Academic literature on the topic 'SILICO ANALYSIS'

Mestrado em Biomedicina Molecular<br>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.<br>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.

Lipid membranes are a fundamental component of living cells, mediating the physical separation of intracellular components from the external environment, as well as the different cellular organelles from cytoplasm. Transmembrane transport proteins confer permeability to lipid membranes, which is essential for nutrient translocation and energy metabolism. Crystallography of transmembrane proteins is a particularly challenging problem. Due to their natural localization and chemical properties only a limited number of structures are to date available at atomic resolution. In silico analysis can be successfully applied to address the structure and to propose testable models of transporters and pores and of their function. My PhD work focused on two main models: Pendrin (SLC26A4) and the Permeability Transition Pore (PTP). These two systems allowed me to investigate different membrane types and permeation mechanisms, i.e. the plasma membrane-specific anion exchange (SLC26A4) and the inner mitochondrial membrane (IMM) unselective PTP. Pendrin mutations are estimated to be the second most common genetic cause of human deafness, but a precise 3D structure of the protein is still missing. Aim of my work was to obviate the absence of structural information for pendrin transmembrane domain and to give a functional explanation for mutations collected in the MORL Deafness Variation Database. The human pendrin 3D model was inferred by homology with SLC26Dg and then validated analyzing the surface distribution of hydrophobic residues. The resulting high quality model was used to map 147 pathogenic human mutations. Three mutation clusters were found, while their localization suggested an innovative 14 transmembrane domain structure for pendrin. The nature of PTP has long remained a mystery. In 2013 Giorgio et. al. suggested dimers of F1FO (F)-ATP synthase to form the pore, however the exact PTP composition and how can a pore form from the energy-conserving enzyme is still matter of debate. PTP opening is triggered by an increased Ca2+ concentration in the mitochondrial matrix, and is favored by oxidative stress. To shed light on PTP function, I investigated the effect of Ca2+ binding to the Me2+ binding site of the F1 domain of F-ATP synthase through molecular dynamics (MD) simulations. A similar approach was also applied to the F-ATP synthase β subunit mutation T163S, which alters the relative affinity for Mg2+ and Ca2+. Experimental data show that Ca2+ binding stiffens the complex structure and that the T163S mutation induces resistance to PTP opening. Further, catalytic site rearrangement induced from different ion occupancy, as well as the mutation T163S, yields relevant variation of the interaction between F1 domain and OSCP subunit. I suggest that an unstructured loop between residues 82-131 of the β subunit transmits the structural rearrangement originated into catalytic site to the OSCP subunit and then to the inner membrane through the rigid lateral stalk. The critical role emerging for OSCP in the PTP regulation opens two parallel questions, i.e. (i) how the OSCP-mediated opening signal is transmitted to the trans-membrane region and (ii) what are the transmembrane PTP components. Variation in pore conductivity among species suggested that the putative pore-forming subunits may be different in different species. Sequence alignment was performed for all the subunits of F-ATP synthase, but we mainly focused on subunits e, g and b due to their localization in the complex and sequence conservation. Specific mutations affecting F-ATP synthase were collected and their functional effect is currently under analysis. In parallel, the presence and features of e, g and f subunits across eukaryotes was investigated by mean of phylogenetic analysis. Protein homologues of these specific subunits were found to be widespread in eukaryotes from yeast to plants while we found that Oomycetes lack subunits e and g and green algae subunit e. This observation suggest an ancient evolution for the F-ATP synthase dimerization subunits and possibly for the PTP. Further analysis and experimental validation are planned to clarify this aspect.<br>Le membrane lipidiche sono una componente fondamentale delle cellule viventi, separano fisicamente le componenti intracellulari dall’ambiente esterno e i diversi organelli del citoplasma. Le proteine di trasporto conferiscono permeabilità alle membrane lipidiche, proprietà essenziale per la traslocazione di nutrienti e la conservazione dell’energia. La cristallografia di proteine transmembrana è problematica a causa della loro localizzazione e proprietà chimiche, e solo un numero piuttosto ridotto di strutture è disponibile. L’analisi in silico può essere applicata con successo per investigare le strutture e il funzionamento proporre modelli testabili di trasportatori e delle loro funzioni. Il lavoro del mio dottorato sì è focalizzato su due modelli: la pendrina (SLC26A4) e il poro di transizione di permeabilità (PTP). Questi due sistemi proteici mi hanno permesso di studiare due differenti tipi di membrana e meccanismi di permeabilità: la membrana plasmatica con scambio specifico di anioni (SLC26A4) e la membrana interna mitocondriale con la permeabilità non selettiva mitocondriale (PTP). Le mutazioni della pendrina sono stimate essere la seconda causa genetica più comune della sordità umana, ma la struttura della proteina non è stata ancora determinata. Scopo del mio lavoro è stato quello di sopperire all’assenza di informazioni strutturali per il dominio transmembrana della pendrina e di dare una spiegazione funzionale per le mutazioni raccolte nel MORL Deafness Variation Database. Il modello 3D della pendrina è basato sull’omologia con SLC26Dg (3) ed è stato validato analizzando la distribuzione sulla superfice dei residui idrofobici. L’alta qualità risultante dal modello è stata usata per mappare 147 mutazioni patologiche umane. Tre cluster di mutazioni sono stati trovati e la loro localizzazione suggerisce per pendrina un innovativa struttura a 14 domini transmembrana. Anche la natura del PTP è rimasta a lungo misteriosa. Nel 2013 Giorgio et al. hanno suggerito che i dimeri di F1FO (F)-ATP sintasi formino il poro, tuttavia l’esatta composizione e il modo in cui il poro di transizione si possa formare è ancora materia di dibattito. L’apertura del PTP è innescata da un aumento della concentrazione di Ca2+ nella matrice mitocondriale ed è favorita dallo stress ossidativo. Per fare luce sul funzionamento del PTP ho studiato l’effetto del legame del Ca2+ al sito per i cationi divalenti (Me2+) nel dominio F1 attraverso la dinamica molecolare (MD). Un approccio simile è stato anche applicato alla mutazione T163S, che fa variare l’affinità relativa per Mg2+ e Ca2+. I dati sperimentali mostrano come la mutazione induca resistenza all’apertura del PTP. La MD ha dimostrato come il legame del Ca2+ irrigidisca la struttura del complesso. Il riarrangiamento del sito catalitico indotto dai differenti ioni che lo occupano, così come la mutazione T163S, causa rilevanti variazioni delle interazioni tra il dominio F1 e la subunità OSCP. Suggerisco che un loop non strutturato tra i residui 82-131 della subunità β trasmetta il riarrangiamento strutturale originato nel sito catalitico a OSCP e quindi alla membrana interna attraverso il rigido stalk laterale. Il ruolo critico che emerge per OSCP nella regolazione del PTP apre due domande collegate: (i) come il segnale di apertura mediato da OSCP venga trasmesso alla regione trans-membrana e (ii) quali siano i componenti transmembrana del PTP. Le variazioni di conduttanza del poro osservate in specie diverse suggeriscono che le subunità che formano il canale debbano avere delle differenze significative. E’ stato prodotto un allineamento di sequenze per tutte le subunità della F-ATP sintasi. I risultati preliminari ci hanno spinto a focalizzarci sulle subunità e, g e b a causa della loro localizzazione e conservazione di sequenza. Basandomi sugli allineamenti multipli ho suggerito mutazioni puntiformi per testare l’importanza di specifici residui ai fini dell’apertura del poro. In parallelo la presenza delle subunità e e g tra gli eucarioti è stata indagata attraverso un analisi filogenetica. Proteine omologhe di queste specifiche subunità sono presenti in tutti gli eucarioti: dai lieviti alle piante, tuttavia gli Oomiceti sono risultati mancanti delle subunità e e g e le alghe verdi della subunità e. Questi risultati suggeriscono un’origine antica per le subunità di dimerizzazione della F-ATP sintasi e probabilmente anche del PTP. Per chiarire questo aspetto saranno necessarie ulteriori analisi e verifiche sperimentali.

Regulation of gene transcription is a complex process involving many different proteins, some of which bind in a sequence-specific manner to DNA motifs in the gene promoter. The need to maintain specific interactions between transcription factors and proteins involved in the RNA polymerase II complex is expected to impose constrains on the relative position and spacing of the interacting DNA motifs. The present work includes the development of a novel approach to identify motifs that show a preferential location in DNA sequences and the implementation of a public web application called PEAKS. We investigated if the arrangement and nature of the most common motifs depended on the breath of expression of the gene. We found differences that serve to illustrate that many key specific regulatory signals may be present in the proximal promoter region in mammalian genes. We also apply other methods for the identification of specific transcription factors (TFs) involved in the co-regulation of a set of genes. Data from experimentally-verified transcription factors binding sites (TFBSs) support the biological relevance of our findings.<br>La regulació de la transcripció dels gens és un procés complex que implica moltes proteïnes diferents, algunes de les quals s'unexien a motius específics d'ADN localitzats a la regió promotora dels gens. S'espera que la necessitat de mantenir les interaccions específiques entre els factors de transcripció i les proteïnes implicades en el complex de la ARN polimerasa II imposi limitacions en la posició relativa i l'espaiat dels motius d'interacció amb l'ADN. La feina presentada en aquesta tesi inclou el desenvolupament d'un nou metode per l'identificació de motius que mostren una localització preferencial en seqüències d'ADN i l'implementació d'una aplicació web pública anomenada PEAKS. Hem investigat si la col·locació i la naturalesa de la majoria dels motius comuns depen del rang d'expresió del gen. Hem trobat diferències que serveixen per il·lustrar el fet que moltes senyals clau de regulació gènica poden estar presents en la regió proximal del promotor dels gens de mamífers. També hem aplicat altres mètodes per a l'identificació de factors de transcripció (TFs) específics involucrats en la co-regulació d'un grup de gens. Dades de llocs d'unio dels TFs (TFBSs) verificats experimentalment recolzen la rellevància biològica dels nostres resultats.

WRINKLED 1(WRI1), a member of AP2/EREBP class of transcription factors regulates carbon allocation between glycolytic and fatty acid biosynthetic pathway. Additionally, among the four WRI1 paralogs in Arabidopsis, WRI3 and 4 but not WRI2, are also able to increase fatty acid content in different tissues. While the role of WRI1 is well established in seeds, the potential or WRI1 or its paralogs as master regulators in oilrich nonseed tissues is poorly understood. Recent transcriptome studies of avocado (Persea americana) mesocarp revealed that the ortholog of WRI2, along with WRI1 and WRI3 was highly expressed during oil accumulation.Through transient expression assays, wefurther demonstrated thatbothPaWRI1 andPaWRI2 can accumulate oil in tobacco leaves. We conducted a comprehensive and comparative in silico analysis of WRI paralogs from a dicot, monocot and a basal angiosperm to identify distinct features associated with function. These data provide insights into the possible evolutionary changes in WRI1 homologs and allow for identification of new targets to enhance oil biosynthesis in diverse tissues.

Mestrado em Biomedicina Molecular<br>Familial amyloid polyneuropathy (FAP) or paramiloidosis is an autosomal dominant neurodegenerative disease with onset on adult age that is characterized by mutated protein deposition in the form of amyloid substance. FAP is due to a point alteration in the transthyretin (TTR) gene and until now more than 100 amyloidogenic mutations have been described in TTR gene. FAP shows a wide variation in age-at-onset (AO) (19-82 years, in Portuguese cases) and the V30M mutation often runs through several generation of asymptomatic carriers, before expressing in a proband, but the protective effect disappear in a single generation, with offspring of late-onset cases having early onset. V30M mutation does not explain alone the symptoms and AO variability of the disease observed in the same family. Our aim in this study was to identify genetic factors associated with AO variability and reduced penetrance which can have important clinical implications. To accomplish this we genotyped 230 individuals, using a directautomated sequencing approach in order to identify possible genetic modifiers within the TTR locus. After genotyping, we assessed a putative association of the SNPs found with AO and an intensive in silico analysis was performed in order to understand a possible regulation of gene expression. Although we did not find any significant association between SNPs and AO, we found very interesting and unreported results in the in silico analysis since we observed some alterations in the mechanism of splicing, transcription factors binding and miRNAs binding. All of these mechanisms when altered can lead to dysregulation of gene expression, which can have an impact in AO and phenotypic variability. These putative mechanisms of regulation of gene expression within the TTR gene could be used in the future as potential therapeutical targets, and could improve genetic counselling and follow-up of mutation carriers.<br>A Polineuropatia amiloidótica familiar (FAP) ou paramiloidose é uma doença neurodegenerativa autossómica dominante com início na vida adulta sendo caracterizada pela deposição da proteína mutada na forma de substância amilóide. A FAP é devida a uma mutação pontual no gene transtirretina (TTR) e até agora mais de 100 mutações amiloidogénicas foram descritas neste gene. A FAP apresenta uma grande variação na idade de início (AO) (19-82 anos, nos casos portugueses) e a mutação V30M pode segregar através de várias gerações de portadores assintomáticos, antes de se expressar num probando. No entanto, este efeito protetor pode desaparecer numa única geração, com os filhos de casos tardios a apresentarem um início precoce. A mutação V30M não explica por si só os sintomas e a variabilidade da AO observada dentro de uma mesma família. O nosso objetivo neste trabalho foi identificar fatores genéticos associados com a variabilidade da AO e a penetrância reduzida. De modo a cumprir este objetivo genotipámos 230 doentes, por sequenciação automática, para identificar possíveis modificadores genéticos dentro do locus da TTR. Após a genotipagem, investigamos uma possível associação dos SNPs encontrados com a AO e realizamos uma intensiva análise in silico de modo a perceber uma possível regulação da expressão génica. Apesar de não termos encontrado nenhuma associação entre os SNPs e a AO, encontrámos resultados não descritos e muito interessantes na análise in silico dado termos observado algumas alterações a nível do mecanismo de splicing, ligação de fatores de transcrição e ligação de miRNAs. Todos estes mecanismos quando alterados podem levar à desregulação da expressão do gene, o que pode ter um impacto na AO e variabilidade fenotípica. Estes mecanismos hipotéticos da regulação da expressão génica no gene da TTR podem ser úteis para no futuro serem aplicados como potenciais alvos terapêuticos, beneficiando o aconselhamento genético e o follow-up dos portadores da mutação.

Soybean seed lectin, Le1, is specifically located in seeds of soybean, Glycine max, (L.) Merr., due to its promoter. Gene homologues of Le1 were previously identified as possibly located in other parts of soybean. We cloned two novel promoters from these genes, and show that they drive reporter gene expression in transgenic Arabidopsis. A total of 1.3kb was isolated from each of the Le2 and Le3 5' promoter regions and fused with the GUS reporter gene. A previously cloned Le1 5' promoter was used as a control and the constructs were introduced into Arabidospis. GUS expression in transformed plants reveals that GUS driven by Le3 is found predominantly in vegetative tissues whereas GUS driven by Le2 show low expression in all tissues examined. The expression patterns resulting from the three different lectin promoters are distinct and consistent with regulatory motifs computationally identified in the sequences.<br>Chez le soja (Glycine max), le promoteur du gene lectine Le1 dirige l'expression spécifique dans les graines. Des homologues de Le1 existent dans le genome du soja et sont exprimées ailleurs dans la plante. Nous avons isolé deux promoteurs de ces homologues de lectine, et décrivons le patron d'expression qu'ils dirigent. Un total de 1.3 kilobase des regions 5' des promoteurs, en amont du gène, a été isolé pour chacune des copies Le2 et Le3, et fusionné avec le gène rapporteur GUS. Le promoteur de Le1 étant déjà connu, il sert de controle. L'Arabidopsis transformée avec ces constructions, montre que le promoteur de Le3 dirige l'expression dans les tissues végétatifs, tandis que le promoteur de Le2 procure un niveau minimal d'expression dans tous les tissus examinés. De plus, des analyses bioinformatiques identifient des motifs spécifiques dans les sequences de promoteurs qui confirment les patrons d'expression que nous avons démontrés.