbapenems and efficiency of a carbapenem-based treatment. Most of the time, KPC-producing K. pneumoniae exhibit a heteroresistant phenotype with carbapenems, but its clinical impact remains unknown. The mechanisms underlying the regulation of carbapenemases expression remain to be explored.The objectives of the thesis are to obtain deeper insights into genomic plasticity of carbapenemase–producing clones, and into the expression of their β-lactamases.The first part of this work was dedicated to the in vivo evolution of a single strain of KPC-producing K. pneumoniae ST258 that colonized a patient for almost 5 years. Genomic comparison of 17 isolates revealed a remarkable diversification with occurrence of several mutations with impact on bacterial virulence and susceptibility to antibiotics.Several studies extensively described the genetic structures of blaKPC-carrying plasmids, but information regarding gene expression at the whole plasmid level are lacking. Accordingly, we performed RNA-seq on Escherichia coli TOP10 transformed with an IncFIIk-IncFI blaKPC-2-carrying plasmid, with or without imipenem exposure. In both conditions, plasmid-encoded genes related to antimicrobial resistance and involved in plasmid replication were the most expressed. Imipenem exposure led to a more general response with overexpression of E. coli numerous chromosome-encoded genes involved in oxidative stress response. In addition, analysis of blaKPC-2 gene expression in several species using 5’RACE revealed the presence of several promoters whose strength depends on the bacterial genetic background. This could promote higher expression of blaKPC-2 gene and explain the association of some isoforms of Tn4401 in different species. The tools developed in the frame of this work were also applied to study a single Enterobacter kobei ST125 clone whose natural cephalosporinase (ACT-28) has increased hydrolytic activity towards imipenem. Finally, genomic analysis of the first ESBL-producing Shewanella sp. was performed. It revealed the presence of blaCTX-M-15 and blaSHV-2 genes carried on an IncA/C plasmid and a new chromosomally-encoded oxacillinase variant of OXA-48 with carbapenemase activity, called OXA-535. OXA-535 was found to be closely related to OXA-436, another carbapenemase which has recently spread in Enterobacteriaceae. Analysis of the genetic environment of both blaOXA-48-like genes confirmed the role of Shewanella spp. as progenitors of class D carbapenemases.Overall, this work contributes to a better comprehension of the diffusion of multi-drug resistant clones and of the mechanisms implicated in β-lactamase expression

L’obesitat i la diabetis tipus 2 (DT2) són dues malalties estretament relacionades que representen un greu problema de salut, social i econòmic per la seva alta prevalença a tot el món. Ambdues malalties també es relacionen amb altres patologies que presenten una mortalitat elevada. Les teràpies disponibles actualment no són del tot efectives. Per tant, el desenvolupament de noves estratègies terapèutiques per a l'obesitat i la DT2 és crucial. El teixit adipós s'ha definit com un òrgan que té un paper central en el control del balanç energètic. El descobriment de funcions endocrines i termogèniques dels adipòcits han tornat a centrar l’interès en l’estudi d’aquest teixit. La termogènesi no associada a tremolor està descrita per dur-se a terme al teixit adipós marró dels ratolins, però sota certs estímuls, com l'exposició prolongada al fred, cèl·lules amb una morfologia similar als adipòcits marrons (adipòcits beix) apareixen en alguns dipòsits de teixit adipós blanc de rosegadors i humans, un procés conegut com a browning. L’activació a través de l’exposició al fred de la termogènesi no associada a tremolor en humans augmenta el consum energètic en repòs, la sensibilitat a la insulina i millora el metabolisme de la glucosa. No obstant, es necessiten més estudis d'expressió gènica per aprofundir en els mecanismes moleculars subjacents a la millora induïda per fred a través de la termogènesi no associada a tremolor, així com per determinar les diferències entre l'activació del teixit adipós marró (BAT) i el browning del teixit adipós blanc (WAT). En aquesta tesi doctoral, es van examinar els canvis transcriptómica dels dipòsits adiposos blancs epidídimal i inguinal (eWAT i iWAT, respectivament), així com la del dipòsit adipós marró interscapular (iBAT) de ratolins exposats a 22ºC o 4ºC durant 4 dies. L’exposició al fred va augmentar l’activitat metabòlica i termogènica de l’iWAT. En aquest dipòsit, els gens relacionats amb la glucòlisi, el cicle de l'àcid tricarboxílic, la lipòlisi i la degradació d'alguns aminoàcids van presentar una expressió incrementada per mantenir la potència protonmotriu per generar calor. A més, l'expressió de gens relacionats amb la termogènesi també va augmentar molt, demostrant una inducció del browning induïda per fred en el iWAT. S'ha descrit que el dipòsit d’eWAT és resistent al browning. Per tant, l’activació metabòlica observada d’aquest dipòsit va ser suau en comparació amb la d’iWAT, i en aquest dipòsit no es va observar cap millora rellevant de la termogènesi no associada a tremolor. Finalment, l’iBAT ja presentava nivells d’expressió de gens termogènics elevats, ja que els ratolins no estaven estabulats a termo-neutralitat. L’observació que els gens termogènics i metabòlics presentaven un patró d’expressió similar entre les mostres va recolzar l’ús d’eines d’anàlisi de patrons per descriure Atp4b i 1700040L02Rik com a nous gens potencialment implicats en la termogènesi. La sobreexpressió d’Atp4b i 1700040L02Rik en el teixit adipós mitjançant l’ús de vectors AAV va produir una reducció del guany de pes corporal, una disminució del pes de l'eWAT i del fetge, la millora de la hipertròfia dels adipòcits blancs i la reducció de la esteatosi hepàtica. Tots aquests resultats van ser potencialment fruit de la termogènesi millorada detectada en iWAT. En general, aquests resultats indiquen un possible nou paper anti-obesogènic d’aquests gens. Els resultats d’aquesta tesi han contribuit a una millor comprensió de la inducció de la termogènesi no associada a tremolor en els dipòsits de teixit adipós de ratolins. Entre els diferents dipòsits de teixit adipos, l’anàlisi exploratori dels nivells d’expressió gènica va determinar que el iWAT era el dipòsit que va respondre de manera més significativa a l’exposició al fred. A més, tal com es va observar en l’anàlisi de l’enriquiment de vies i d’ontologia gènica, aquesta resposta va ser altament coordinada, presentant un elevat nombre de gens relacionats amb rutes metabòliques que presentaven una expressió altament incrementats. Igualment, l’estudi detallat de les vies metabòliques va destacar una gran inducció de la termogènesi no associada a tremolor, revelar que els mecanismes de producció i de consum d’energia estaven altament sincronitzats. Aquest estudi detallat del teixit adipós també va permetre la identificació de nous gens potencialment implicats en la termogènesi no associada a tremolors.
Obesity and type 2 diabetes (T2D) are two closely related diseases that represent a serious health, social and economic problem due to their high prevalence worldwide. Both diseases are also associated with other pathologies that present high mortality. The currently available therapies are not entirely effective. Thus, the development of new therapeutic strategies for obesity and T2D is crucial. Adipose tissue has been defined as an organ that plays a central role in the control of energy balance. The proved endocrine and thermogenic functions of adipocytes has renewed interest in the study of this tissue. Non-shivering thermogenesis has been described as occurring in brown adipose tissue of mice, but under certain stimuli, such as prolonged cold exposure, brown fat-like cells (beige adipocytes), appear in some white adipose tissue depots of rodents and humans. The activation of non-shivering thermogenesis in humans through cold-exposure increases resting energy expenditure, whole-body glucose disposal, insulin sensitivity, and ameliorates glucose metabolism independently of BMI. However, more gene expression studies to gain insight into the molecular mechanisms underlying the cold-induced enhancement of non-shivering thermogenesis, as well as to determine differences between BAT activation and browning of WAT, are needed. In this study, the transcriptomic response of epididymal and inguinal white adipose depots (eWAT and iWAT, respectively) as well as that of the interscapular brown adipose depot (iBAT) of mice either exposed to 22ºC or 4ºC for the period of 4 days were examined. Cold exposure increased the metabolic and thermogenic activity of iWAT. In this depot, genes related to glycolysis, tricarboxylic acid cycle, lipolysis, and the degradation of some amino acids presented a high upregulation to maintain the protonmotive power to generate heat. Moreover, the expression of thermogenic-related genes was also highly increased, demonstrating a cold-induced browning of iWAT. The eWAT depot has been reported to be resilient to browning. Thus, the observed metabolic activation of this depot was mild in comparison with that of iWAT, and no relevant enhancement of non-shivering thermogenesis was observed in this depot. Finally, iBAT already presented high expression levels of thermogenic genes because mice were not housed at thermoneutrality. The observation that genes related to thermogenesis and metabolism presented a similar expression pattern among samples endorsed the utilization of pattern matching analysis tools to unravel Atp4b and 1700040L02Rik as novel genes potentially involved in thermogenesis. The overexpression of Atp4b and 1700040L02Rik in adipose tissue by means of AAV vectors produced a body weight gain reduction, decreased eWAT, and liver weight, amelioration of white adipocytes hypertrophy, and reduced hepatic steatosis potentially as a result of the detected enhanced thermogenesis in iWAT. Overall, these results indicate a new potential anti-obesogenic role for these genes. The results from this thesis contributed to a better understanding of the induction of non-shivering thermogenesis in adipose tissue depots in mice. Among the different adipose depots, exploratory data analysis of the gene expression levels of mice exposed from 22ºC to 4ºC determined that iWAT was the depot that responded most significantly to cold exposure. Moreover, as observed in the pathway enrichment and gene ontology analysis, this response was highly coordinated, presenting a high number of genes related to metabolic pathways highly affected. The detailed study of the metabolic pathways led to the detection of a high induction of non-shivering thermogenesis, revealing that both energy production and energy consumption mechanisms were highly synchronized. This in detail study of the adipose tissue also allowed the identification of novel genes potentially involved in non-shivering thermogenesis.

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid advances have enormously expanded sequencing opportunities and applications, but also imposed heavy strains on steps prior to sequencing, as well as the subsequent handling and analysis of the massive amounts of sequence data that are generated, in order to exploit the full capacity of these novel platforms. The work presented in this thesis (based on six appended papers) has contributed to balancing the sequencing process by developing techniques to accelerate the rate-limiting steps prior to sequencing, facilitating sequence data analysis and applying the novel techniques to address biological questions.   Papers I and II describe techniques to eliminate expensive and time-consuming preparatory steps through automating library preparation procedures prior to sequencing. The automated procedures were benchmarked against standard manual procedures and were found to substantially increase throughput while maintaining high reproducibility. In Paper III, a novel algorithm for fast classification of sequences in complex datasets is described. The algorithm was first optimized and validated using a synthetic metagenome dataset and then shown to enable faster analysis of an experimental metagenome dataset than conventional long-read aligners, with similar accuracy. Paper IV, presents an investigation of the molecular effects on the p53 gene of exposing human skin to sunlight during the course of a summer holiday. There was evidence of previously accumulated persistent p53 mutations in 14% of all epidermal cells. Most of these mutations are likely to be passenger events, as the affected cell compartments showed no apparent growth advantage. An annual rate of 35,000 novel sun-induced persistent p53 mutations was estimated to occur in sun-exposed skin of a human individual.  Paper V, assesses the effect of using RNA obtained from whole cell extracts (total RNA) or cytoplasmic RNA on quantifying transcripts detected in subsequent analysis. Overall, more differentially detected genes were identified when using the cytoplasmic RNA. The major reason for this is related to the reduced complexity of cytoplasmic RNA, but also apparently due (at least partly) to the nuclear retention of transcripts with long, structured 5’- and 3’-untranslated regions or long protein coding sequences. The last paper, VI, describes whole-genome sequencing of a large, consanguineous family with a history of Leber hereditary optic neuropathy (LHON) on the maternal side. The analysis identified new candidate genes, which could be important in the aetiology of LHON. However, these candidates require further validation before any firm conclusions can be drawn regarding their contribution to the manifestation of LHON.
QC 20111115

Introduction Septic shock, also defined as distributive shock, is a complication of sepsis, characterized by pronounced hypotension, followed by anomalous distribution of blood at vessels, organs and tissues. The hemodynamic, cellular and metabolic alterations described in septic shock patients lead to a mortality that is at present around 40%. Septic shock patients develop dysfunctions or failure to multiple organs (MOF) but the molecular mechanisms triggering tissue injury remain largely undetermined and a specific treatment for septic shock is still not available. Aim This work is part of the European Project ShockOmics, a multicentric, prospective, observational study, whose aim is to identify with a multiscale approach, molecular biomarkers in septic shock patients who develop acute heart failure. The specific aim of the present Research project is to investigate the modifications induced by septic shock on transcriptional profile, using blood cells as RNA source. This investigation is performed at different timepoints starting from admission of the patient to the intensive care unit (ICU). Materials and Methods Septic shock patients were recruited in the ICUs of Geneva and Bruxelles University Hospitals, that are Partners of ShockOmics Project. Blood samples were collected in the acute phase of the disease at ICU admission (T1), after the appropriate pharmacological intervention (T2 ) and at steady state on day 7 of the ICU stay (T3). RNA was extracted from whole blood and RNA sequencing was used to evaluate the expression level of genes, long non coding RNAs and microRNAs. We explored the dataset using PCA and unsupervised hierarchical clustering and we identified differentially expressed genes and microRNAs across conditions. Gene Ontology analysis was used to identify relevant biological processes involved in shock. We identified microRNA regulatory targets with an in silico target prediction. Results We identified two main gene expression profiles corresponding to the acute phase of shock and to the condition of steady state. Between the acute phase of shock (day 1) and the steady state condition (day 7) we observed in patients at day 7 a downregulation of pathways of the innate immune response (Toll-like receptor and C-type lectin receptors pathways) and of acute inflammation (IL-1 receptor family and alarmins) and the upregulation in the same patients of genes of the adaptive immunity related to B and T lymphocytes activation. A transcriptional regulation was observed also for genes with antimicrobial function and protease activity and for genes involved in carbohydrate metabolism, lipid inflammatory pathway, transport of vesicles and protein synthesis. miR-125a-5p and miR-150-5p, with a predicted regulatory role in the MAPK pathway, and miR-193a-3p were differentially expressed in the acute and steady state condition. Conclusion We observed a significant modulation of multiple classes of genes involved in defense response to pathogens, immunity, inflammation and metabolism. From these results it appears that in septic shock a relevant change in the transcriptomic profile of blood cells is induced, in order to counteract the pathogens and as a consequence of the hemodynamic changes underlying the circulatory failure. The transcriptomic profile of septic shock patients showed inter patient variability reflecting the complexity of the shock condition and of the individual response to treatment. Specific signatures could turn out by combining clinical data and expression profile and could be used to better classify septic shock patients.

Dans la première partie j'ai analysé des jeux de données de RNA-seq de transcriptome de petits ARNs disponibles dans les bases de données publiques. J'y ai observé 2 points intrigants : - une grande partie des lectures (bien que courtes) ne peux pas être alignée sur le génome de référence sans discordance et cette fraction non-alignable est parfois majoritaire. - de nombreuses lectures ont des tailles autours de 15-18nt qui ne correspondent à aucun type de petits ARNs connues, cette fraction est également majoritaires dans certains cas. Ces expériences sont souvent conçues pour la détection des miRNAs et l'analyse bioinformatique de ces données passent toujours par un alignement sur le génome de référence ou sur des séquences connues pour donner des petits ARNs. J'ai donc simplement éliminé la contrainte d'alignement dans l'analyse de ces données et effectué un regroupement des lectures par similarité (à la manière des ESTs). Ce regroupement donne une vision différente des données dans laquelle la notion de position génomique n'est plus centrale et ouvre la possibilité d'y découvrir des phénomènes non-standard. La deuxième partie est tirée d'une collaboration avec le laboratoire U675 INSERM. J'ai fait l'analyse bioinformatique des gènes dérégulés par la répression par RNAi du gène REST dans une lignée de neuroblastome de souris (N18). Ce gène est un facteur de transcription qui réprime les gènes neuronaux dans les cellules non neuronales. Ce répertoire de gènes dérégulés est potentiellement constitué de gènes clefs dans la biologie des neurones
In first part of this thesis I have analysed small RNA-seq transcriptome data. I have noticed : - a large fraction of reads can't be aligned perfectly on reference genome - lot of reads are very short (15-18 nt) and don't match on previously known functionnal small RNAs. These experiments are designed for miRNA discovery and bioinformatics analysis of these data use alignments on genome or on known small RNA precursors sequences. I have eliminated the alignment and I have clustered these sequences. This clustering let me to observe these data with a new view in wich the genomic location is not central and open the gate to discover unconventional events. The second part is the analysis of deregulate genes by the silencing of the gene REST/NRSF in mouse N18 cell line. This gene is a transcription factor and it works as a repressor of neuronal genes in non neuronal cells. This deregulate genes repertoire potentially contains key genes in neuron biology. We found in this repertoire a network of genes centered on SWI/SNF complex including SMARCA2. This gene was associated to schizophrenia (SZ) in association studies and structural variation studies. In this network we found another genes associated to SZ. We show that these genes exhibit positive evolution in primate compare to rodents

Les maladies cardiovasculaires représentent un important problème de santé publique à travers le monde. Parmi ces pathologies, l’infarctus du myocarde (IM) a fait l’objet de nombreux essais visant à diminuer sa sévérité. Néanmoins peu ont remporté leur pari. Cet échec peut avoir plusieurs composantes parmi lesquelles la méconnaissance de la complexité des mécanismes moléculaires impliqués. Notre compréhension de l’IM a cependant été nettement améliorée grâce à des méthodes utilisées pendant les dernières décennies et qui consistaient à étudier séparément un nombre limité d’acteurs moléculaires impliqués dans un mécanisme simple et linéaire. Cependant, l’échec des essais cliniques basés sur ces approches réductionnistes ont montré leurs limites. L’émergence de la biologie des systèmes, ces dernières années, a stimulé les recherches visant à mieux intégrer et comprendre la nature complexe et stochastique des réseaux moléculaires et leur dynamique dans la progression des pathologies. L’objectif général de cette thèse a consisté en le développement et l’amélioration de méthode d’analyses et de combinaison des données spatio-temporelles issus d’expériences réalisées sur un modèle d’infarctus du myocarde chez la souris. L’objectif scientifique visait à caractériser les principaux signaux dynamiques au cours de la séquence d’ischémie-reperfusion. A cet effet, nous avons tout d’abord développé une chaîne de méthode utilisant la clarification d’organe et la microscopie de fluorescence permettant de quantifier, en 3 dimensions, la zone du myocarde soumise au choc oxydant lors de la reperfusion. Dans une seconde partie, nous avons développé une nouvelle chaîne analytique pour caractériser la dynamique d’expression des transcrits. Appliquée aux animaux contrôles (soumis à la chirurgie et l’anesthésie), nous mettrons, grâce à cette chaîne de méthode, le rôle majeur de la voie de l’interleukine 6 dans le développement de la réponse immunitaire et nous concluons ainsi sur la nécessité de réaliser une analyse dynamique du modèle expérimental pour caractériser sa réponse à l’échelle moléculaire et éviter toute surinterprétation de la réponse à l’IM
Cardiovascular diseases represent a major health burden worldwide. According to the World Health Organization, 17 million people are dying each year by heart diseases which account to 31% of total deaths globally. Among these diseases is myocardial infarction (MI). Several efforts have been made to decrease the associated mortality rates but unfortunately, only few has succeeded to date. This failure is contributed to several factors, among them is the misunderstanding of the mechanism responsible for the progression of the disease.Our understanding of the MI pathology has been greatly improved by the approaches that have been widely used in the previous decades, relying mainly on studying molecules/pathways separately. However, this knowledge was not enough to make a difference clinically. Therefore, deciphering the interconnections between molecules has become an urge for better understanding of the diseases’ progression. In this regard, the work in this doctoral thesis involves different aspects of the MI pathology. The general aim of this work is to improve the dynamic analytical approach using systems biology tools, where new mechanistic information is decoded. Firstly, in a 3D heart model, we propose a chain of methods using clarified mouse heart and fluorescence microscopy to molecularly characterize the area at risk in the myocardium of IR and cardioprotected mice based on its redox state. In addition, we aim to develop a new analytical approach using dynamical large-scale transcriptomic data for characterizing the dynamic transcripts expression. Applying this approach on a control mouse model (mice subjected to anesthesia and surgical interventions), we show that Il-6 is a major mediator of the activated inflammatory reaction. In conclusion, this analytical approach highlights the necessity of the sapatio-temporal analysis to characterize the molecular events occurring in response to MI

La tesi di Giorgio Bertolazzi è incentrata sullo sviluppo di nuovi algoritmi per la predizione dei legami miRNA-mRNA. In particolare, un algoritmo di machine-learning viene proposto per l'upgrade del web tool ComiR; la versione originale di ComiR considerava soltanto i siti di legame dei miRNA collocati nella regione 3'UTR dell'RNA messaggero. La nuova versione di ComiR include nella ricerca dei legami la regione codificante dell'RNA messaggero.
Bertolazzi’s thesis focuses on developing and applying computational methods to predict microRNA binding sites located on messenger RNA molecules. MicroRNAs (miRNAs) regulate gene expression by binding target messenger RNA molecules (mRNAs). Therefore, the prediction of miRNA binding is important to investigate cellular processes. Moreover, alterations in miRNA activity have been associated with many human diseases, such as cancer. The thesis explores miRNA binding behavior and highlights fundamental information for miRNA target prediction. In particular, a machine learning approach is used to upgrade an existing target prediction algorithm named ComiR; the original version of ComiR considers miRNA binding sites located on mRNA 3’UTR region. The novel algorithm significantly improves the ComiR prediction capacity by including miRNA binding sites located on mRNA coding regions.

Orientadores: Anete Pereira de Souza, Sindélia Freitas Azzoni
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Buscando contribuir com o desenvolvimento da tecnologia de produção do etanol de segunda geração, o presente estudo analisa o transcriptoma de T. harzianum IOC-3844 utilizando técnicas de sequenciamento high-thoughput. O principal objetivo dessas análises foi identificar, caracterizar e catalogar os transcritos expressos por T. harzianum relacionados com a degradação de substratos complexos, como o bagaço de cana de açúcar, revelando o conjunto de genes envolvidos na degradação da biomassa. A análise do transcriptoma do fungo Trichoderma harzianum sob condições que induzem a degradação da biomassa permitiu a identificação de sequências de genes potencialmente eficazes no processo de biodegradação, uma etapa essencial à compreensão do processo de hidrólise enzimática. O sequenciamento resultou em 246 milhões de sequências com 72 pb, o que corresponde a 14,7 GPB analisados. Após a montagem , 32.494 contigs foram gerados, submetidos à identificação e classificados de acordo com sua identidade. Todas as sequências de contigs foram comparados com o banco de dados do NCBI, Gene Ontology (GO terms), Enciclopédia de Genes Kyoto (KEGG), Carbohydrate Active-Enzymes (CAZYmes). Foram identificados 487 CAZymes no transcriptoma, inclusive aquelas ligadas as reações químicas de despolimerização de celulose e hemicelulose. As sequências classificadas como atividade catalítica (6.975) e atividade reguladora (143) podem estar envolvidas com esse tipo de reação.A análise permitiu definir o principal conjunto de genes envolvidos na degradação da celulose e de hemicelulose do T. harzianum , e genes acessórios relativos à despolimerização de biomassa. Uma análise dos níveis de expressão permitiu determinar os conjuntos de genes diferencialmente expressos em diferentes condições de cultivo. Os resultados obtidos acrescentam conhecimento sobre a constituição do genoma, as atividades de expressão gênica do fungo Trichoderma harzianum e fornece informações importantes a respeito dos mecanismos genéticos de degradação de biomassa que o fungo utiliza. As informações obtidas poderão ser utilizadas para outras espécies de fungos filamentosos com potencial para a biodegradação
Abstract: In order to contribute to the development of second-generation ethanol technology, this study analyzes the transcriptome of T. harzianum IOC-3844 using high-thoughput sequencing techniques. The main objective of this analysis was to identify, characterize and catalog the transcripts expressed by T. harzianum related to the degradation of complex substrates such as sugar cane bagasse, revealing the set of genes involved in the degradation of biomass. The analysis of the transcriptome of the fungus Trichoderma harzianum under conditions that induce the degradation of biomass allowed the identification of genes potentially effective in the biodegradation process, an essential step for understanding the enzymatic process. Sequencing resulted in 246 million sequences with 72 bp, which corresponds to 14.7 GBP analyzed. After assembly, 32,494 contigs were generated, identified and classified according to their identity. All sequence contigs were compared with NCBI database, Gene Ontology (GO terms), Kyoto Encyclopedia of Genes (KEGG), Carbohydrate Active-Enzymes (CAZYmes). 487 CAZymes were identified in the transcriptome, including those related to reactions of cellulose and hemicellulose depolymerization. Sequences classified as catalytic activity (6,975) and regulatory activity (143) may be involved with this type of reaction. This analysis define the set of genes involved in the degradation of cellulose and hemicellulose of T. harzianum, and accessories genes related to depolymerization of the biomass. An analysis of expression levels was used to calculate the set of differentially expressed genes in different culture conditions. The results add to knowledge about the composition of the genome and gene expression activity of the fungus Trichoderma harzianum, and provides important information regarding the genetic mechanisms of biomass degradation that the fungus uses. The information obtained may be used for other species of filamentous fungi with potential for biodegradation
Doutorado
Genetica Vegetal e Melhoramento
Doutora em Genética e Biologia Molecular

Orientadores: Anete Pereira de Souza, Renato Vicentini dos Santos
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A cana-de-açúcar é uma das espécies de maior importância econômica no mundo devido ao seu potencial bioenergético. No entanto, o seu alto nível de complexidade genética é um desafio para a aplicação de ferramentas moleculares no melhoramento. Os recentes avanços das tecnologias de sequenciamento e genotipagem indicam o potencial de aumentar o nosso entendimento sobre a genética e a biologia molecular desta espécie. As sequências genômicas e de transcriptomas são valiosa fonte de informação para o desenvolvimento de ferramentas moleculares que permitam a identificação de regiões no genoma que estejam relacionadas com características de interesse para o melhoramento. O uso das novas tecnologias de sequenciamento de alto desempenho tem grande potencial de impacto nestas pesquisas. A presente tese objetivou analisar o transcriptoma de seis variedades comerciais e dados genômico da variedade R570, com a finalidade de identificar genes potencialmente úteis para o desenvolvimento de marcadores moleculares. A partir do método RNA-Seq, foram geradas mais de 400 milhões de sequências, as quais permitiram obter um total de 72.269 transcritos representados por uma única isoforma montados com auxílio do programa Trinity. Estes transcritos foram alinhados com sequências de Viridiplantae, gramíneas, e exclusivamente contra proteínas de sorgo, arroz, milho e transcriptoma de cana-de-açúcar, depositados em banco de dados público. Esta análise permitiu identificar o conjunto de genes de cana-de-açúcar compartilhados com outras gramíneas, bem como levou à identificação de novos transcritos que não haviam sido catalogados para cana-de-açúcar, além de longos RNAs não codificantes. Os transcritos foram anotados no Cluster of Orthologous Groups (COG) e no Gene Ontology (GO), com posterior análise de enriquecimento dos termos GO, a partir da qual foram anotados os transcritos, possivelmente relacionados a genes que conferem características de importância agronômica. No transcriptoma foram identificados mais de 700 mil SNPs e aproximadamente cinco mil regiões microssatélites. Analisando um total de 32 Mbp de sequências genômicas da variedade R570 foram identificados 4.342 microssatélites, com frequência média de sete SSR/Kb. As sequências geradas e exploradas neste trabalho são valiosa fonte de informações para entender a arquitetura genética da cana-de-açúcar, principalmente para o desenvolvimento de marcadores moleculares, os quais podem ser utilizados no mapeamento genético
Abstract: The sugarcane is one of the most economically important species in the world, due to their energy potential. However, high level of genetic complexity has been a major challenge for the use of molecular tools applied to improvement of this crop. Recent advances in sequencing and genotyping technologies indicate the potential to increase our understanding of the genetics and molecular biology of this specie. The genomic and transcriptomic are valuable sources of information for the molecular tools development that allow identification of regions in the genome that are related to characteristics of interest for the improvement. The high-throughput sequencing technologies have great impact of this research. This thesis aimed to analyze the transcriptome of six commercial varieties and genomic sequencing from R570 variety, in order to identify genes potentially useful for the molecular markers development. From RNA-Seq method were generated over 400 million sequences, which allowed obtain a total of 72,268 transcripts representing a single isoform assembled by Trinity. These transcripts were aligned against Viridiplantae, grasses, and exclusively against sorghum, rice and maize proteins, and sugarcane transcriptome available in the public database. This analysis allowed identifying a set of shared genes with other grasses, new transcripts that had not yet been cataloged for sugarcane and long non-coding RNAs. The transcripts were also annotated using the COG (Cluster of Orthologous Groups) and GO (Gene Ontology) database, followed by enrichment analysis for GO terms, from which it was possible to identify genes that play roles, possibly related to traits of agronomic importance. In the transcriptome were identified over 700 thousands SNPs and five thousands microsatellites regions. In the genomic sequences from R570 variety, in a total of 32 Mbp were identified 4,342 microsatellites, with an average frequency of seven SSR / Kb. The sequences generated and explored in this work is a valuable source to understand the genetic architecture of the sugarcane, mainly for molecular markers development, which can be used in genetic mapping
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular

Yersinia pestis, l'agent de la peste, est une bactérie à Gram-négatif classée comme agent pathogène ré-émergent et potentielle arme de bioterrorisme. De plus, l'apparition d'une souche multi-résistance de cette bactérie souligne la nécessité de mieux comprendre comment cette bactérie hyper-virulente interagit avec son hôte. Afin d'identifier des facteurs génétiques de vulnérabilité à la peste, notre laboratoire travaille sur la réponse de souris résistantes versus sensibles à Y. pestis. Notre stratégie pour identifier les facteurs génétiques impliqués dans la résistance/sensibilité à la peste combine une approche de cartographie de QTL (Quantitative Trait Locus) et d'analyse d'expression génique. Nous avons précédemment décrit la lignée SEG/Pas, issue de Mus spretus, comme la première résistante à une souche virulente de Y. pestis, alors que la plupart des lignées murines de laboratoire, telle que la lignée C57BL/6J, sont extrêmement sensible à la bactérie. Des croisements entre SEG/Pas et C57BL/6J nous ont permis d'identifier trois QTL impliqués dans la résistance à Y. pestis, localisés sur les chromosomes 3, 4 et 6. Deux des QTL (situés sur les chromosomes 4 et 6) ont pu être confirmés par l'analyse de lignées congéniques. Plus de 40 % des femelles bi-congéniques hétérozygotes pour ces deux QTL ont survécu à l'infection, alors que tous les témoins C57BL/6J ont succombé. La dissection de ces deux QTL par l'analyse de lignées sous-congéniques, nous a permis d'affiner l'architecture génétique de la résistance à la peste chez SEG/Pas. Nous avons conclu qu'un minimum de quatre facteurs génétiques, au sein de ces deux QTL, sont nécessaires pour augmenter la résistance à Y. pestis chez la Souris. Cependant, la production de plusieurs lignées congéniques portant le QTL situé sur le chromosome 3, dont une lignée triple congénique, ne nous a pas permis de confirmer l'existence de ce QTL. En parallèle de l'analyse génétique, nous avons déterminé que les macrophages de SEG/Pas et de C57BL/6J présentaient des caractéristiques différentes après exposition à Y. pestis. Une analyse différentielle du profil transcriptionnel des macrophages de ces deux lignées a été réalisée à l'aide de puces à ADN. Nos résultats montrent une forte activation de la production cytokinique dans les macrophages de SEG/Pas en réponse à la bactérie, activation qui n'est pas observée dans la lignée C57BL/6J. Ces résultats suggèrent que les souris SEG/Pas sont capables de mettre en place une réponse immune innée plus forte ou peut-être plus précoce que C57BL/6J. Nous avons ensuite étudié par qRT-PCR l'expression en cinétique de 44 gènes dans des macrophages de SEG/Pas, C57BL/6J et des bi-congéniques portant les QTL sur les chromosomes 4 et 6. Cette étude nous a permis de confirmer que les souris SEG/Pas sont capables se mettre en place une forte réponse inflammatoire lors de l'infection. Cependant, aucune différence significative n'a été observée entre la lignée bicongénique et la lignée parentale C57BL/6J. D'autres expériences seront nécessaires afin de mieux comprendre les mécanismes biologiques impliqués dans la résistance intermédiaire de cette lignée. La dissection génétique associée à l'analyse de l'expression génique de ces lignées résistante et sensible permet d'augmenter notre compréhension de la réponse de l'hôte à Y. pestis
Yersinia pestis, the agent of plague, is a deadly gram-negative bacterium classified as a re-emerging pathogen and class A biological weapon. The appearance of a multi-resistant strain highlights the need to better understand how this pathogen kills its host. To identify genetic factors of host susceptibility to plague, our laboratory is investigating the response of resistant versus susceptible mice to Y. pestis. Our strategy to decipher genetic determinants involved in resistance to plague combines Quantitative Trait Loci (QTL) mapping with gene expression analysis. We previously described the Mus spretus-derived SEG/Pas strain as the first to resist fully virulent Y. pestis, while most inbred strains, such as C57BL/6, are highly susceptible. Crosses between these two strains identified three QTLs (located on chromosome 3, 4 and 6) contributing to resistance. Two of the QTLs (on chromosome 4 and 6) were confirmed through creation of congenic mice. Up to 40% of the congenic mice heterozygous at these two QTLs, on a C57BL/6J background, survived the infection while all C57BL/6J mice died. Further dissection of these two QTLs, through the use of subcongenic strains, enabled us to refine the genetic architecture of resistance to plague in SEG/Pas mice. We concluded that a minimum of four genetic factors, within these two QTLs, are required to increased resistance to Y. pestis in mice. Despite production of numerous congenic strains, including triple congenic mice, we were not able to confirm the existence of the third QTL identified on chromosome 3. In parallel to genetic studies, we determined that SEG/Pas and C57BL/6J macrophages exhibit distinct characteristics upon in vitro exposure to Y. pestis. The underlying molecular differences were investigated by using microarrays. Our results show strong activation of cytokines in SEG/Pas macrophages in response to Y. pestis, which is not found in C57BL/6J macrophages. These results suggest that SEG/Pas mice are able to better activate innate immune response to Y. pestis than C57BL/6J mice.We further studied the expression of 44 genes in a kinetic study on macrophages in vitro of SEG/Pas, C57BL/6J and bicongenic mice (carrying QTLs on chromosome 4 and 6). This study confirmed that SEG/Pas mice are able to build a stronger inflammatory response at early time of infection. Nevertheless no significant differences were observed in the bicongenic strain compared to C57BL/6J. Further studies will be required to understand the mechanisms involved in the intermediate resistance of this strain. This combination of genetic dissection and gene expression analysis of resistant and susceptible mouse strains will enhance our ability to better understand the host response to plague

Thesis: M.B.A., Massachusetts Institute of Technology, Sloan School of Management, 2016. In conjunction with the Leaders for Global Operations Program at MIT.
Thesis: S.M. in Engineering Systems, Massachusetts Institute of Technology, Department of Biological Engineering, 2016. In conjunction with the Leaders for Global Operations Program at MIT.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 71-75).
Building biological understanding of the Chinese Hamster Ovary (CHO) system used to manufacture therapeutic proteins is paramount to efficient CHO bioprocess optimization. This understanding can be built by analyzing and synthesizing biological data; such as transcriptomic (gene expression), proteomic (protein levels), or metabolomic (metabolite levels). This thesis describes a streamlined workflow for analyzing transcriptomic data. This streamlined workflow not only reduced the barrier to conducting the analysis but also reduced the analysis cycle time. With the use of this workflow, a number of historical Amgen microarray datasets were mined to identify gene expression signatures indicative of productivity. The result of this mining identified key biological pathways specific to a highly productive Amgen cell line. This work suggests that these pathways are critical to heightened levels of protein production. Using this information to engineer future cell lines could enable Amgen to improve cellular protein production by over 30%, impacting costs associated with drug substance manufacturing. More broadly, this example of streamlining and standardizing transcriptomic data provides a framework for how Amgen Process Development can leverage biological data to improve CHO systems understanding and achieve operational impacts.
by Kerry Rachel Weinberg.
M.B.A.
S.M. in Engineering Systems

Embryonic stem cells (ESCs) are a special type of cell marked by two key properties: The capacity to create an unlimited number of identical copies of themselves (self-renewal) and the ability to give rise to differentiated progeny that can contribute to all tissues of the adult body (pluripotency). Decades of past research have identified many of the genetic determinants of the state of these cells, such as the transcription factors Pou5f1, Sox2 and Nanog. Many other transcription factors and, more recently, epigenetic determinants like histone modifications, have been implicated in the establishment, maintenance and loss of pluripotent stem cell identity. The study of these regulators has been boosted by technological advances in the field of high-throughput sequencing (HTS) that have made it possible to investigate the binding and modification of many proteins on a genome-wide level, resulting in an explosion of the amount of genomic data available to researchers. The challenge is now to effectively use these data and to integrate the manifold measurements into coherent and intelligible models that will actually help to better understand the way in which gene expression in stem cells is regulated to maintain their precarious identity. In this thesis, I first explore the potential of HTS by describing two pilot studies using the technology to investigate global differences in the transcriptional profiles of different cell populations. In both cases, I was able to identify a number of promising candidates that mark and, possibly, explain the phenotypic and functional differences between the cells studied. The pilot studies highlighted a strong requirement for specialised software to deal with the analysis of HTS data. I have developed GeneProf, a powerful computational framework for the integrated analysis of functional genomics experiments. This software platform solves many recurring data analysis challenges and streamlines, simplifies and standardises data analysis work flows promoting transparent and reproducible methodologies. The software offers a graphical, user-friendly interface and integrates expert knowledge to guide researchers through the analysis process. All primary analysis results are supplemented with a range of informative plots and summaries that ease the interpretation of the results. Behind the scenes, computationally demanding tasks are handled remotely on a distributed network of high-performance computers, removing rate-limiting requirements on local hardware set-up. A flexible and modular software design lays the foundations for a scalable and extensible framework that will be expanded to address an even wider range of data analysis tasks in future. Using GeneProf, billions of data points from over a hundred published studies have been re-analysed. The results of these analyses are stored in an web-accessible database as part of the GeneProf system, building up an accessible resource for all life scientists. All results, together with details about the analysis procedures used, can be browsed and examined in detail and all final and intermediate results are available and can instantly be reused and compared with new findings. In an attempt to elucidate the regulatory mechanisms of ESCs, I use this knowledge base to identify high-confidence candidate genes relevant to stem cell characteristics by comparing the transcriptional profiles of ESCs with those of other cell types. Doing so, I describe 229 genes with highly ESC-specific transcription. I then integrate the expression data for these ES-specific genes with genome-wide transcription factor binding and histone modification data. After investigating the global characteristics of these "regulatory inputs", I employ machine learning methods to first cluster subgroups of genes with ESC-specific expression patterns and then to define a "regulatory code" that marks one of the subgroups based on their regulatory signatures. The tightly co-regulated core cluster of genes identified in this analysis contains many known members of the transcriptional circuitry of ESCs and a number of novel candidates that I deem worthy of further investigations thanks to their similarity to their better known counterparts. Integrating these candidates and the regulatory code that drives them into our models of the workings of ESCs might eventually help to refine the ways in which we derive, culture and manipulate these cells - with all its prospective benefits to research and medicine.

Ticks are important vectors of a wide variety of pathogens including protozoa, bacteria and viruses. Many of the viruses transmitted by ticks are of medical or veterinary importance including tick-borne encephalitis virus (TBEV) and Crimean- Congo hemorrhagic fever virus causing disease in humans, and African swine fever virus and Nairobi sheep disease virus affecting livestock. Although several studies have elucidated tick antimicrobial mechanisms including cellular immune responses such as nodulation, encapsulation and phagocytosis and humoral immune responses such as the JAK/STAT pathway, complement-like proteins, antimicrobial peptides, lectin like pattern-recognition molecules and lysozymes, very little is known about the innate immune response of ticks towards viral infection. This study therefore aimed to identify molecules that might be involved in the response of ticks to viral infection. The hypothesis was that TBEV infection leads to changes in the expression of immunity-related transcripts and proteins in Ixodes spp. tick cells and that at least some of these might be antiviral. Ixodes scapularis-derived cell lines IDE8 and ISE6 were chosen since I. scapularis is currently the only tick species with a sequenced genome and an Ixodes ricinus-derived cell line, IRE/CTVM19, was used because I. ricinus is the natural vector of TBEV. Basic parameters required to study the responses of tick cells to infection were determined, including levels of virus infection, kinetics of virus replication and production, formation of replication complexes and uptake of dsRNA or siRNA. The cell lines IDE8, ISE6 and IRE/CTVM19 were infected with either of two tick-borne flaviviruses, TBEV and Langat virus (LGTV), or with the mosquito-borne alphavirus Semliki Forest virus (SFV). Infection was characterised using techniques including plaque assay, luciferase assay, immunostaining and conventional, confocal and electron microscopy. Two time points for transcriptomics and proteomics analysis of TBEVinfected IDE8 and IRE/CTVM19 cells were selected: day 2 post-infection (p.i.) when virus production was increasing and day 6 p.i. when virus production was decreasing. RNA and protein were isolated from TBEV-infected and mock-infected tick cells at days 2 and 6 p.i. and RNA-Seq and mass spectrometric technologies were used to identify changes in, respectively, transcript and protein abundance. Differential expression of transcripts was determined using the data analysis package DESeq resulting in a total of 43 statistically significantly differentially expressed transcripts in IDE8 cells and 83 in IRE/CTVM19 cells, while differential protein representation using Χ2 test statistics with Bonferroni correction in IDEG6 software resulted in 76 differentially represented proteins in IDE8 cells and 129 in IRE/CTVM19 cells. These included transcripts and proteins which could affect stages of the virus infection, including virus entry, replication, maturation and protein trafficking, and also innate immune responses such as phagocytosis, RNA interference (RNAi), the complement system, the ubiquitin-proteasome pathway, cell stress and the endoplasmic reticulum (ER) stress response. After verification of sequencing data by qRT-PCR, the ability of several of the identified transcripts or proteins to affect virus infection was determined by knockdown experiments in IDE8 and IRE/CTVM19 cells using wild type LGTV, LGTV replicons or TBEV replicons. Knockdown of genes encoding proteins including the ER chaperone gp96 and the heat-shock protein HSP90 resulted in increased virus production in both cell lines, hinting at an antiviral role. In contrast, knockdown of calreticulin, another ER chaperone, resulted in a decrease in virus production in IRE/CTVM19 cells but not in IDE8 cells, implying a requirement for virus production. This functional genomics approach has identified possible novel genes/proteins involved in the interaction between flaviviruses and tick cells and also revealed that there might be antiviral innate immune pathways present in ticks additional to the exogenous RNAi pathway.

An important hallmark of aging is the loss of proteostasis, which can lead to the formation of protein aggregates and mitochondrial dysfunction in neurons. Although it is well known that protein synthesis is finely regulated in the brain, especially at synapses, where mRNAs are locally translated in activity-dependent manner, little is known as to the changes in the synaptic proteome and transcriptome during aging. Therefore, this work aims to elucidate the relationship between transcriptome and proteome at soma and synaptic level during aging. Cerebral cortices were isolated from 3 weeks-old mice, 5 months-old and 18 months-old mice and synaptosomal fraction was extracted by ultracentrifugation on discontinuous sucrose gradient. The fraction was then analyzed by Data Independent Analysis (DIA) Mass Spectrometry and the resulting data were analyzed using Spectronaut software. RNA was also extracted and analyzed by ribo-zero RNA-seq. Data were analyzed and combined with R software. Proteomic and transcriptomic data analysis revealed that, in young animals, proteins and transcripts are correlated and synaptic regulation is driven by changes in the soma. During aging, there is a decoupling between transcripts and proteins and between somatic and synaptic compartments. For example, there is an increase of ribosomal proteins at synapses that is not mirrored by a concomitant increase at somatic level. Furthermore, soma-synapse gradient of ribosomal genes changes upon aging, i.e. ribosomal transcripts are less abundant and ribosomal proteins are more abundant in synaptic compartment of old mice with respect to younglings. Mass spectrometry analysis of synaptic protein aggregates revealed that they are particularly rich in ribosomal proteins and also of some components of lysosomes and proteasome, suggesting that loss of proteostasis and inefficient degradation leads to aggregation of ribosomes in synaptic compartment. Strikingly, Desmoplakin, a structural constituent of desmosomes, was also highly abundant in synaptic aggregates. This study suggests that aging affects both the local translational machinery and the trafficking of transcripts and proteins.

Sturgeons are a group of Condrostean fishes with very high evolutionary, economical and conservation interest. The eggs of these living fossils represent a luxury delicacy and are one of the most valuable foods of animal origin. The intense exploitation of wild stocks for the harvesting of caviar caused in the last decades a dramatic decline of their distribution and abundance leading, in 2010, the International Union for Conservation of Nature to list them as the more endangered group of species. As a direct consequence, world-wide efforts have been made to develop sturgeon aquaculture programmes for caviar production. In this context, selective farming of females would increase the economical profits and the characterisation of genes involved in sex determination becomes a major issue. The 454 sequencing of four normalised cDNA libraries from gonads and brain of A naccarii and A. stellatus, one male and one female full-sib per species, yielded 182,066 and 167,776 reads for A. naccarii which after a strict quality control were iteratively assembled together, giving more then 55,000 high quality Expressed sequence tags (ESTs). A total of 184,374 and 169,286 raw reads were instead produced for A. stellaus male and female libraries respectively, resulting in 63,606 ESTs after two round assembly. It was estimated the joint assembly of A. naccarii was able to cover about 80% of its total transcripts expressed in both gonad and brain with a mean contigs coverage of 4X. Similarly 86% transcriptome coverage was achieved by assembling both sex specific libraries of A. stellatus, with 3.6X as mean contig coverage. The Multi-step annotation process finally results in 16% and 15% successfully annotated sequences, with GO terms, respectively in A. naccarii and A. stellatus. Both transcriptomes were screened for 32 sex related genes and highlighted 5 and 2 genes that are potentially specifically expressed in A. naccarii male and female, at the first life stage at which sex is histologically identifiable. The screening in A. stellatus is currently at preliminary stage and further filtering steps are required. Both sturgeon transcriptomes were also compared with those of other fish species for which relevant genomic informations were available. Finally, 21,791 putative EST-linked Single Nucleotide Polymorphisms (SNPs) and 5,295 Single Sequence Repeats (SSRs) were identified in A. naccarii, while 15,449 and 5,696 were putatively classified in A. stellatus assembly. This study represents the first characterisation of transcriptomes from two high endangered sturgeon species. Most of the information acquired for A. naccarii were well organised into the public database AnaccariiBase, freely available at http://compgen.bio.unipd.it/anaccariibase/, while the information obtained for A stellatus will be released soon. This study represents a precious source of information for more focussed studies aimed at characterising or comparing genes, deciphering molecular mechanisms or genetic pathways in this group of species, or discovering hundreds of EST-linked markers with several possible applications in sturgeon conservation.
Gli storioni sono un gruppo di pesci Condrostei, di elevato interesse evoluzionistico, economico e di conservazione. Le uova di questi fossili viventi costituiscono uno degli alimenti di origine animale più preziosi sul mercato. L'intenso sfruttamento delle popolazioni selvatiche per la raccolta del caviale ha causato, negli ultimi decenni, un calo drammatico della loro distribuzione ed abbondanza che ha portato, nel 2010, l'Unione Internazionale per la Conservazione della Natura ad indicarli come il gruppo di specie a maggior rischio di estinzione. Come diretta conseguenza, sono stati compiuti sforzi notevoli, in tutto il mondo, per sviluppare programmi finalizzati alla produzione di caviale via acquacoltura. In questo contesto, l'allevamento selettivo delle femmine aumenterebbe i profitti economici e la caratterizzazione dei geni coinvolti nella determinazione del sesso, diventa decisiva. Il sequenziamento 454 di quattro librerie di cDNA normalizzate, costruite a partire da gonadi e cervello di A naccarii e A. stellatus, un maschio ed una femmina (fratelli) per specie, ha prodotto 182,066 e 167,776 reads grezze rispettivamente per i due sessi di A naccarii, che dopo un severo controllo di qualità sono state assemblate insieme attraverso un processo iterativo, risultando in più di 55,000 Expressed Sequence Tags (ESTs) di qualità elevata. Per la specie A. stellatus, invece, sono state prodotte 184,374 reads per la libreria maschile e 169,286 per quella femminile, allineate in 63,606 ESTs dopo due giri di assemblaggio. E’ stato stimato che, l’assemblaggio di A. naccarii contenga i tag di circa l’80% dei trascritti totali espressi in gonadi e cervello, in questa specie, con una copertura media dei contigs pari a 4X. La copertura del trascrittoma di A. stellatus è stata stimata in circa l’86%, con 3.6X come media di copertura dei contigs. Il processo di annotazione multi-fase, ha portato ad annotare correttamente, con termini GO circa 16% ed il 15% delle sequenze, rispettivamente in A. naccarii ed A. stellatus. Entrambi i trascrittomi sono stati interrogati alla ricerca di 32 geni legati al sesso e sono stati evidenziati 5 geni potenzialmente espressi in modo specifico nel maschio e 2 nella femmina di A. naccarii, nel primo stadio di sviluppo, in cui il sesso è istologicamente identificabile. La ricerca nel trascrittoma di A. stellatus è attualmente preliminare e sono necessarie ulteriori fasi di filtraggio. Entrambi i trascrittomi sono stati confrontati con quelli di altre specie di pesci, per la quali erano disponibili rilevanti informazioni genomiche. Infine, 21.791 putativi Single Nucleotide Polymorphisms (SNPs) e 5.295 Single Sequence Repeats (SSR) EST-linked sono stati identificati in A. naccarii, mentre 15.449 e 5.696 sono stati rispettivamente classificati nell’assemblaggio di A. stellatus. Questo studio rappresenta la prima caratterizzazione dei trascrittomi di due specie di storione ad elevato rischio di estinzione. Gran parte delle informazioni acquisite per la specie A. naccarii sono state organizzati all’interno della banca dati pubblica AnaccariiBase, liberamente disponibile all’indirizzo http://compgen.bio.unipd.it/anaccariibase/, mentre le informazioni ottenute per A stellatus saranno rilasciate al più presto. Questa analisi rappresenta una preziosa fonte di informazioni per ulteriori studi più mirati, volti a caratterizzare o confrontare geni, a decifrare i meccanismi molecolari o le vie genetiche in questo gruppo di specie, o a scoprire centinaia di marcatori associati ad ESTs per diverse applicazioni nella conservazione dello storione.

INTRODUCTION: Breast cancer is the most common cancer in women worldwide and metastatic dissemination is the principal factor related to death by this disease. Breast cancer stem cells (bCSC), defined in this work as the ALDH1high/LIN-/ESA+ population, are thought to be responsible for metastasis and chemoresistance. The objective of this work is to find gene master regulators, in particular transcription factors (TFs), which are controlling the bCSC phenotype. METHODS: We used in this work two groups of datasets with transcriptome data, the discovery dataset group contains one dataset obtained by ourselves containing three paired samples comparing the bCSC and the bulk of the tumor (My Data - bCSC/Bulk dataset), a dataset with eight paired samples comparing the bCSC and cancer cells (Wicha - bCSC/CC dataset) and a dataset with 115 samples of breast cancer tissue (clinical response dataset). The second group, validation datasets, contains the BRCA-TCGA dataset with information of 621 samples, 4142 breast cancer samples of the Kmplot tool, 17 primary samples of BasL subtype and their information of grafting in patient derived xenografts and analyzes of cell lines (MF10A and HMLE). For the analyzes we used the paired t-test in the Limma R package, the ARACNE algorithm for the inference of regulons in the clinical response dataset, MRA-FET to define the master regulators of the bCSC phenotype, and GSEA to identify the biological meaning of the findings in the different datasets. RESULTS: We identified 12 TFs as master regulators of the bCSC phenotype, with nine of them forming two highly interconnected networks, one positively related with the bCSC phenotype formed by SNAI2, TWIST, PRRX1, BNC2 and TBX5 with its regulons, defined here as the mesenchymal transcription network and one negative correlated to the phenotype formed by SCML4, ZNF831, SP140 and IKZF3, defined as the immune response transcription network, totally unknown in the context of breast cancer in the literature. Although still with weak evidence, ZEB1 seems to control the two networks and can be responsible for the expression of ALDH1 and of the three remaining TFs: ID4, HOXA5 and TEAD1. As their names portray, our data showed in the different datasets, and independently of the molecular subtype and of the platform used, that the mesenchymal transcription network seems to be responsible for the bCSC phenotype and the immune response transcription network to the adaptive immune response in the tumor and a better prognosis for the patients. We also defined 10 membrane proteins as new markers and/or therapeutic targets of the bCSC. CONCLUSION: We found and described two TF networks that seem to control the bCSC phenotype, one of them totally unknown until now and correlated to a good prognosis. Our findings have a clear potential for clinical use.
INTRODUÇÃO: O cancer de mama é no mundo o câncer mais comum em mulheres e a disseminação metastática é o principal fator relacionado com a morte pela doença. Acreditasse que as células tronco do câncer de mama - bCSC, na sigla em inglês e definida neste trabalho com a população ALDH1high/LIN-/ESA+ - é responsável pela metástase e pela quimioresistência. O objetivo deste trabalho é encontrar genes que são essenciais para o controle do fenótipo das bCSC, em particular fatores de transcrição. MATERIAIS E MÉTODOS: Nesse trabalho nós utlizamos dois grupos de datasets com dados do transcriptoma, o grupo de datasets de descoberta contém um dataset gerado por nós com 3 amostras pareadas comparando as bCSC com o tumor total (My Data - bCSC/Bulk dataset), um dataset com 8 amostras pareadas comparando as bCSC com as células cancerígenas (Wicha - bCSC/CC dataset) e um dataset com 115 amostras de tecido de câncer de mama (Clinical Response dataset). O segundo grupo, grupo de validação, contém o dataset BRCA-TCGA com 621 amostras, as 4142 amostras de câncer de mama da ferramenta Kmplot, as 17 amostras humanas primárias do subtipo BasL e sua informação sobre a geração, ou não, de tumores em camundongos imunosuprimidos e a análise de linhagens celulares (MF10A e HMLE). Para a análise dos dataset utilizamos o test-t pareado no pacote Limma da liguagem R, o algoritmo ARACNE para a inferência de regulons no dataset Clinical Response, a análise MRA-FET para definir os Reguladores Mestres para o fenótipo das bCSC e a análise GSEA para identificar o significado biológico de nosso achados nos diferentes datasets. RESULTADOS E DISCUSSÃO: Nós identificamos 12 TFs como reguladores mestres, com 9 deles formando duas redes altamente conectadas, uma positivamente relacionada ao fenótipo bCSC formada por SNAI2, TWIST, PRRX1, BNC2 e TBX5 com seus regulons, e definida aqui como a rede de transcrição mesenquimal, e uma rede correlacionada negativamente, formada por SCML4, ZNF831, SP140 e IKZF3, definida aqui como a rede de transcrição da resposta imune e totalmente desconhecida da literatura no contexto do câncer de mama. Embora ainda com fraca evidencia, ZEB1 para controlar as duas redes e ser responsável pela expressão de ALDH1 e dos 3 TFs restantes: ID4, HOXA5 e TEAD1. Como mostram seus nomes, e independente do dataset, do subtipo molecular ou da plataforma utilizada, a rede de transcrição mesenquimal, parece ser responsável pela manutenção do fenótipo de células tronco cancerígenas e a rede de transcrição da resposta imune pela resposta imune adaptativa ao tumor e a um bom prognóstico para as pacientes. CONCLUSÃO: Nós encontramos e descrevemos duas redes de fatores de transcrição que parecem controlar o fenótipo das bCSC, uma delas totalmente desconhecida até agora e relacionada a um bom prognóstico. Nosso achados possuem um claro potencial para uso clínico.

L'analyse de données d'expression montre qu'il est avantageux d'analyser les processus biologiques en termes de modules plutôt que simplement considérer les gènes un par un. Dans ce projet nous avons conduit une analyse non supervisée des données d'expression de gènes de plusieurs cohortes de tumeurs urothéliales en appliquant la méthode d'Analyse en Composantes Indépendantes. Plusieurs études ont démontré les meilleures performances de l'ACI par rapport à l'ACP et les méthodes de clustering, pour obtenir une décomposition plus réaliste des données d'expression en patterns d'expression pertinents et associés avec le phénotype d'intérêt.Les tumeurs urothéliales apparaissent et évoluent selon deux voies distinctes dont la probabilité de progression en cancer musculo-invasif diffère radicalement. Excepté la mutation de FGFR3 dans le groupe le moins agressif, les processus moléculaires sous-jacents n'ont pas été complètement identifiés. Le principal objectif de cette thèse était dédié aux interprétations biologiques des différentes composantes indépendantes pour aider à confirmer et étendre la liste des processus biologiques connus pour être impliqués dans le cancer de vessie.Chaque composante indépendante est caractérisée par une liste de projections de gènes et de contributions pondérées d'échantillons tumoraux . En combinant expertise biologique et comparaison des listes de gènes à des voies existantes et en étudiant conjointement l'association des composantes aux annotations cliniques et moléculaires, nous avons pu différencier les CIscausées par des facteurs techniques, tels que le prélèvement chirurgical de celles ayant des interprétations biologiques pertinentes. De plus, parmi les signaux pertinents biologiquement, cette analyse nous a permis de différencier les signaux provenant du stroma, comme la réponse immunitaire médiée par les lymphocytesB&T, de ceux produits par les tumeurs elles-mêmes, comme les signaux reliés à la prolifération ou à la différenciation. La classification des tumeurs selon leurs contributions à certaines CIs a pu être étroitement associée à des annotations anatomo-cliniques, et a mis en évidence de nouveaux sous-types de tumeur spotentiels, qui suggèrent l'existence de voies de progression supplémentaires dans le cancer de vessie. De façon similaire, l'étude des contributions de groupes de tumeurs basés sur des annotations cliniques ou moléculaires a montré différents niveaux de contamination par le stroma entre les tumeurs mutées et nonmutées pour FGFR3. La reproductibilité des composantes a été étudiée en utilisant des graphes de corrélation. La majeure partie des CIs interprétées a été validée sur trois jeux de données indépendants, et plusieurs d'entre elles ont aussi détectées dans un jeu de données de lignées cellulaires.Une deuxième étude sur le rétinoblastome a montré que nous pouvions tirer partie de l'ACI dans des contextes variés. Le rétinoblastome est initié par la perte des deux alléles du gène suppresseur de tumeur RB1. D'autres évènements génomiques non identifiés sont nécessaires à la progression de la maladie. Nous avons observé une association entre âge des patients et altérations génomiques. Les patients jeunes présentant moins d'altérations que les patients âgés, ces derniers présentant des gains du 1q et des pertes du 16q. Cette séparation des tumeurs selon l'âge est également observée sur les données d'expression, notamment en appliquant l'ACI dont l'une des composantes discrimine les patients selon leur âge. Ces résultats suggèrent l'existence de deux voies de progression dans le rétinoblastome. L'analyse des données à haut débit fournit de nombreuses listes de gènes. Afin de les interpréter, une possibilité est d'extraire les dernières publications groupées par sujets prédéfinis (fonction, localisation,...)
Practice of gene expression data analysis shows that it is advantageous to analyze biologicalprocesses in terms of modules rather than simply consider gene one by one. In this project, we conducted anunsupervised analysis of the gene expression data of several cohorts of urothelial tumors, applying theIndependent Component Analysis method. Several studies demonstrated the outperformance of ICA overPCA and clustering-based methods in obtaining a more realistic decomposition of the expression data intoconsistent patterns of coexpressed genes associated with the studied phenotypes[1, 2, 3, 4].Urothelial tumors arise and evolve through two distinct pathways which radically differ on their probabilityof progression to muscle invasion. Except the mutation of FGFR3 in the less aggressive group, theunderlying molecular processes have not been completely identified. The first and main objective of the PhDthesis was dedicated to the biological interpretation of the different independent components to help toconfirm and extend the list of biological processes known to be involved in bladder cancer.Each independent component (IC) is characterized by a list of gene projections on the one hand and weightedcontributions of tumor samples on the other hand. By combining biological expertise and comparison of theassociated list of genes to known pathways, and jointly studying the association of the components tomolecular and clinical annotations, we have been able to differentiate components that were caused bytechnical factors, such as surgical sampling, from those having consistent biological interpretationin terms of tumor biology. Moreover, among the biologically meaningful signals, this analysis allowed us todifferentiate the signals from stroma of the tumor, like immune response mediated by B- and T-lymphocytes,from the signals produced by the tumors themselves, like signals related to proliferation, or differentiation.The clustering of the tumor samples according to their contributions on some ICs can be closely associated toanatomo-clinical annotations, and highlighted new potential subtypes of tumors which suggest existence ofadditional pathways of bladder cancer progression. Similarly, the study of the contributions of preestablishedgroups of tumors based on clinical or molecular criteria showed different levels of stromacontamination between FGFR3 non-mutated and mutated tumors. The reproducibility of the components wasinvestigated using correlation graphs. The major part of the interpreted ICs was validated on threeindependent bladder cancer datasets, and several of them were also identified in an urothelial cancer celllines data set.A second study about retinoblastoma gave us the occasion to show that we can take advantage ofICA in various contexts. Retinoblastoma is initiated by the loss of both alleles of the RB1 tumor suppressorgene. Although necessary for initiation, other genomic events, that remain to be identified, are needed for theprogression of the disease [5]. We observed, as it was previously described [6], an association between age ofthe patients and levels of genomic aberrations, the younger patients having fewer alterations than the olderpatients, which generally present gain of 1q and loss of 16q. We showed that this tendency of the tumors tobe clustered into two groups of age can also be observed on the expression data by applying ICA whose oneof the component was highly correlated to the age of the patients. These results suggest the existence of twopathways of progression in retinoblastoma.The analysis of high throughput data provides many lists of genes. To interpret them, a possibility isto study the latest related publications grouped by pre-defined group of topics (function, cellular location...).To that aim, in a third study, we introduced a web-based Java application tool named GeneValorization whichgives a clear and handful overview of the bibliography corresponding to one particular gene list [7]

Dans un contexte actuel qui tend vers une réduction d’intrants dans les systèmes de culture et une relance de la production de protéines végétales pour réduire la dépendance alimentaire de la France, la culture des légumineuses constitue une alternative. Les légumineuses à graines offrent une source riche en protéines pour l’alimentation animale et humaine. Au sein de l'UMR1347 Agroécologie, le pôle "déterminismes Génétiques et Environnementaux de l’Adaptation des Plantes à des Systèmes de culture Innovants" (GEAPSI) étudie via une approche multidisciplinaire (génétique, écophysiologie, physiologie moléculaire) l’adaptation des espèces végétales, et notamment des légumineuses aux contraintes environnementales. Ces travaux de thèse ont été réalisés au sein de l'équipe "Étude des Mécanismes Moléculaires" qui s'intéresse plus particulièrement à des critères de qualité de la graine (teneur en protéine, taille de graine) et au déterminisme génétique de ces caractères chez la plante modèle Medicago truncatula en vue d'un transfert de connaissances vers l'espèce cible Pisum sativum. Chez les légumineuses, la taille de la graine est déterminée par la capacité de l’embryon à se diviser lors de l’embryogenèse et à accumuler des réserves lors du remplissage. Aux stades précoces du développement, l’assimilation de nutriments est réalisée majoritairement par les tissus qui entourent l’embryon: l’albumen et le tégument. Les recherches menées visent à évaluer la contribution de l’albumen dans le développement de la graine chez M. truncatula. Nous avons révélé plusieurs gènes DOF (DNA-binding with One zinc Finger) comme étant exprimé dans ce tissu. Ils appartiennent à une large famille de facteurs de transcription impliqués dans de nombreux processus biologiques mais dont les rôles restent à préciser. Un de ces gènes, nommé DASH pour Dof Affecting Seed embryogenesis and Hormone metabolism, est exprimé préférentiellement dans l’albumen lors de l’embryogenèse. Des mutants TILLING et Tnt-1 isolés pour ce facteur de transcription sont affectés dans le développement de la graine (avortement à environ 10 jap). La cytologie du développement aux stades précoces (6 à 10 jap) a révélé que l’expression de ce gène dans l’albumen est requise pour un développement normal de l’embryon, démontrant le rôle de l’albumen dans le contrôle de l’embryogenèse chez les légumineuses. Une étude comparative du transcriptome des gousses de dash vs sauvage a permis d’émettre des hypothèses quant à la fonction du gène DASH. Une dérégulation du métabolisme hormonal, en particulier de l’auxine, a été mise en évidence et plusieurs gènes cibles potentiels de ce facteur de transcription ont été sélectionnés. Une comparaison du transcriptome des trois tissus de la graine à 12 jap a été réalisée chez la lignée sauvage de référence (Jemalong, A17). Elle a permis de préciser la localisation tissulaire de ces gènes cibles putatifs, de mettre en évidence des voies métaboliques plus spécifiques de l’albumen et de proposer des hypothèses quant à la fonction de ce tissu dans le développement de la graine
In the current context, which necessitates a reduction in inputs in crop systems and boosting of production of plant proteins to reduce France’s dependency on feed imports,, growing legumes represents an alternative. Grain legumes are major sources of proteins for animal and human nutrition. In the UMR1347 Agroécologie, the objectives of the study group "déterminismes Génétiques et Environnementaux de l’Adaptation des Plantes à des Systèmes de culture Innovants" (GEAPSI) are to promote legume cultivation and adaptation to environmental stresses, via multidisciplinary approaches (genetics, ecophysiology, molecular physiology). This thesis project was carried out in the "Étude des Mécanismes Moléculaires" team, particularly interested in seed quality traits such as protein content or seed size and identification of genes implicated in variations of these trait s. Experiments were performed using Medicago truncatula as a model species for legumes with a view to transferring the information to the target crop species Pisum sativum.Legume seed size is determined by the embryo’s capacity to divide during embryogenesis and to accumulate reserves during seed filling. At early developmental stages, nutrient assimilation occurs predominantly in embryo-surrounding tissues: the endosperm and seed coat. This thesis project aims to evaluate the endosperm contribution to seed development in M. truncatula. We have shown several DOF (DNA-binding One Zinc Finger) genes to be expressed in this tissue. They belong to a large family of transcription factors implicated in numerous biological processes, but whose role remain to be elucidated. One of these genes, termed DASH for Dof Affecting Seed embryogenesis and Hormone metabolism, is expressed preferentially in the endosperm during embryogenesis. TILLING and TnT1 mutants isolated for this transcription factor are affected in seed development (abortion at 10 DAP). The cytology of development at early stages (6 to 10 DAP) revealed that the expression of this gene in the endosperm is required for the normal development of the embryo, demonstrating the role of the endosperm in the control of embryogenesis in legumes. A comparative transcriptomics study of dash vs wild-type pods allowed us to suggest hypothesis about the function of the DASH gene. Evidence for a deregulation of hormone metabolism, in particular for auxin, was obtained, and several potential target genes of this transcription factor were selected. A comparison of the transcriptome of the three tissues of the seed at 12 DAP was carried out for the reference wild-type line (Jemalong A17). This allowed the tissue localization of the target genes, to reveal metabolic pathways preferentially expressed in the endosperm, and to propose hypotheses about the role of this tissue during seed development

The vasculature of many solid tumours is highly distinct from that of its host tissue, both structurally and in terms of functional protein expression. These differences offer an opportunity for specific targeting of therapeutics against the tumour vasculature. This thesis describes the investigation of this tumour vascular profile, in clinical samples from renal cell carcinoma, colorectal cancer and colorectal liver metastases, as well as murine breast tumours resistant to the antiangiogenic drug, sunitinib. This analysis allowed the identification of three tumour endothelial markers in renal and colorectal malignancies, MCAM, LAMA4 and GRIN2D. The expression of each of these markers was linked with patient survival with these malignancies, suggesting their utility for prognostication. MCAM and GRIN2D showed highly tumour specific expression profiles in multi-organ tissue array analysis, highlighting them as promising candidates for the targeting of therapies to the tumour vasculature. The specific localisation of monoclonal anti-MCAM antibodies to renal tumour vasculature was demonstrated, further supporting this suggestion. Putative vascular markers of tumour resistance to antiangiogenic therapy were also identified. Aquaporin-1 (AQP1) was found to be up-regulated in cases of acquired resistance, mammalian target of rapamycin (mTOR) in innate resistance and pleiotrophin (PTN) in both, highlighting their potential as diagnostic candidates for predicting therapy response, or as targets to circumvent resistance. The need for effective diagnostic tests in this indication, was demonstrated by the finding that metastasis is enhanced by sunitinib therapy, in innately resistant tumours.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references.
Systems biology represents a powerful method to describe and manipulate phenotypes of interest by incorporating biological information from various levels of cellular organization. Such an approach is illustrated from a library of both rationally-directed and combinatorial gene knockout strains of E. coli recombinantly producing the small molecule lycopene. Global genomic and proteomic expression changes associated with increased lycopene production of mutant E. coli constructs were discovered using whole-genome DNA microarrays and a novel LC-MS technique, respectively. While most genes and proteins showed few expression changes, key differences were identified, including targets distal to the non-mevalonate and precursorsupplying pathways. Based upon the expression data sets, it was hypothesized that the following may be associated with lycopene overproduction: histidine biosynthesis (hisH); the quinone pool (wrbA); acid resistance (ydeO and gadE); the glyoxylate pathway (iclR); NADPH redox balance (pntB); growth rate reduction; and membrane composition. In the pre-engineered background strain, deleting pntB (~20-25%) and ydeO (~30%) each led to moderately increased production; overexpressing wrbA led to 50-100% more production at 8 hours and 5-15% more production at later time points; deleting iclR caused small production increases (~5-10%); and supplementing media with histidine caused the parental and mutant strains to have similar production.
(cont.) From these observations, several themes emerged. First, reduced cellular growth and energy conservation appear to be important tradeoffs for increasing lycopene production. Second, reducing overflow metabolism to acetate and corresponding acid stress as well as providing a gluconeogenic flux to increase lycopene precursors appeared beneficial. Next, NADPH availability and balance seemed to be critical production factors. The sS factor is known to affect lycopene accumulation, and it was observed to have far-reaching effects on both the transcriptomic and proteomic data sets. While expression changes were not strictly additive between the five mutant strains examined in comparison to the pre-engineered background strain, a number of these common factors appear to be responsible for the high lycopeneproduction phenotype. This work serves as an important example of incorporating multiple layers of complementary biological information to define a basis for an observed phenotype, demonstrating a powerful paradigm for realizing production increases via systems metabolic engineering.
by Brian E. Mickus.
Ph.D.

Neospora caninum is an economically significant parasitic protozoan causing the disease neosporosis in cattle and dogs. Although a close relative of the zoonotic apicomplexan Toxoplasma gondii, the two organisms exhibit differing host ranges and infection dynamics. T. gondii is a model organism that has been much studied, and a great deal is known about the genes and proteins involved when it invades a host cell. This thesis explores protein expression in the proliferative and invasive tachyzoite stage of N. caninum, in particular the expression of proteins pertaining to the apical complex of organelles; those responsible for entry and establishment within a host cell. Almost 20 % of the predicted proteome has been identified by this analysis to be expressed in the tachyzoite stage, with approximately 50 % of the predicted repertoire of apical proteins being detected. The discovery of differences between these two parasites’ highly syntenic genomes could lead to a better understanding of the process by which T. gondii is able to cause disease in humans, while N. caninum has not been observed to do so. One finding of the recent genome sequencing and annotation project in N. caninum was that a key T. gondii virulence determinant, rhoptry gene 18 (ROP18) was pseudogenised in N. caninum. This finding was investigated further in this thesis to demonstrate that the pseudogenisation of ROP18 was conserved across a range of N. caninum isolates and that in vitro, N. caninum was not able to subvert the murine interferon-gamma (IFN-γ) immune response using ROP18 in the way that virulent T. gondii tachyzoites do. The tissue-dwelling Coccidia have a multi-stage life cycle which includes a latent tissue cyst-encapsulated stage called the bradyzoite. Tachyzoites convert to this more quiescent form when induced by cellular stress, and are able to remain as such for long periods, even years. At times of weakened host immunity, bradyzoites can recrudesce to produce an active infection, which can cross the placenta in a pregnant animal to infect the foetus. This a major route by which N. caninum infection is maintained within cattle herds, therefore the biology of stage conversion from tachyzoite to bradyzoite and vice-versa is of interest to researchers. An RNA-Seq analysis of cultured tachyzoites and bradyzoites identified a marked reduction in rhoptry gene expression, and differing expression profiles of other invasion-related genes from the micronemes and dense granules. Overall, these data identify proteins released from the apical organelles in N. caninum and give an insight into the different repertoires expressed by the tachyzoite and bradyzoite life stages. Furthermore, a comparison between N. caninum and T. gondii predicted apical proteomes indicates that although most genes are shared in a one-to-one orthologous relationship between the two organisms, there are a small number of differences which may turn out to be important to the biology of the parasite, as in the case of ROP18.

Next generation sequencing (NGS) technology is currently employed to explore the molecular profiles associated to different biological contexts. The application of this technology provides at same time a high-resolution and global view of the genome and epigenome phenomena, enabling us to study the molecular events underlying many human diseases, including cancer. Our lab tries to exploit the utility of high throughput sequencing technologies generating genomic, transcriptomic and epigenomic data from patient's cohort to study the underlying molecular mechanisms that characterize the specific diseases and map the key regulators that can be critical targets for relevant therapeutic measures. I take the advantage of this technology to mainly understand two aggressive cancers: Ovarian Cancer (OC) and Glioblastoma multiforme (GBM). OC is a leading cause of cancer-related death for which no significant therapeutic progress has been made in the last decades. Also, in this case, despite multimodal treatment its prognosis remains extremely poor. This is due to the fact that the molecular mechanisms underlying OC tumorigenesis and progression are still poorly understood (Vaughan et al., 2011). GBM is the most common and aggressive primary brain malignancy with very poor prognosis (Frattini et al., 2013). The median survival rate is of 12-15 months (Singh et al., 2012) with 5-year survival that is less than 5% despite the multimodal treatment which include surgery, radiotherapy and chemotherapy. To this end, I will be integrating various genomic and transcriptomic analysis to define the key regulatory actors that characterize the disease progression paving. This integrated analysis has been devised in form of a computational workflow that gives way for a discovery pipeline for physiopathologically meaningful epigenetic targets that can lead to therapies.

Las enfermedades complejas se encuentran entre las más comunes y son causadas por una combinación de factores genéticos y ambientales (contaminación ambiental, estilo de vida, etc). Entre las enfermedades complejas que se pueden destacar se encuentran la obesidad, el asma, la hipertensión o la diabetes. Diversos estudios científicos sugieren que el hecho de padecer enfermedades complejas está condicionado a la aparición o acumulación de determinados factores ambientales. Asimismo, se ha descrito que los factores ambientales son unos de los principales contribuyentes a la carga mundial de morbilidad. Todo esto nos lleva a definir el término exposoma como el conjunto de factores ambientales a los que un individuo se ve expuesto desde la concepción hasta la muerte. El estudio de la mecánica subyacente que vincula el exposoma con la salud es un campo de investigación emergente con un fuerte potencial para proporcionar nuevos conocimientos sobre la etiología de las enfermedades. La primera parte de esta tesis se centra en la exposición a la radiación ultravioleta. La exposición a la radiación ultravioleta proviene de fuentes tanto naturales como artificiales. La radiación ultravioleta incluye tres subtipos de radiación según su longitud de onda (UVA 315-400 nm, UVB 315-295 nm y UVC 295-200 nm). Si bien la principal fuente natural de radiación ultravioleta es el Sol, la UVC no llega a la superficie de la Tierra debido a su absorción por la capa estratosférica de ozono. En consecuencia, la exposición a radiación ultravioleta a la que estamos usualmente sometidos consisten en una mezcla de UVA (95 %) y UVB (5 %). Los efectos de la radiación ultravioleta en humanos pueden ser beneficiosos o perjudiciales dependiendo de su cantidad y forma. Los efectos perjudiciales y agudos de la radiación ultravioleta incluyen eritema, oscurecimiento del pigmento, retraso en el bronceado y engrosamiento de la epidermis. Repetidas lesiones en la piel producidas por radiación ultravioleta pueden predisponer, en última instancia, a efectos crónicos de fotoenvejecimiento, inmunosupresión y fotocarcinogénesis. El mayor efecto beneficioso de la radiación ultravioleta es la síntesis cutánea de la vitamina D. La vitamina D es necesaria para mantener el calcio fisiológico y del fósforo para la mineralización ósea y para prevenir el raquitismo, la osteomalacia y la osteoporosis. El paradigma del exposoma es trabajar con múltiples exposiciones a la vez en vez centrarse en una sola exposición. Este enfoque permite tener una visión más parecida a la realidad que vivimos. Luego, la segunda parte se centra en las herramientas para explorar cómo caracterizar y analizar el exposoma y cómo probar sus efectos en múltiples capas biológicas intermedias para proporcionar información sobre los mecanismos moleculares subyacentes que vinculan las exposiciones ambientales a los resultados de salud.
Most common diseases are caused by a combination of genetic, environmental and lifestyle factors. These diseases are referred to as complex diseases. Examples of this type of diseases are obesity, asthma, hypertension or diabetes. Several empirical evidence suggest that exposures are necessary determinants of complex disease operating in a causal background of genetic diversity. Moreover, environmental factors have long been implicated as major contributors to the global disease burden. This leads to the formulation of the exposome, that contains any exposure to which an individual is subjected from conception to death. The study of the underlying mechanics that links the exposome with human health is an emerging research field with a strong potential to provide new insights into disease etiology. The first part of this thesis is focused on ultraviolet radiation (UVR) exposure. UVR exposure occurs from both natural and artificial sources. UVR includes three subtypes of radiation according to its wavelength (UVA 315-400 nm, UVB 315-295 nm, and UVC 295-200 nm). While the main natural source of UVR is the Sun, UVC radiation does not reach Earth's surface because of its absorption by the stratospheric ozone layer. Then, exposures to UVR typically consist of a mixture of UVA (95%) and UVB (5%). Effects of UVR on human can be both beneficial and detrimental, depending on the amount and form of UVR. Detrimental and acute effects of UVR include erythema, pigment darkening, delayed tanning and thickening of the epidermis. Repeated UVR-induced injury to the skin, may ultimately predispose one to the chronic effects photoaging, immunosuppression, and photocarcinogenesis. The beneficial effect of UVR is the cutaneous synthesis of vitamin D. Vitamin D is necessary to maintain physiologic calcium and phosphorous for normal bone mineralization and to prevent rickets, osteomalacia, and osteoporosis. But the exposome paradigm is to work with multiple exposures at a time and with one or more health outcomes rather focus in a single exposures analysis. This approach tends to be a more accurate snapshot of the reality that we live in complex environments. Then, the second part is focused on the tools to explore how to characterize and analyze the exposome and how to test its effects in multiple intermediate biological layers to provide insights into the underlying molecular mechanisms linking environmental exposures to health outcomes.

With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function.

This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis.

More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses.

Keywords:functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.

Schistosomiasis, a disease of humans caused by helminth parasites of the genus Schistosoma, kills 200 to 500 thousand people annually, endangering over 600 million people world-wide with 200 million people infected in 2003 [1, 2]. Three species of schistosome are primarily responsible for human infections, namely, Schistosoma haematobium, endemic to Africa, India, and the Middle East, S. mansoni, endemic to Africa / South America, and S. japonicum endemic to China and the Philippines [3]. The major pathological effects of schistosomiasis result from the deposition of parasite ova in human tissues and the subsequent intense granulomatous response induced by these eggs. There is a high priority to provide an effective sub-unit vaccine against these schistosome flukes, using proteins encoded by cDNAs expressed by the parasites at critical phases of their development. One technique that may expedite this gene identification is the use of microarrays for expression analysis. A 22,575 feature custom oligonucleotide DNA microarray designed from public domain databases of schistosome ESTs (Expressed Sequence Tags) was used to explore differential gene expression between the Philippine (SJP) and Chinese (SJC) strains of S. japonicum, and between males and females. It was found that 593, 664 and 426 probes were differentially expressed between the two geographical strains when mix sexed adults, male worms and female worms were compared respectively. Additionally, the study revealed that 1,163 male- and 1,016 female-associated probes were differentially expressed in SJP whereas 1,047 male- and 897 female-associated probes were differentially expressed in SJC [4]. Further to this, a detailed real time PCR expression study was used to explore the differential expression of eight genes of interest throughout the SJC life cycle, which showed that several of the genes were down-regulated in different life cycle stages. The study has greatly expanded previously published data of strain and gender-associated differential expression in S. japonicum. Further, the new data will provide a stepping stone for understanding the complexities of the biology, sexual differentiation, maturation, and development of human schistosomes, signaling new approaches for identifying novel intervention and diagnostic targets against schistosomiasis [4].

High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling. RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2>0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics. The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research.

QC 20170109

Orientador: Marcelo Brocchi
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-23T15:34:06Z (GMT). No. of bitstreams: 1 Lima_BrunadeAraujo_D.pdf: 3794605 bytes, checksum: 178a4bf447e5292f789a4713d5500b38 (MD5) Previous issue date: 2013
Resumo: A violaceína é um pigmento violeta produzido por algumas espécies bacterianas de origem ambiental, tais como Chromobacterium violaceum e Janthinobacterium lividum. Esta molécula apresenta várias propriedades biológicas incluindo antibacteriana, antifúngica, antiviral, antiprotozoária e antitumoral, apesar de sua função exata na fisiologia dos micro-organismos que a produz, ainda é desconhecido. No presente trabalho, a atividade antimicrobiana da violaceína produzida comercialmente, o extrato semi purificado e nanopartículas de vanadato de prata foram avaliados contra espécies de bacterianas gram-positivas e gram-negativas. A violaceína exibiu efeito antimicrobiano contra Staphylococcus aureus resistente à meticilina (MRSA) e Enterococcus resistente à vancomicina (VRE), que são micro-organismos frequentemente relacionados com infecções adquiridas em hospitais. Os valores de MIC (concentração inibitória mínima) e MBC (concentração bactericida mínima) da violaceína produzida comercialmente foram de 0,625 ?M e 1,25 ?M respectivamente e, análise de curvas de crescimento e tempo-morte revelaram um efeito antibacteriano durante 12 horas contra MRSA. A microscopia eletrônica de transmissão mostrou os efeitos da violaceína com alterações morfológicas e ultra estruturais, incluindo alterações na parede celular e formação de septos de divisão anormais. Nos resultados obtidos das análises de proteômica e transcriptoma a violaceína afetou a expressão de várias classes funcionais de proteínas e genes em MRSA, incluindo processos biológicos em biossíntese da parede celular e divisão celular que corroboram as alterações ultra estruturais visualizadas. Em conclusão, a violaceína produzida comercialmente demonstrou atividade antimicrobiana para S. aureus MRSA e pela primeira vez, os efeitos da violaceína sobre o metabolismo de S. aureus foram descritos, indicando possíveis alvos e vias metabólicas afetadas por esta droga. No seu conjunto, estes dados indicam a violaceína como uma droga potencial para o tratamento de infecções provocadas por MRSA
Abstract: Violacein is a violet pigment produced by some bacterial species of environmental source, such as Chromobacterium violaceum and Janthinobacterium lividum. This molecule has numerous biological properties including antibacterial, antifungal, antiviral, antiprotozoal and antitumor activity, although the exact role in the physiology of producing microorganisms is still unknown. In this study, the antimicrobial activity of violacein produced commercially, semi purified extract and silver vanadate nanoparticles were evaluated against several species of gram-positive and gram-negative bacteria. Violacein exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), microorganisms that are often related to hospital-acquired infections. MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of violacein produced commercially were 1.25 ?M mM and 0.625 ?M respectively, and analysis of growth and time-kill curves showed an antibacterial effect against MRSA for 12 hours. The transmission electron microscopy showed the effects of violacein with morphological and ultra-structural changes, including changes in cell wall formation and abnormal division septum. The results obtained from the analysis of proteomic and transcriptomic revealed that violacein affects the expression of several functional classes of proteins and genes in MRSA, including biological processes in cell wall biosynthesis and cell division, supporting ultra-structural changes. In conclusion, violacein produced commercially demonstrated antimicrobial activity against S. aureus MRSA and the effects on the metabolism of S. aureus have been described, indicating possible targets and pathways affected by this drug. These data indicate violacein as a potential drug for the treatment of infections caused by MRSA
Doutorado
Microbiologia
Doutora em Genética e Biologia Molecular

This study explores the handling and analyzing of big data in the field of bioinformatics. The focus has been on improving the analysis of public domain data for Affymetrix GeneChips which are a widely used technology for measuring gene expression. Methods to determine the bias in gene expression due to G-stacks associated with runs of guanine in probes have been explored via the use of a grid and various types of cloud computing. An attempt has been made to find the best way of storing and analyzing big data used in bioinformatics. A grid and various types of cloud computing have been employed. The experience gained in using a grid and different clouds has been reported. In the case of Windows Azure, a public cloud has been employed in a new way to demonstrate the use of the R statistical language for research in bioinformatics. This work has studied the G-stack bias in a broad range of GeneChip data from public repositories. A wide scale survey has been carried out to determine the extent of the Gstack bias in four different chips across three different species. The study commenced with the human GeneChip HG U133A. A second human GeneChip HG U133 Plus2 was then examined, followed by a plant chip, Arabidopsis thaliana, and then a bacterium chip, Pseudomonas aeruginosa. Comparisons have also been made between the use of widely recognised algorithms RMA and PLIER for the normalization stage of extracting gene expression from GeneChip data.

Bioinformatics has acquired a lot of importance especially with the advent of genomic approaches. The large amount of data produced by ``omics'' experiments requires appropriate frameworks to handle, store and mine the information and to derive appropriate work hypotheses. Transcriptome is defined as the whole amount of RNA molecules produced by a cell that provides the bridge between the genome and proteins. RNA molecules can be divided in two major classes: protein coding RNAs or messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). While the first class has been the most studied in the last decades, ncRNAs were recently discovered demonstrating their importance in cell regulatory processes. The most important class of the ncRNAs is composed by the micro RNAs (miRNAs) that have been related to several pathologies, including cancer, because of their ability to regulate oncogenes or oncosuppresors and mRNAs involved in the cell cycle. Here, I am presenting a work that aims at following and providing the appropriate structure for the interpretation and storage of the transcriptomics data. In this regard, I devised a tool to integrate expression levels from microarray experiments with gene annotation data like the genome localization and organization in biological pathways. The tool was devised and tuned using two datasets: the first one concerning expression profiles of patients with acute myeloid leukemia (ALL), the second one regarding muscular dystrophies. The application of this new tool to these datasets was very promising, especially regarding meta-analysis studies (muscular dystrophies). For this reason I applied the new tool to analyze public and in-house produced datasets of expression profiles of patients with inflammatory myopathies. This analysis allowed generating the hypothesis of the involvement of JAK-STAT and interferon type I signaling pathways in myopathies. The inferred results were validated using qRT-PCR and the presences of specific proteins produced by validated mRNAs were tested by ELISA and proteomic analysis. To complete and extend the knowledge of the muscle physiology, I used the pig as a new model organism to develop a framework aiming at the integration of miRNA expression and the regulation of their mRNA-target. It was important to develop the appropriate experimental instruments to perform the expression analyses. I developed two microarray platforms to perform the expression profiles of both miRNA and mRNA purified from the same sample. Then, with the expression data, I computationally analyzed aspects of miRNA biogenesis and performed the data integration leading to the production of regulatory networks specific of the studied tissues, including skeletal-muscle. Our miRNA sequences (mature and hairpin) were crossed with public data from RNA-seq experiments demonstrating that there is an important overlap between our results and the sequences identified by RNA-seq, confirming the goodness of our approach
Con l’avvento degli approcci genomici la bioinformatica ha acquisito un importanza sempre maggiore nello studio della biologia. Infatti, gli approcci “omici” permettono di produrre un enorme quantitativo di dati che deve essere archiviato in corrette strutture (database). L’archiviazione del dato comporta la necessità di permettere l’accesso e la manipolazione dello stesso al fine di svolgere gli studi appropriati. Sono quindi richiesti strumenti appropriati che consentano l’ispezione e la manipolazione dei database fine di formulare delle ipotesi coerenti con la problematica biologica che si sta studiando. Il trascrittoma è definito come l’insieme delle molecole di RNA che sono prodotte da una cellula e rappresentano un passaggio necessario nel processo che dal gene porta alla produzione della proteina. Le molecole di RNA possono essere suddivise in due grandi gruppi: gli RNA codificanti o messaggeri e gli RNA non codificanti. Mentre la prima classe è stata oggetto di ampi studi negli ultimi decenni, gli RNA non codificanti sono stati scoperti solo di recente e associati a funzioni puramente regolative. La classe più importante coinvolta nel processo regolativo degli RNA messaggeri è quella dei micro RNA (miRNA) che sono stati oggetto di un studio intenso che li ha messi in relazione con lo sviluppo di patologie come il cancro in quanto coinvolti nella regolazione fine dell’espressione genica di oncogeni, oncosoppressori o geni del ciclo cellulare. In questa tesi presento una serie di soluzioni bioinformatiche mirate a fornire le strutture appropriate per condurre gli esperimenti e le analisi dei dati di trascrittomica. Nel corso del periodo di dottorato, ho sviluppato un metodo che consente l’integrazione dei livelli di espressione genica ottenuti da esperimenti di microarray con informazioni riguardanti la localizzazione degli stessi nei cromosomi o la loro organizzazione in processi biologici. Questo metodo è stato messo a punto e raffinato nel suo funzionamento usando due gruppi di dati disponibili nei database pubblici: il primo riguarda dati di espressione genica ottenuti da esperimenti di microarray su leucemia mieloide acuta; il secondo riguarda l’espressione genica di distrofie muscolari derivanti sempre da dati di microarray. I risultati di questo nuovo metodo si sono dimostrati molto promettenti, in particolare nell’applicazione della meta-analisi che consiste nell’integrare dati provenienti da differenti laboratori. Forte di questo primo risultato, ho applicato questo metodo di analisi anche all’ispezione dei processi sregolati nelle miopatie infiammatorie affiancando ai dati disponibili prodotti nel laboratorio di Genomica Funzionale diretto dal Prof. G. Lanfranchi quelli depositati nei database pubblici. La meta-analisi da me implementata ha permesso di studiare questa serie di dati sfruttando, per la prima volta, la localizzazione dei geni e raggruppandoli per la funzione permettendo di generare ipotesi sui meccanismi patologici. Grazie a questa tipologia di analisi ho ipotizzato il coinvolgimento nelle miopatie infiammatorie delle vie di segnale che fanno capo a JAK/STAT e agli interferoni. Le ipotesi generate analizzando i dati sono state confermate andando a validare i geni coinvolti nelle vie di segnale appena menzionate usando la qRT-PCR. Inoltre, usando approcci di proteomica, in collaborazione con la Prof. C. Gelfi (Università di Milano) e la tecnica ELISA, è stata anche validata la presenza delle proteine coinvolte in queste vie di segnale nei pazienti affetti da miopatie infiammatorie. Nella parte conclusiva del mio dottorato, mi sono occupato di completare ed estendere la conoscenza della fisiologia muscolare. Per far questo mi sono spostato sul maiale, un organismo modello molto importante per lo studio di patologie umane e per la produzione di componenti biologiche che possono essere utilizzate per sostituire quelle degradate nell’uomo (valvole aortiche per esempio). Usando il maiale ho sviluppato un sistema per integrare l’espressione dei miRNA e la regolazione che questi esercitano nei messaggeri target. Come prima cosa ho sviluppato le piattaforme di microarray per eseguire l’analisi dell’espressione genica di 14 tessuti di maiale. In particolare ho sviluppato due tipi di piattaforme per eseguire l’analisi dell’espressione dei trascritti e dei miRNA purificati dallo stesso campione. Con questi dati di espressione ho condotto analisi per delucidare alcuni aspetti inerenti la biogenesi dei miRNA. Infine, la completezza dei dati prodotti mi ha permesso di costruire delle reti di regolazione specifiche per ogni tessuto analizzato. Per confermare la validità del nostro approccio ho analizzato il grado di sovrapposizione tra le sequenze derivate dal nostro studio e le sequenze prodotte dai vari esperimenti di RNA-seq. Con questa analisi ho confermato la validità del mio approccio in quanto è stato rivelato una sovrapposizione importante tra le nostre sequenze e quelle derivate da RNA-seq

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

Le microenvironnement des tumeurs est composé de cellules immunitaires, de fibroblastes et de cellules endothéliales, ainsi que d’autres cellules non-malignes. Son étude a permis d’établir des classifications qui ont une valeur pronostique et théranostique, ainsi que de développer des traitements modulant la composition et l’orientation fonctionnelle du microenvironnement. En parallèle, des classifications moléculaires des tumeurs ont proposé des taxonomies stratifiant les cancers humains en sous-groupes associés à des différences de survie des patients et leur réponse aux traitements. Des études récentes suggèrent que le phénotype de la cellule cancéreuse est un facteur critique dans le façonnement du microenvironnement tumoral, suggérant un possible consensus entre les classifications immunitaires et moléculaires. Le but de cette thèse était donc de caractériser le microenvironnement des sous-groupes moléculaires de tumeurs humaines. Le cancer colorectal a été le premier cancer humain dans lequel il a été mis en évidence qu’une réponse immunitaire adaptative était associée à un contrôle de la croissance tumorale, et représente ainsi un exemple type pour l’immunologie des tumeurs. A l’inverse, le carcinome du rein à cellules claires est une exception vis-à-vis de l’immunologie des tumeurs, puisqu’une forte réponse immunitaire adaptative y est associée à des tumeurs plus agressives. Des classifications transcriptomiques ont été récemment établies pour ces deux cancers, qu’à première vue tout oppose sur le plan immunitaire. Dans ce travail, je propose une méthode permettant l’étude du microenvironnement tumoral à partir de données transcriptomiques, et décris son application à l’étude du contexte immunitaire des cancers colorectaux et du rein à cellules claires. Ces analyses suggèrent qu’une unification des classifications moléculaires et immunitaires des tumeurs humaines est possible, remettent en cause notre conceptualisation des liens entre la composition du microenvironnement tumoral et le pronostic du patient, et évoque des pistes immunothérapeutiques potentiellement adaptées à certains sous-groupes de patients dans ces cancers
Tumors grow within a complex microenvironment composed of immune cells, fibroblasts, endothelial cells and other non-malignant cells. The study of the composition of tumor microenvironments has led to classifications with prognostic and theranostic values, as well as the discovery of treatments modulating the composition and the functional orientation of the microenvironment. Concurrently, molecular classifications of tumors have proposed taxonomies within cancers that define groups of patients with different prognoses and are associated with response to treatments. Recent evidence suggest that the phenotype of the malignant cell is a critical determinant in the shaping of its microenvironment, suggesting potential correlations between immune and molecular classifications. The goal of this PhD project was therefore to analyze the microenvironment of molecularly-classified human tumors. Colorectal cancer represents a paradigm for tumor immunology, as it is the humancancer in which it was exemplified that an adaptive immune response can control tumor Growth and metastasis. Conversely, clear-cell renal cell carcinoma represents an exception in tumor immunology, as an extensive adaptive immune response is associated with more aggressive diseases. Molecular transcriptomic classifications were recently proposed for both of these apparently immunologically contrasted cancers. In this work, I propose a methodology that enables the characterization of the tumor microenvironment using transcriptomic data, and apply it to describe the immune contexture of molecular subgroups of colorectal and clear-cell renal cell carcinomas. These analyses argue in favor of the unification of molecular and immune classifications of human cancers, challenge our current views of the relationship between the composition of the tumor microenvironment and patient’s prognosis, and suggest immunotherapeutic approaches that could benefit subgroups of patients in these two cancers

The impact that zinc oxide nanoparticles (ZnONP) have on plant physiological responses was evaluated by comparing gene expression changes after Arabidopsis thaliana plants were exposed to ZnONP, in comparison with ionic Zn2+ (ZnSO4) and non-treated controls. After treatment with ZnONP (concentration10 μg L−1), ionic Zn2+ (applied as ZnSO4 at a concentration of 19.7 μg/ L −1), expression analyses via RNA sequencing revealed that 369 genes were down regulated and 249 were upregulated (p < [FDR] 0.05, expression difference < 3). The downregulated genes in ZnONP treated seedlings compared to the Zn +2 ion and untreated controls were mainly abiotic stress (oxidative stress, low temperature) and biotic stress such as defense responsive genes based on the gene ontology analysis. The upregulated genes in response to ZnONP treated plants compared to the Zn +2 ion control plants were mainly photosynthesis, light harvesting complex, and hormone responsive genes such as abscisic acid.

Orientador: Paulo Arruda
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-06T21:19:28Z (GMT). No. of bitstreams: 1 Ferreira_NataliaCristinaVerza_D.pdf: 9495810 bytes, checksum: 18cf9ae055a2b9f39de109abc6de469c (MD5) Previous issue date: 2006
Resumo: O seqüenciamento de ESTs (etiquetas de seqüências expressas) e a sua organização em bancos de dados constituem poderosas ferramentas para identificar genes de interesse expressos em determinados tecidos e/ou tipos celulares. Neste trabalho criou-se um banco de seqüências expressas chamado MAIZESTdb, que contém ESTs de diversos tecidos de milho, porém enriquecido com seqüências provenientes do endosperma de milho em desenvolvimento. O MAIZESTdb contém 227.431 ESTs vindos de mais de 30 órgãos e tecidos de milho diferentes, 30.531 seqüenciados em nosso laboratório a partir de bibliotecas construídas com RNA mensageiro de endosperma. Estas seqüências representam uma grande contribuição na identificação de novos genes expressos no endosperma. A análise deste banco de ESTs possibilitou a identificação de 4.032 transcritos preferencialmente expressos no endosperma, e a sua anotação revelou uma ampla variedade de prováveis genes novos envolvidos no desenvolvimento e no metabolismo do endosperma. O banco MAIZESTdb foi utilizado neste trabalho para a identificação de fatores de transcrição (TFs) expressos no endosperma de milho, e, especialmente, na identificação de fatores preferencialmente expressos no endosperma, que podem desempenhar papéis regulatórios importantes durante a formação da semente. Foram identificados 1.233 TFs expressos em milho, 414 dos quais expressos no endosperma em desenvolvimento. Foram identificados ainda, através de análises in silico, 113 TFs preferencialmente expressos no endosperma, conjunto este que representa 9.2% dos TFs expressos identificados em milho, e que possivelmente contém reguladores importantes dos processos de especificação celular e desenvolvimento do endosperma de milho. Esta é a maior coleção de fatores de transcrição já descrita para este tecido, e representa uma fonte de dados importante para identificação de reguladores dos principais processos relacionados ao desenvolvimento do endosperma, como metabolismo de nitrogênio e carboidratos e controle da massa da semente. Uma das famílias mais representadas entre os TFs preferencialmente expressos no endosperma foi a família NAC de fatores de transcrição. Esta família apresentou 12 membros preferencialmente expressos no endosperma de milho. Um novo membro da família NAC, chamado de EPN-1 (Endosperm Specific NAM 1), teve seu perfil de expressão caracterizado. Sua expressão pode ser detectada desde os 5 DAPs, embora o pico de expressão ocorra entre 20 e 25 DAP, e ele apresenta expressão preferencial no endosperma. O promotor do gene EPN-1 foi clonado, seqüenciado e analisado quanto aos seus possíveis elementos CIS regulatórios; foram encontrados elementos conservados relacionados à endosperma-especificidade, elementos relacionados à regulação por ácido abscísico e giberelinas, e elementos conservados presentes nos promotores de a-amilases, indicando uma possível relação deste gene com o processo de transição entre a maturação e a germinação da semente. Ensaios de expressão transitória com o promotor do gene EPN-1 revelaram que sua expressão está dirigida à camada de aleurona do endosperma de milho, o que constitui mais uma evidência de sua possível função na regulação de genes relacionados aos processos de maturação e germinação da semente
Abstract: The sequencing of ESTs (expressed sequence tags) and its organization in databases constitute powerful tools to identify genes of interest in certain tissues and/or cell types. In this work we have created MAIZESTdb, a database of ESTs expressed in diverse maize tissues. The importance of this database, however, is that it is enriched with sequences from developing maize endosperm. The MAIZESTdb contains 227,431 ESTs coming from more than 30 different maize tissues and organs, 30,531 of which sequenced from endosperm cDNA libraries constructed in our laboratory. These sequences represent a great contribution for the identification of novel genes expressed in endosperm. The analysis of this ESTs database led to the identification of 4,032 transcripts preferentially expressed in the endosperm, and its annotation revealed a great variety of new genes involved in endosperm metabolism and development. The MAIZESTdb was then used to identify transcription factors (TFs) expressed in maize endosperm, and, mainly, in the identification of TFs preferentially expressed in the endosperm. We identified 1,233 TFs expressed in diverse maize tissues, 414 of which expressed in developing endosperm. We also identified, through in silico comparison of transcript abundance and library source, 113 TFs with preferential expression in endosperm, representing 9,2% of the TFs identified in this work. This dataset probably contains important regulators of cellular specification of the endosperm development. This is the biggest TFs collection reported for this tissue, and represents an important source of data for identification of regulators for main processes related to the endosperm development such as nitrogen and carbohydrate metabolism and control of seed mass. One of the most represented families among the TFs preferentially expressed in endosperm was the NAC family of transcription factors. This family presented 12 members with preferential expression in the endosperm. A new member of the NAC family, called EPN-1 (Endosperm Specific NAM 1), was characterized. Its expression
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular

Mestrado em Biomedicina Molecular
As lesões na medula espinal são uma desordem neurológica comum com um impacto significativo na sociedade moderna do ponto de visto físico, psicosocial e socioeconómico. Apesar de vários vertebrados serem capazes de regenerar lesões do sistema nervoso central, nomeadamente da medula espinal (ex. Rã, Peixe-zebra, Salamandra), está bem estabelecido que os seres humanos, e outros mamíferos adultos, não o conseguem fazer. Como tal, em consequência de lesões traumáticas no cérebro ou medula espinal, há incapacidade dos axónios crescerem extensivamente no tecido lesado. No entanto, um estudo importante realizado no virar do século por Ramón y Cajal, comprovou que a incapacidade das fibras nervosas regenerarem “deriva de condições externas, da presença ou ausência de fatores auxiliares que são indispensáveis para o processo regenerativo”, trazendo assim esperança que a neuroregeneração possa ser alcançada por modulação de condições celulares e moleculares. Esta dissertação tem como objetivo adquirir uma melhor e mais extensa compreensão dos genes e processos fisiológicos que são cruciais durante a regeneração da medula espinal, usando estudos de expressão genómica de modelos regenerativos, tais como Xenopus laevis, Xenopus tropicalis e Danio rerio, estabelecendo-se simultaneamente um paralelismo com os respetivos ortólogos humanos com o objetivo de encontrar genes candidatos no genoma humano passíveis de serem modulados com vista a alterar o estatuto não-regenerativo dos mamíferos adultos.
Spinal cord injuries are a common neurologic disorder that have devastating impacts on modern society, be it from physical, psychosocial, or socioeconomic point of view. Although many small vertebrates are capable of regenerating lesions to the central nervous system, namely the spinal cord, (e.g. frog, zebrafish, salamander) it is well established that humans and other adult mammals cannot. As so, failure of axons to grow extensively through damaged central nervous system (CNS) tissues is a common consequence of injury to the brain and spinal cord on adult mammals. However, an important study made at the turn of the century by Ramón y Cajal, proved that the failure of central fibers to regrow “derives from external conditions, the presence or absence of auxiliary factors that are indispensable to the regenerative process”, thus bringing hope that neuroregeneration can be achieved by modulating cellular and molecular conditions. Through this dissertation, we aim to get a better understanding of the involvement of the genes and physiological processes that are crucial during regeneration of the spinal cord, using genome wide expression studies of regenerative models such as Xenopus laevis, Xenopus tropicalis, and Danio rerio, while drawing parallel to its human orthologues. Being our goal to find perfect gene candidates in the human genome that are predictably capable of being modulated so we can alter the non-regenerative status of the adult mammals.

The Human Protein Atlas is an open-source database containing information about protein expression and location in the human cells,tissues and organs. The aim is to map all the proteins in humans using various biotechnology techniques such as antibody-based imaging, andRNA sequencing etc. Based on previous transcriptome analysis, 173 genes were shown to have an elevated expression in ovary compared to all other major tissue types in the human body. There is however no information regarding the expression in ovary during the reproductive years versus the post-menopausal years. In this thesis, the gene expression in ovaries of women in reproductive age was compared with women in post-menopausal age. 509 genes were found to have an at least 2-fold higher mean value RNA expression in the reproductive age group. 14 of these genes were analyzed further with antibody staining and multiplex immunofluorescence staining to localize the corresponding proteins. The results show that these genes are expressed in a variety of structures in the ovarian tissue, such as the oocyte, the granulosa cells and the corpus luteum. This thesis has demonstrated how data analysis can be used to find genes important for the ovary of women in reproductive age and in the future, this could aid research in female fertility.

Doutoramento em Biologia
O conhecimento de mecanismos de genómica funcional tem sido maioritariamente adquirido pela utilização de organismos modelo que são mantidos em condições laboratoriais. Contudo, estes organismos não reflectem as respostas a alterações ambientais. Por outro lado, várias espécies, ecologicamente bem estudadas, reflectem bem as interacções entre genes e ambiente mas que, das quais não existem recursos genéticos disponíveis. O imposex, caracterizado pela superimposição de caracteres sexuais masculinos em fêmeas, é induzido pelo tributilestanho (TBT) e trifenilestanho (TPT) e representa um dos melhores exemplos de disrupção endócrina com causas antropogénicas no ambiente aquático. Com o intuito de elucidar as bases moleculares deste fenómeno, procedeu-se à combinação das metodologias de pirosequenciação (sequenciação 454 da Roche) e microarrays (Agilent 4*180K) de forma a contribuir para um melhor conhecimento desta interacção gene-ambiente no gastrópode Nucella lapillus, uma espécie sentinela para imposex. O trancriptoma de N. lapillus foi sequenciado, reconstruído e anotado e posteriormente utilizado para a produção de um “array” de nucleótidos. Este array foi então utilizado para explorar níveis de expressão génica em resposta à contaminação por TBT. Os resultados obtidos confirmaram as hipóteses anteriormente propostas (esteróidica, neuroendócrina, retinóica) e adicionalmente revelou a existência de potenciais novos mecanismos envolvidos no fenómeno imposex. Evidência para alvos moleculares de disrupção endócrina não relacionados com funções reprodutoras, tais como, sistema imunitário, apoptose e supressores de tumores, foram identificados. Apesar disso, tendo em conta a forte componente reprodutiva do imposex, esta componente funcional foi a mais explorada. Assim, factores de transcrição e receptores nucleares lipofílicos, funções mitocondriais e actividade de transporte celular envolvidos na diferenciação de géneros estão na base de potenciais novos mecanismos associados ao imposex em N. lapillus. Em particular, foi identificado como estando sobre-expresso, um possível homólogo do receptor nuclear “peroxisome proliferator-activated receptor gamma” (PPARγ), cuja função na indução de imposex foi confirmada experimentalmente in vivo após injecção dos animais com Rosiglitazone, um conhecido ligando de PPARγ em vertebrados. De uma forma geral, os resultados obtidos mostram que o fenómeno imposex é um mecanismo complexo, que possivelmente envolve a cascata de sinalização envolvendo o receptor retinoid X (RXR):PPARγ “heterodimer” que, até à data não foi descrito em invertebrados. Adicionalmente, os resultados obtidos apontam para alguma conservação de mecanismos de acção envolvidos na disrupção endócrina em invertebrados e vertebrados. Finalmente, a informação molecular produzida e as ferramentas moleculares desenvolvidas contribuem de forma significativa para um melhor conhecimento do fenómeno imposex e constituem importantes recursos para a continuação da investigação deste fenómeno e, adicionalmente, poderão vir a ser aplicadas no estudo de outras respostas a alterações ambientais usando N. lapillus como organismo modelo. Neste sentido, N. lapillus foi também utilizada para explorar a adaptação na morfologia da concha em resposta a alterações naturais induzidas por acção das ondas e pelo risco de predação por caranguejos. O contributo da componente genética, plástica e da sua interacção para a expressão fenotípica é crucial para compreender a evolução de caracteres adaptativos a ambientes heterogéneos. A contribuição destes factores na morfologia da concha de N. lapillus foi explorada recorrendo a transplantes recíprocos e experiências laboratoriais em ambiente comum (com e sem influência de predação) e complementada com análises genéticas, utilizando juvenis provenientes de locais representativos de costas expostas e abrigadas da acção das ondas. As populações estudadas são diferentes geneticamente mas possuem o mesmo cariótipo. Adicionalmente, análises morfométricas revelaram plasticidade da morfologia da concha em ambas as direcções dos transplantes recíprocos e também a retenção parcial, em ambiente comum, da forma da concha nos indivíduos da F2, indicando uma correlação positiva (co-gradiente) entre heritabilidade e plasticidade. A presença de estímulos de predação por caranguejos estimulou a produção de conchas com labros mais grossos, de forma mais evidente em animais recolhidos de costas expostas e também provocou alterações na forma da concha em animais desta proveniência. Estes dados sugerem contra-gradiente em alterações provocadas por predação na morfologia da concha, na produção de labros mais grossos e em níveis de crescimento. O estudo das interacções gene-ambiente descritas acima demonstram a actual possibilidade de produzir recursos e conhecimento genómico numa espécie bem caracterizada ecologicamente mas com limitada informação genómica. Estes recursos permitem um maior conhecimento biológico desta espécie e abrirão novas oportunidades de investigação, que até aqui seriam impossíveis de abordar.
Our understanding of functional genomic mechanisms is largely acquired from model organisms through laboratory conditions of exposure. Yet, these laboratory models typically have little environmental relevance. Conversely, there are numerous “ecological” model species that present important geneenvironment interactions, but lack genomic resources. Imposex, the superimposition of male sexual characteristics in females, is caused by tributyltin (TBT) and triphenyltin (TPT) and provides among the most widely cited ecological examples of anthropogenically-induced endocrine disruption in aquatic ecosystems. To further elucidate the functional genomic basis of imposex, combinations of 454 Roche pyrosequencing and microarray technologies (Agilent 4*180K) were employed to elucidate the nature and extent of gene-environment interactions in the prosobranch gastropod, Nucella lapillus, a recognized sentinel for TBT-induced imposex. Following transcriptome characterization (de novo sequencing, assembly and annotation), microarray fabrication and competitive hybridizations, differential gene expression analyses provided support for previously suggested hypotheses underpinning imposex (steroid, neuroendocrine, retinoid), but also revealed potential new mechanisms. Evidence for endocrine disruption (ED) targets such as the immune system, apoptosis and tumour suppressors other than reproduction-related functions were found; however, given the ED nature of imposex, primary focus was on gender-differentiation pathways. Among these, transcription factors and lipophilic nuclear receptors as transducers of TBT toxicity along with mitochondrial functions and deregulation in transport activity suggested new putative mechanisms for the TBT-induced imposex in N. lapillus. Particularly, up-regulation of a putative nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) homolog was evident, and its role was further confirmed by inducing imposex in vivo using Rosiglizatone, a well-known vertebrate PPARγ ligand. Our analyses show that TBT-induced imposex is a complex mechanism, but is likely to act through the retinoid X receptor (RXR):PPARγ heterodimer signalling pathway, hitherto not described in invertebrates. Moreover, collectively, our findings support a commonality of signalling between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption. The genomic resources generated here largely contribute to the molecular understanding of imposex, yielding valuable insights for further examination of responses to TBT contamination exposure. Additionally, we anticipate that the new genomic resources described herein will contribute to the further exploration of adaptive responses of dogwhelks to environmental variation. N. lapillus was also used to explore adaptive shell shape morphology in response to natural variation in wave-action and crab predation. Knowledge of the contributions of genotype, plasticity and their interaction to phenotypic expression is crucial for understanding the evolution of adaptive character traits in heterogeneous environments. We assessed contributions of the above factors by reciprocal transplantation of snails between two shores differing in exposure to wave action and predation, and rearing snails of the same provenance in a laboratory common garden experiment with crab-predation odour, complemented by genetic analysis. The two target populations are genetically different but maintain the same karyotype. Truss-length and morphometric analyses revealed plasticity of shell shape in reciprocal transplants, but also the partial retention of parental shape by F2 snails in common garden controls, indicating co-gradient variation between heritable and plasticity components. Crab-predation odour influenced shell shape of snails from exposed-site origin and stimulated the production of thicker shell lips with greater response in snails of exposed-site ancestry. We interpret these data as countergradient variation on predator-induced changes in shell shape and increased thickening of the shell lip as well as on growth rates. The above exploration of gene-environment interactions demonstrates the feasibility, insights and novel opportunities that can now be addressed in a species that is well characterised ecologically, but hitherto constrained by the general lack of genomic tools and archived resources. Notably, a greater focus on detailed responses of a single species facilitates the comparative approach, as illustrated by the apparent commonality in regulation of endocrine disruption processes in invertebrates and vertebrates.
FCT; FSE - SFRH/BD/27711/2006

Polyploidization instantly doubles all genome content by combining two genomes that have markedly different methylation and gene expression levels. This process may be accompanied by genetic and epigenetic changes in each genome. Sequencing of the transcriptome (RNA-seq) and the methylome (bisulfite treated libraries whole genome libraries) were used to measure gene expression and methylation levels of genic regions of allopolyploid cotton petals and petals of their diploid relatives. Many differentially expressed genes detected by RNA-seq were consistent with expression levels previously detected by microarrays. RNA-seq results also reconfirmed the presence of general polyploid gene expression trends like expression level dominance and homoeologous expression biases in Gossypium polyploid species. Expression biases between A- and D-genome homoeologs and expression level dominance was characterized for thousands of genes in tetraploids and a diploid F1-hybrid. Unlike the results of microarray study previously done we found a slightly greater number of genes showing A-genome bias vs genes showing D-genome bias. More commonly the overall expression level from homoeologs of polyploid is heterotic i.e the expression level is greater than the average of the expression levels from the two parent genomes. In addition, genome methylation (CG, CHG, and CHH contexts) of each genome was assessed in the diploid and tetraploid samples. The A- and D-genomes had distinct levels of DNA methylation for each context. DNA methylation may be independently regulating homoeologous expression levels of a small number of genes.

Gene expression is tightly regulated by complex transcriptional regulatory mechanisms to achieve specific expression patterns, which are essential to facilitate important biological processes such as embryonic development. Dysregulation of gene expression can lead to diseases such as cancers. A better understanding of the transcriptional regulation will therefore not only advance the understanding of fundamental biological processes, but also provide mechanistic insights into diseases. The earlier versions of high-throughput expression profiling techniques were limited to measuring average gene expression across large pools of cells. In contrast, recent technological improvements have made it possible to perform expression profiling in single cells. Single-cell expression profiling is able to capture heterogeneity among single cells, which is not possible in conventional bulk expression profiling. In my PhD, I focus on developing new algorithms, as well as benchmarking and utilising existing algorithms to study the transcriptomes of various biological systems using single-cell expression data. I have developed two different single-cell specific network inference algorithms, BTR and SPVAR, which are based on two different formalisms, Boolean and autoregression frameworks respectively. BTR was shown to be useful for improving existing Boolean models with single-cell expression data, while SPVAR was shown to be a conservative predictor of gene interactions using pseudotime-ordered single-cell expression data. In addition, I have obtained novel biological insights by analysing single-cell RNAseq data from the epiblast stem cells reprogramming and the leukaemia systems. Three different driver genes, namely Esrrb, Klf2 and GY118F, were shown to drive reprogramming of epiblast stem cells via different reprogramming routes. As for the leukaemia system, FLT3-ITD and IDH1-R132H mutations were shown to interact with each other and potentially predispose some cells for developing acute myeloid leukaemia.

The Brown Plant Hopper (BPH) is a serious pest of rice in Asia. Development of novel control strategies can be facilitated by a comparison of BPH feeding behaviour on varieties exhibiting natural genetic variation, and then an elucidation of the underlying mechanisms of resistance. We began by understanding BPH feeding behaviour on 12 rice varieties with different resistance background using Electrical Penetration Graph (EPG) and honeydew clock experiments. Seven feeding behaviours (waveforms) were identified and could be classified into two phases, feeding and non- feeding. Cluster analysis has separated the 12 varieties into 3 main groups, resistant, moderate and susceptible. Then, we undertook microarray analysis on all varieties to identify candidate genes which may contribute to resistance. The results reveal the difference between resistant and susceptible varieties. The data agree with EPG and honeydew clock experiments. A total of 21556 probes passed filter in statistical analysis using quantile method (in Genespring) and 239 probes significantly contributed to the difference between resistant versus susceptible (Volcano analysis). Some of them were found to be highly correlated with EPG data and could therefore be potential resistance candidate genes against BPH such as gene encoding hexose transporter, protein kinases, Alpha-DOX2 and peroxidases.