Rozprawy doktorskie na temat „SILICO ANALYSIS”

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.

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.
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.
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
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.
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.
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.

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 oil-rich 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, we further demonstrated that both PaWRI1 and PaWRI2 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.

DNA barcoding is a method used for the identification and discovery of animal species. It usually involved a 648 base pair fragment of the mitochondrial cytochrome c oxidase subunit I, known as COI. This work is focused on the study of the genetic identification in the families belonging to the order Pleuronectiformes, commonly known as flatfish, and the accuracy of the genetic marker most used for the study of their DNA barcodes. The results indicate possible existence of taxonomical mistakes because several families do not show a gap between maximum intraspecific distance - which is the maximum distance within a specie - and the minimum interspecific distance - which is the minimum distance between a species and its nearest neighbor (NN), meaning that the marker in use cannot reliably distinguish among those species. This study uses a bioinformatic approach to design new Pleuronectiformes barcodes and compares their coverage and resolving power with that of existing barcodes. The new primers, proposed by the program EcoPrimer, are based on two indices that estimate the resolution capacity of the barcodes and the taxonomic coverage of them, for the amplification. The performances of both barcoding regions already in use (COI and 16 rDNA genes), and the new primer pairs designed, were performed through a ‘in silico PCR’. The results show that the new primer pairs, located in a different regions of 16S rDNA gene compared to the universal barcode region used in fishes, present best resolution capacity and taxonomic coverage than the others already in use. This is an essential complement for future barcoding studies.

Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. This thesis presents the results of several computational and experimental studies of the CTLD structure, function and evolution.¶ Core structural properties of the CTLD fold were explored in a comparative analysis of the 37 distinct CTLD structures available publicly, which demonstrate significant structural conservation despite low or undetectable sequence similarity. Pairwise structural alignments of all CTLD structures were created with three different methods (DALI, CE and LOCK) and analysed manually and using a computational algorithm developed for this purpose. The analysis revealed a set of conserved positions and interactions, which were classified based on their role in CTLD structure maintenance.¶ The CTLD family is large and diverse. To organize and annotate the several thousand of known CTLD-containing protein sequences and integrate the information on their evolution, structure and function a local database and a web-based interface to it were developed. The software is written in Perl, is based on bioperl, bioperl-db and Apache::ASP modules, and can be used for collaborative annotation of any collection of phylogenetically related sequences.¶ Several studies of CTLD genomics were performed. In one such study, carried out in collaboration with the RIKEN structural genomics centre, CTLD sequences from the Caenorhabditis elegans genome were identified and clustered into groups based on similarity. The most representative members of the groups were then selected, which if characterized structurally would tell most about the C. elegans CTLDs and provide templates for homology modelling of all C. elegans CTLD structures.¶ In the other whole-genome study, the CTLD family in the puffer fish Fugu rubripes was analysed using the draft genome sequence. This work extended and complemented three genome-level surveys on human, C. elegans and D. melanogaster reported previously. The study showed that the CTLD repertoire of Fugu rubripes is very similar to that of mammals, although several interesting differences exist, and that Fugu CTLD-encoding genes are selectively duplicated in a manner suggesting an ancient large-scale duplication event. Another important finding was the identification of several new CTLDcps, which had mammalian orthologues not recognized previously.¶ CBCP, a novel CTLD-containing protein highly conserved between fish and mammals with previously unknown domain architecture, was predicted in the Fugu study based solely on ab initio gene models from the Fugu locus and cross-species genomic DNA alignments. To test if the prediction was correct, a full-length cDNA of the mouse CBCP was cloned, its tissue distribution characterized and untranslated regions determined by RACE. The full-length mCBCP transcript is 10 kb long, encodes a protein of 2172 amino acids and confirms the original prediction. The presence of a large N-terminal NG2 domain makes CBCP a member of a small but very interesting family of Metazoan proteins.

Thesis (Ph.D.)--Australian National University, 2004.
"This CD contains the software (mostly written in Perl) used for comparative structure analysis reported in Chapter 4 (str_comp directory), and for the CTLD database system described in Chapter 2 (CTLD_DB directory)."

This thesis collects the results of three different research projects carried out in collaboration with the University of Parma. In chapter 2 the identification and characterization of antimicrobial compounds active as protein-protein interaction inhibitors of bacterial RNA polymerase subunit β' and transcription initiation factor σ is described. This peculiar protein-protein interaction has been identified as a promising target for antibacterial research and some small molecules were identified as disruptors of this interaction. In this project, a virtual screening campaign, aimed to the identification of novel antimicrobial molecules acting disrupting β'-σ interaction, was carried out. The newly identified compounds were experimentally tested in different assays, confirming their activity as PPI disruptors and highlighting their antimicrobial potential. A pharmacophore model was built for the most promising compounds to identify common patterns of interactions which were necessary for the activity and a binding hypothesis was proposed for each active compound that could be exploited as starting point for further optimization and for the discovery of new molecules. In the third chapter modelling studies were applied to NAPE-PLD, an enzyme responsible for the synthesis of bioactive lipids involved in the regulation of several physiological and pathological conditions. Modulation of NAPE-PLD activity could represent a promising therapeutic strategy for a wide range of diseases but, despite the important role played by this enzyme, no new active molecules have been reported so far. This work describes the identification and characterization of the first small molecule inhibitor of NAPE-PLD. Docking studies were performed, thanks to the availability of NAPE-PLD crystal structure, highlighting the binding pose of the compound and they were confirmed by SAR studies and mutagenesis experiments. Despite its low potency, this molecule represents a molecular probe which can help in better characterize and investigate NAPE-PLD mechanisms and its role in pathologic and physiological processes. NAPE-PLD together with other lipases, is characterized by a mechanism called “interfacial activation” which affects enzyme conformation and its catalytic machinery. The understanding of this phenomenon could be crucial to gain insight into enzyme’s behavior highlighting aspects of the catalytic mechanism that couldn’t be revealed by the X-ray structure alone. Molecular dynamics simulations were thus performed on the apo form of NAPE-PLD to better understand the conformational changes of the enzyme in both aqueous and membrane environment. Results highlights that the membrane environment stabilizes the open conformation, which is compatible with substrate recruitment, while water stabilizes a closed that block the access of the substrate to the active site, confirming the interfacial activation phenomenon. The last chapter of this thesis describes the statistical analysis that was performed on a large database of activity data collected by testing 400 compounds against Schistosoma mansoni, a parasite responsible for schistosomiasis, a tropical neglected disease. Three organizations, the University of California San Diego (UCSD), the Swiss Tropical and Public Health Institute (STPH) and Fiocruz (Fundação Oswaldo Cruz) Foundation in Brazil have experimentally tested the Pathogen Box, a collection of molecules active against different pathogens, monitoring the effects on the helminth. Different assays were performed and the results were collected using different numerical ranges, thus a mathematical transformation of the data was applied to the dataset to obtain activity values in the same scale. This was necessary to perform a statistical analysis to evaluate coherence of the collected data and to identify the most promising compounds.
Questa tesi di dottorato raccoglie i risultati di tre diversi progetti di ricerca che ho svolto in collaborazione con l’Università di Parma. In particolare, il capitolo 2 descrive l’identificazione e la caratterizzazione di composti con attività antimicrobica che agiscono come inibitori dell’interazione proteina-proteina tra subunitá β' e fattore di trascrizione σ nell’RNA polimerasi batterica. Evidenze sperimentali hanno dimostrato che l’interfaccia tra le due proteine rappresenta un sito di legame per piccole molecole e composti che legano questa regione sono in grado di inibire l’interazione proteina-proteina arrestando la trascrizione e mostrando un’attività antibatterica. L’obiettivo di questo lavoro è stato identificare, tramite un protocollo di virtual-screening, all’interno di una libreria di composti, un sottoinsieme di molecole attive come inibitori dell’interazione β'-σ. Test sperimentali sono poi stati eseguiti sui composti più promettenti confermando l’attività per alcuni e provando la loro efficacia come composti antibatterici. Un modello farmacoforico è stato poi costruito per razionalizzare la relazione tra struttura e attività e si è inoltre ipotizzata una modalità di legame per i composti attivi con la subunità target β' che potrà essere utilizzata come punto di partenza per lo screening di nuove librerie o per la progettazione di nuovi composti. Nel capitolo 3 vengono invece descritti studi di modellistica molecolare effettuati sull’enzima NAPE-PLD. Questa lipasi di membrana è responsabile della produzione di lipidi bioattivi coinvolti in diversi processi fisiologici e patologici e la sua modulazione risulta essere particolarmente importante nel trattamento di diverse patologie. La disponibilità della struttura cristallografica dell’enzima umano ha reso possibile effettuare studi di docking per ipotizzare le modalità di legame della prima molecola capace di inibire NAPE-PLD, identificata mediante HTS. La posa di docking ottenuta risulta compatibile con i dati SAR in nostro possesso ed è inoltre confermata da studi di mutagenesi. Nonostante l’attività inibitoria limitata del composto esso può essere considerato il punto di partenza per sviluppare nuovi inibitori. Sono poi state effettuate simulazioni di dinamica molecolare per valutare i cambiamenti conformazionali dell’enzima in due ambienti differenti: in presenza di solvente acquoso oppure in presenza di membrana cellulare per testare l’ipotesi di “attivazione interfacciale”, un fenomeno caratteristico delle lipasi. Questo meccanismo è caratterizzato dall’equilibrio conformazionale tra uno stato definito “aperto”, compatibile con l’accesso del substrato e uno stato “chiuso” in cui l’accesso del substrato è bloccato. Durante il tempo di una simulazione di dinamica molecolare si sono potuti evidenziare cambiamenti conformazionali della proteina compatibili con questa teoria permettendoci di ipotizzare un meccanismo di “recruitment” del substrato quando l’enzima si trova in presenza della membrana cellulare. L’ultimo capitolo descrive il progetto di ricerca che ho seguito presso la University of California San Diego: qui ho svolto un lavoro di analisi di dati di attività biologica ottenuti testando 400 composti di una libreria chiamata Pathogen Box sul platelminta Schistosoma mansoni per identificare composti attivi contro la schistosomiasi, una parassitosi comune nei paesi sottosviluppati. Tre organizzazioni, la University of California San Diego (UCSD), il Swiss Tropical and Public Health Institute di Basilea (STPH) e la fondazione brasiliana Fiocruz (Fundação Oswaldo Cruz) hanno eseguito diversi saggi collezionando una grande quantità di dati di attività che ho successivamente analizzato per verificare diversi aspetti tra cui l’identificazione dei composti più potenti e la coerenza dei dati raccolti tra le diverse organizzazioni.

A potential path to the development of small-molecule inhibitors is to identify small-molecules that mimic the interactions of short peptides with proteins. The present study uses Perl scripts and a MySQL database to build a unique dataset of 258 protein-peptide interactions (ProPep) from structures contained in the Protein Data Bank. The physiochemical and structural nature of protein-peptide interfaces were analysed in part using a novel amino acid pictogram analysis alongside accessible surface area, residue pairing and amino acid composition analysis. The results indicate that, for the peptide, proline residues and tyrosine residues play specific roles in protein-peptide interfaces. Furthermore it was observed that the peptide residues are significantly more buried than the residues of the cognate protein surface. The virtual screening program LIDAEUS was used to mine chemical databases to identify novel peptidomimetic compounds that have evincible binding to protein targets of therapeutic interest. Target-based and fragment-based virtual screening identified a series of potential compounds targeting the interaction between p21 and PCNA. Whilst the docking results were promising, results from testing in biological assays were inconclusive. A target-based virtual screening approach to identify small-molecule mimics of the interaction between the GnRH peptide and GnRH receptor yielded two promising compounds that demonstrated weak binding in biological assays. A third study to identify small-molecules binding to the SH3 domain of PSD-95 produced some promising hit compounds that as yet have not been tested in binding assays.

N-acylethanolamines (NAEs) are a family of signaling lipids derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine (NAPE). In Arabidopsis, NAE mediates physiological functions such as seedling growth, flowering, and response to stress via abscisic acid (ABA) –dependent and –independent signaling pathways. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Studies in model plant Arabidopsis showed the significant role of NAEs that makes it relevant to elucidate the conserved metabolic pathway of NAEs in crop species such as tomato. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To test this hypothesis, AtFAAH was used as a template to identify putative FAAH sequences in tomato, using BLASTX. Six SlFAAH sequences with the conserved amidase signature sequence and the catalytic triad, formed by Lys205, Ser281, and Ser305 in AtFAAH, were identified. Phylogenetic analysis of putative SlFAAH homologs and other FAAH family proteins (Arabidopsis, rice and moss), using CLUSTALW, revealed the two sequences that are closely related to the functionally characterized AtFAAH1. Using molecular visualization system (PyMOL), protein structures of putative SlFAAH1and 2 were predicted and compared with AtFAAH; both sequences showed similar domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Furthermore, transcript levels for SlFAAH1 and SlFAAH2 will be quantified and correlated with the NAE levels in various tissues to predict their role in tissue-specific NAE hydrolysis. Together, these molecular and biochemical characterization studies in tomato are expected to further validate the conserved nature of NAE metabolic pathway in plants.

Megasatellites are polymorphic tandem repetitive sequences with repeat-units longer than or equal to 1000 base pairs. The novel algorithm Megasatfinder predicts megasatellites in the human genome. A structured method of analysing the algorithm is developed and conducted. The analysis method consists of six test scenarios. Scripts are created, which execute the algorithm using various parameter settings. Three nucleotide sequences are applied; a real sequence extracted from the human genome and two random sequences, generated using different base probabilities. Usability and accuracy are investigated, providing the user with confidence in the algorithm and its output. The results indicate that Megasatfinder is an excellent tool for the detection of megasatellites and that the generated results are highly reliable. The results of the complete analysis suggest alterations in the default parameter settings, presented as user guidelines, and state that artificially generated sequences are not applicable as models for real DNA in computational simulations.

The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer. Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form. A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes. A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place. The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization. In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand. Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor. In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs. In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR. The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA. The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article "Decreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.

The androgen receptor (AR) fulfils important roles for both sexes. By mediating the biological function of androgens, the AR has remained the target for endocrine therapies treating prostate cancer. The AR also determines the effectiveness of medroxyprogesterone acetate (MPA) in treating AR positive breast cancer. Every man will be affected by prostate cancer if he lives long enough. Prostate cancer continues to be a leading cause of death for males despite research into this cancer covering more than 60 years since Huggins' seminal 1941 study showing that androgens play a key role in this cancer. Unfortunately, significant advances have not been forthcoming and the effect of treatment has remained largely the same over past decades, whereby initial treatment provides temporary remission but eventually advanced cases become refractory to further intervention and the disease recurs in a more aggressive form. A plethora of factors are exquisitely sensitive to minute changes in the AR's structural profile, which can be altered by a single mutation, resulting in aberrant activity. A principal feature of these variant ARs associated with prostate cancer, is enhanced capacity to bind a number of molecules other than its cognate ligand, dihydrotestosterone (DHT). The promiscuous activity of this receptor leads to continued AR signalling and stimulus for the cancer despite low androgen levels induced by treatment regimes. A key question is whether mutations occurring within the AR occur as a result of cancer or contribute to the propagation of the cancer. Recent research has demonstrated that treatments incorporating anti-androgens such as flutamide, which are designed to impede prostate cancer progression by inhibiting AR activity, may actually provide selective pressure favouring somatic mutation of the receptor to take place. The specific changes to the AR which are responsible for gains of function have not been resolved as their crystal structures, which are used to provide conformational analysis of proteins, are tremendously problematic to produce with little success found in literature. Generating representative crystals of the AR protein involves producing soluble recombinant protein. Unfortunately the AR is prone to aggregation and is highly unstable, especially in the presence of antagonistic molecules or absence of a stabilising ligand, preventing the protein from being maintained in the soluble state required for crystallization. In order to produce sufficient quantities of soluble material for crystallization, the androgen receptor's ligand binding domain (LBD) was produced as a recombinant protein in Escherichia coli bacteria strain BL21 (DE3) in the presence of DHT, flutamide, as well as in the absence of ligand. Since soluble unbound AR-LBD has not been produced until now, the bacterial culture containing no ligand was further processed to the stage of cleaving the purification tag from the recombinant protein and represents considerable progress into producing soluble material for crystallizing the troublesome yet considerably important AR in the absence of ligand. Although distinct from prostate cancer in males, AR activity in breast tissue is also a factor determining the action of drugs, such as MPA, included in therapies aimed at breast cancer. The use of MPA has declined primarily due to its adverse effects including unsuccessful generation of a biological response, as well as the advent of other drugs administered for hormonal therapies treating breast cancer. Alternative drugs are needed when breast cancer therapies fail as tumours develop resistance to primary drugs. Although there are a number of drugs on the market, success would be maximised if the determined therapy is matched with the patient, based for example, on their genetic makeup. There is a conundrum whereby some patients with an AR do not respond to MPA, a drug normally recognised by the receptor. In clinical trials it was discovered that an AR with threonine instead of methionine at residue 780 (M780T) fails to activate in response to MPA, but the exact mechanism has remained elusive and needs to be answered at the molecular level. The X-ray crystallographic studies that generate 3D images of macromolecules and wet chemistry, which have traditionally been used to provide insight into science in these dimensions, are incorporated with computer based molecular simulation. This is both complementary and distinct to traditional scientific methodologies, enabling further elucidation of protein-protein interactions, and the influence applied to such inter-relations by natural and drug ligands. This approach has been used, and is continually developed, to understand the binding mechanisms of current drugs as well as designing new drugs. In order to produce a receptor representing the M780T variant, the crystal structure representing the AR-LBD was mutated in silico, into which MPA was then docked. It was found that MPA binds into the M780T AR-LBD with considerably more spatial displacement compared to the position of DHT in the crystal structure, and is predicted to be the primary reason for the inability of MPA to activate this variant AR. The clarification of MPA binding and failure to elicit a response from the variant AR is significant for a cohort of breast cancer patients, as not only does the presence of an AR in the tumour determine the effectiveness of MPA, but correct composition of the AR, specifically, the absence of a M780T mutation. In the absence of this AR mutation, MPA could effectively be used either as an alternative to primary drugs, or in secondary therapies when primary therapies fail. Aberrant activity of variant ARs in response to MPA should also be taken into consideration when analysing drug studies about the effectiveness of MPA. The findings on the loss of response to MPA by the M780T variant AR have been included in the journal article "Decreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate" appearing in the September 2005 issue of Cancer Research journal.

Today new drugs are tested on cell cultures in wells to minimize time, cost, andanimal testing. The cells are studied using microscopy in different ways and fluorescentprobes are used to study finer details than the light microscopy can observe.This is an invasive method, so instead of molecular analysis, imaging can be used.In this project, phase-contrast microscopy images of cells together with fluorescentmicroscopy images were used. We use Machine Learning to predict the fluorescentimages from the light microscopy images using a strategy called In-Silico Labeling.A Convolutional Neural Network called U-Net was trained and showed good resultson two different datasets. Pixel-wise regression, pixel-wise classification, andimage classification with one cell in each image was tested. The image classificationwas the most difficult part due to difficulties assigning good quality labels tosingle cells. Pixel-wise regression showed the best result.

High grade serous (HGS) ovarian cancer and pancreatic ductal adenocarcinoma (PDAC) are poor prognosis neoplasms that are characterised by high levels of genomic instability, DNA damage repair (DDR) defects, and drug resistance which ultimately results in poor survival rates. This study aims to use markers of genomic instability, in the form of copy number variations (CNVs) and gene expression changes, to identify DDR gene aberrations which contribute to poor patient prognosis and drug resistance in HGS ovarian cancer and PDAC, and which may be exploited for therapeutic benefit. Bioinformatic analyses of HGS ovarian cancer patient datasets (TCGA, Genome Institute of Singapore) were used to determine whether gene pairs co-amplified together, where one of the partners is a DDR gene, associated with poor patient prognosis. RECQL, a DNA helicase, was found to be co-amplified with 6 genes from the 3q12 locus, CPOX, MINA, TMEM45A, TOMM70A, CLDND1 and PTPLB, and all of these co-amplifications were found to significantly associate with reduced PFS in ovarian cancer patients. Functional validation, using siRNA to inhibit gene expression, was carried out to assess the role of these genes in regulating survival. Using a cell line (PEO4) model of chemotherapy resistant HGS ovarian cancer, no induction of apoptosis or changes in long term survival was observed upon silencing of any of the genes. In comparison, gene silencing of RECQL and PTPLB was found to induce an apoptotic response in PDAC cell lines (Panc1 and MiaPaCa2). Long term clonogenic assays also revealed that TMEM45A, TOMM70A and CLDND1 are regulators of Panc-1 cell survival. Together this indicates that in PDAC, the survival advantages of RECQL co-amplification with the 3q12 chromosomal region may be related to the effects of more than one gene. However, the PTPLB:RECQL co-amplification may be a driver of PDAC cell survival. Bioinformatic analyses were also carried out on HGS ovarian cancer and PDAC patient datasets (TGCA and ICGC) as well as cell line datasets (Sanger Institute) aimed at determining single genes within DDR pathways whose aberrations in copy number and gene expression impacts upon patient survival or drug response to cisplatin or gemcitabine. 6 genes, TSTA3, RECQL4, ESRP1, NBN, SUMO3 and EP300, were identified that affected one of the above parameters, of which, EP300 functionally validated as a potential therapeutic target. SiRNA-mediated silencing of this was found to induce apoptosis in cell line models of HGS ovarian cancer (SKOV3) and PDAC (Panc-1). In addition, loss of EP300 increased the apoptotic response of SKOV3 cells to treatment with cisplatin, gemcitabine, doxorubicin, paclitaxel and the DNA-PKcs inhibitor NU7441. In Panc1 cells, only response to gemcitabine and paclitaxel was significantly increased with EP300 loss. Mechanistic studies to define how EP300 regulates SKOV3 cell survival found that EP300 gene silencing induced dysregulated DNA damage recognition, and cell cycle arrest at the G2/M phase. RPPA proteomic analysis identified Wee1, Plk1, cyclin B, MAPK1 and histone 3 as potential mediators of the cell cycle arrest observed. Many changes to apoptotic signalling were also observed after loss of EP300, although this counter-intuitively, increased the levels of anti-apoptotic proteins, such as Bcl-xl, Bcl-2 and pS136 Bad. Also, pro-survival Akt activity, determined as phosphorylation at S473, was reduced by EP300 gene silencing. This indicates a novel link between p300 and Akt-mediated cell survival of HGS ovarian cancer. Overall, this study has combined in silico analyses of patient and cell line datasets with functional in vitro validation to identify RECQL and EP300 as potential therapeutic targets for HGS ovarian cancer and PDAC. Future work to validate these in additional cell line models, including those with defined genomic backgrounds, will further progress these as novel therapeutic strategies.

The intrinsic stochasticity of biomolecular systems is a well studied phe- nomenon. Less attention has been paied to other sources of variability, so called extrinsic noise. While the precise definition of extrinsic noise de- pends on the system in question, it affects all cells and its significance has been demonstrated experimentally. Information theory provides a rigorous mathematical framework for quan- tifying both the amount of information available to a signalling system and its ability to transmit this information. Intracellular signal transduction re- mains a relatively unexplored frontier for the application of information theory. In this thesis, we rely on a metric called mutual information to quantify in- formation flow in models of biochemical signalling systems. After briefly discussing the theoretical background and some of the practical difficulties of estimating mutual information in Chapter 2, we apply it in the context of simplified models of intracellular signalling, referred to as motifs. Using a comprehensive set of two-node motifs we explore the effects of extrinsic noise, model parameters and various combinations of interaction, on the system's ability to transmit information about an input signal, repre- sented by a telegraph process. Our results illustrate the importance of the system's response time and demonstrate a trade-off in transmitting infor- mation about the current state of the input or its average intensity over a period of time. In Chapter 4, we address the problem of determining the magnitude of ex- trinsic noise in the presence of intrinsic stochasticity. Using the Approximate Bayesian Computation - sequential Monte Carlo algorithm, together with published experimental data, we infer parameters describing extrinsic noise in a model of E. coli gene expression. Lastly, in Chapter 5, we construct and analyse models of bacterial two- component signalling, bringing together insights gleaned from earlier work. The results show how the abundances of different molecular species in the system may transmit information about the input signal despite its stochas-tic nature and considerable variation in the numbers of protein molecules present.

In this decade, establishing structure-function relationships in human brain has become one of the most influential concepts in modern cognitive neuroscience since interactions among cerebral components are fundamental to explain cortical activities ([1]; [2]; [3]). In literature such relationships have been defined in terms of structural, functional and effective connectivity. This distinction, mainly focused on the theoretic concept, is also related to the different measurement instruments and analytical tools used for acquiring and processing the data. The structural connectivity refers to a pattern of anatomical links among brain regions. Its analysis aims to characterize the architecture of complex networks underlying the cerebral functional organization. Magnetic Resonance Imaging and especially Diffusion Tensor Imaging can be used to convey information concerning the physical connection between neuronal populations. Functional/effective connectivity aims at identifying the presence and the strength of connections in terms of statistically significant dependency. The former is defined as the temporal correlation between neurophysiological events occurring in distributed neuronal groups and areas. The latter describes the causal influence that one neural system exerts over another either directly or indirectly in terms of temporal precedence and physical control ([4];[5]). Functional and effective connectivity can be estimated exploiting both Functional Magnetic Resonance Imaging (fMRI) and electrophysiological signals, such as Electroencephalography (EEG) and Magnetoencephalography (MEG), with different advantages and drawbacks, respectively. fMRI provides high spatial resolution (mm) but poor temporal precision (s) while EEG/MEG has more limited spatial resolution (cm) and higher temporal precision (ms). Because functional and effective connectivity are largely estimated over time, EEG and MEG are more suitable for calculating such connectivity. In literature several methods have been developed to characterize brain connectivity in terms of network topology, connections strength and causality, following two main approaches: the data-driven, where topology, causality and strength are all inferred from data, and the neural model-based, where the model topology is postulated from a priori knowledge and only the connections strength is estimated from the data. - Data driven approach. The data driven approach includes linear, non-linear and information-based techniques. The linear ones provide a battery of indices derived by multivariate autoregressive models (MVAR) based on Granger causality principles ([6]) or MVAR frequency response ([7]). Such are Ordinary Coherence, Partial Coherence, Directed Transfer Function (DTF) and Partial Directed Coherence (PDC). These indexes measure the strength of the linear coupling between two signals; in addition DTF and PDC provide information about causal influence ([8]). o Among the non-linear techniques, phase synchronization has been shown to be very effcient in detecting interactions between oscillators. The phase locking values approach assumes that two dynamic systems may have their phases synchronized even if their amplitude are zero correlates ([9]). o The most representative information-based technique is the cross mutual information that measures the mutual dependence between two signals by quantifying the amount of information gained about one signal from measuring the other, as a function of delay between these two signals ([10]). - Neural model based approach. Representative methods are the Structural Equations Modelling (SEM) and the Dynamic Causal Modelling (DCM) ([11]; [12]). They are multivariate technique used to test hypothesis regarding the influences among interacting variables, but different concepts underlies these two methods. SEM approach assumes that neuronal dynamics are very fast in relation to signals uctuations and, hence, is based on a static neuronal model. This case, the neuronal activity has reached steady-state and changes in connectivity are led directly by changes in the covariance structure of the observed time series ([13]). On the other hand, in DCM the observed time series are modelled as a deterministic dynamical system in which external inputs causes changes in neural activity and therefore in connectivity values ([14]). Most approaches, like those based on Granger causality principles, have been examined in literature to quantify their ability in revealing cerebral connections ([15];[16]; [11]) but their simulation studies do not provide a comprehensive analysis because they use in silico data generated by self-referential linear methods which do not reproduce the complexity of brain. To overcome this issue, an innovative simulation approach has been developed in this work, based on a nonlinear neural mass model ([17]) totally independent of SEM and MVAR linear equation and able to address the complexity of neural networks. This no-self referential approach was exploited to generate in silico network data to be used as a benchmark, to quantitatively compare obtained results with true connections. The main objective of this work was to understand limits and advantages of MVAR indexes and SEM by exploiting the simulation study. Thus, it mainly serves as a proof-of-concept for connectivity measures under ideal conditions. Our purpose was to derive from simulation results some practical procedures in order to classify different brain states to support both cognitive research and clinical activity. First, research activity was focused to address connectivity on simulated data obtained on three regions networks characterized by different strength connections and based on different levels of non linearity. Second, a dataset, made available by Department of Medicine, University of Padova was used to explore application of these methods to real data by applying the simulation study suggestions. This thesis consists of three main section. The ffrst one includes Chapter 1-2-3 describing in detailed the considered connectivity measures, such are those based on Multivariate Autoregressive models and the Structural Equation Modelling, and the simulation study. The second part depicts in silico results and the application to EEG data. Finally, comments are reported in Discussion and Conclusions. Chapter 1 explains how the connecting parameters of MVAR and SEM models are identified on EEG data and describes procedures commonly exploited to analyse connectivity. Chapter 2 reports an overview about the principal models used to generate in silico data, namely the neural mass models, and described the neural mass model exploited in this work. Finally, it characterizes network models adopted to simulate data and lists the procedure followed to generate in silico datasets. Chapter 3 summarizes the computations implemented to have more insights on our data by analysing the output of each methods. It describes the procedure used to evaluate the statistical signiffcance of each index results, such are the F-test for Granger causality index and the null distribution threshold using surrogate data for MVAR frequency indexes. Chapter 4 illustrates the results obtained with the simulation study. First, we reported the complete analysis for a representative subset of experiments, then for all datasets we showed topology and strength estimates. Chapter 6 delineates the procedure followed to study the connectivity in case of hepatic encephalopathy. Chapter 7 covers the Discussion and Conclusions. The Appendix is a parallel work aimed to understand the meaning of connectivity indexes computed via Structural Equation Modelling. By exploiting the neural mass model used to simulate cortical data, the objective is to quantify which measure its estimates represent. We demonstrated that Granger causality is a good estimator with high values both of sensitivity and specificity, while frequency indexes, DTF and PDC, are too much affected by the threshold choice and their interpretation in terms of absolute strength connection is not clear. As regard SEM, we proved the difficulty of its approach to describe just simple situations. Even if SEM is based on linear regression as well as MVAR models, it differently assumes there is no connection with past information, as if brain connectivity could describe time series relationships by the instant we observe it. Hence, it is not sufficiently robust to characterize neuronal dynamic activity.
Negli ultimi decenni le varie tecniche e metodiche sviluppate per lo studio dell'attività cerebrale hanno dimostrato che le diverse regioni neuronali del cervello non operano in isolamento ma interagiscono tra loro formando una complessa rete di connessioni. Lo studio di queste relazioni/connessioni esistenti tra le diverse regioni corticali, tramite l'elaborazione sia di segnali elettrofisiologici, come l'EEG, sia di immagini, come l'fMRI, è generalmente denominato come studio della connettività. La definizione di connettività può essere classificata in tre principali categorie: anatomica, funzionale ed effettiva. La connettività anatomica e strettamente associata alla presenza di connessioni assoniche tra i vari neuroni; la connettività funzionale e definita come la correlazione temporale tra eventi neurofisiologici appartenenti a diverse regioni neuronali; la connettività effettiva è definita come l'influenza che una regione neuronale esercita attraverso una relazione causaeffetto su un'altra regione. In letteratura sono presenti due principali approcci per lo studio della connettività: l'uno di tipo esplorativo, basato esclusivamente sui dati da cui estrarre informazioni sia sulla topologia sia sulla forza; l'altro che prevede la conoscenza a priori di un modello di rete per ottenere informazioni circa l'intensità degli accoppiamenti. L'obiettivo di questa tesi si e focalizzato sulla validazione e implementazione di alcuni dei metodi pi u utilizzati: quelli basati sui modelli autoregressivi multivariati(MVAR), come la Directed Transfer Function (DTF), la Partial Directed Coherence (PDC), e sui principi della causalità di Granger e il metodo detto Structural Equation Modeling (SEM). Questi metodi sono ampiamente esaminati in letteratura per quantificare la loro capacità di rilevare le connessioni cerebrali, ma gli studi di simulazione proposti sono basati su modelli di generazione dei dati in silico che semplificano molto la reale complessità del cervello [15] e che si basano sui modelli autoregressivi stessi. Per superare questo problema e stata sviluppata una simulazione con un approccio innovativo basato sull'utilizzo di un Neural Mass Model[17]. L' obiettivo consiste nel generare dati simulati completamente indipendenti dalle equazioni lineari dei metodi che poi si vanno a testare e, al contempo, in grado di simulare la complessità delle reti neurali. Brevemente, la simulazione consiste delle seguenti fasi: - diversi set di dati in silico sono simulati utilizzando il modello neurale di massa con diversi modelli di topologia, livelli di non linearità e intensità di connessioni; - per ogni set dei suddetti parametri, 100 realizzazioni di segnali di 2 secondi vengono generati; - le reti stimate a partire dai parametri di connettività calcolati con i metodi considerati vengono confrontate con le reti vere. Per analizzare le prestazioni dell'indice di causalità di Granger e degli indici infrequenza Directed Transfer Function (DTF) e Partial Directed Coherence (PDC)sono state effettuate simulazioni Monte Carlo in modo da ottenere una statistica delle performance. Si è osservato che l'indice di Granger è il più affidabile con elevata percentuali di sensibilità e bassa frequenza di falsi positivi e negativi. Per analizzare la stima delle forze, sono stati confrontati i valori dei pesi imposti coni risultati degli indici dei metodi MVAR e le stime ottenute dal SEM mediante regressione lineare. Si e osservato che il SEM e il metodo meno affidabile, mentre i risultati ottenuti con gli indici MVAR presentano una buona correlazione lineare con i pesi veri. Anche in questo caso l'indice di Granger dà i migliori risultati correlando sempre con R > 0:99. I risultati hanno rivelato che l'indice di causalità di Granger è un accurato stimatore della topologia di rete in quanto si e dimostrato in accordo con le reti vere nella maggior parte degli esperimenti simulati, mentre DTF e PDC, oltre a presentare alcune imprecisioni, risultano più difficili da interpretare in termini di forze assolute. Questi risultati suggeriscono di utilizzare l'indice di causalità di Granger come strumento esplorativo per definire sia la topologia della rete sia l'intensità delle forze. Poi, le informazioni in frequenza provenienti dai diversi metodi (DTF, PDC) devono essere integrate per migliorare l'affidabilità dei risultati sulle intensità delle connessioni. L'obiettivo principale di questo studio di simulazione e quello di fornire una procedura robusta da usare per l'analisi della connettività del cervello umano, in grado di classificare i diversi stati del cervello in supporto sia della ricerca in ambito cognitivo e sia dell'attività clinica. L'analisi effettuata sui segnali EEG riportata e un esempio di applicazione a dati reali, in cui si esamina l'effetto dell'iperammonemia indotta da un carico amminoacidico su pazienti cirrotici e soggetti sani sulla riorganizzazione funzionale del segnale EEG (Dott. Amodio, Dipartimento di Medicina, Università degli Studidi Padova). Questa tesi si sviluppa in sei capitoli di seguito brevemente riassunti. Nel Capitolo 1 si definiscono sia i modelli multivariati autoregressivi e gli indici derivati per stimare la connettività in termini di causalità di Granger e nel dominio della frequenza, sia il metodo SEM. Nel Capitolo 2 si presenta il modello utilizzato perla generazione dei dati simulati analizzati in questa tesi e si descrivono le caratteristiche principali delle reti di simulazione considerate. Nel Capitolo 3 vengono descritti sia i metodi impiegati per la valutazione della significatività statistica dei vari stimatori sia la procedura per valutare l'accuratezza delle stime con il confronto sulle reti vere. Nel Capitolo 4 si presentano i principali risultati dello studio della connettività corticale ottenuti mostrando dapprima l'intera analisi su un sottoinsieme di simulazioni, poi sintetizzando i risultati su tutti i dataset. Nel Capitolo 5 viene presentata una possibile applicazione dei metodi prima esposti su un problema di tipo clinico, riguardante l'analisi di EEG su pazienti affetti da Encefalopatia epatica. Infine, nel Capitolo 6 si discutono i risultati presentati nel capitolo 5 evidenziando limiti e vantaggi dei vari metodi e il loro range di applicabilità in modo da visualizzare globalmente le loro prestazioni. L'Appendice riporta un lavoro parallelo eseguito per studiare il significato dei coefficienti di connettività stimati con il metodo SEM utilizzando le equazione del neural mass model descritto in precedenza.

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Essa dissertação teve o apoio financeiro do Edital Universal 14/2011 CNPq (nº processo: 483036/2011-0) e do Edital PROEX nº 04/2011.
Na tentativa de obter novas substâncias com atividade antibacteriana, o objetivo desse trabalho foi avaliar o potencial antibacteriano de quatro peptídeos sintetizados, dos quais dois possuem sequência derivada da fragmentação in silico do G-CSF humano, enquanto os outros dois foram planejados teoricamente, verificando, assim, seu interesse como novos agentes terapêuticos na saúde humana. A avaliação foi realizada em duas etapas: análise in silico, que consistiu em predições de propriedades e parâmetros associados à ação antibacteriana, por meio de ferramentas computacionais; e o experimento in vitro para a determinação da concentração inibitória mínima (MIC) dos peptídeos contra bactérias Gram positivas e negativas. A maioria das predições foi favorável para os quatro peptídeos, pois os resultados determinados para hidrofobicidade, anfipaticidade, tamanho, estrutura secundária, carga líquida, potencial de ligação em membrana, meia-vida e índice de Boman encontram-se dentro de valores desejados para o potencial antibacteriano. Na análise in silico, apenas a predição algorítmica da atividade antimicrobiana gerou resultados desfavoráveis para os peptídeos com sequências derivadas do G-CSF (peptídeos 1 e 2), porém essa mesma predição foi positiva para os outros dois. O ensaio in vitro demonstrou que até a concentração mais alta utilizada dos quatro peptídeos (500 μg/mL) foi insuficiente para a determinação de sua concentração inibitória mínima, porém, observou-se considerável diminuição no crescimento de E. coli (58,7%) pelo peptídeo 4 e de E. fecalis (86,1%) e E. coli (54,9%) pelo peptídeo 3, em comparação com o controle de viabilidade. Esses valores indicam a presença de ação antibacteriana por parte dos peptídeos planejados teoricamente (peptídeos 3 e 4), corroborando com as predições computacionais. Dessa forma, é possível concluir que a análise in silico foi de suma importância para a seleção dos peptídeos a serem sintetizados, os quais apresentaram nos ensaios in vitro resultados em concordância com a predição computacional de atividade antimicrobiana.
In attempt to obtain new substances with antibacterial activity, the aim of this study was to evaluate the antibacterial potential of four synthesized peptides, where two of them have sequence derived from human G-CSF in silico fragmentation, while the other two were theoretically planned, allowing the verification of their interest as new therapeutic agents at human health. The evaluation was performed in two stages: in silico analysis, consisting of predictions of properties and parameters associated with antibacterial effect, through computational tools; and the in vitro experiment for determination of the minimum inhibitory concentration (MIC) of the peptides against Gram positive and negative bacteria. Most predictions was favorable for all four peptides, showed by determined results of hydrophobicity, amphipathicity, size, secondary structure, net charge, membrane binding potential, half-life and Boman Index, considered as desirable values for antibacterial potential. In the in silico analysis, only algorithmic prediction of antimicrobial activity revealed unfavorable results for peptides with sequences derived from G-CSF (peptides 1 and 2), nonetheless, the predictions were positive for the other two. The in vitro assay showed that up to the highest concentration used of the four peptides (500 μg/mL) was insufficient for determination of minimum inhibitory concentration, however it was possible to observe significant growing decrease of E. coli (58.7%) by peptide 4 and E. fecalis (86.1%) and E. coli (54.9%) by peptide 3, when compared with the viability control. These values indicate the presence of antibacterial activity in the theoretically planned peptides (peptides 3 and 4), confirming the computational predictions. Thus, it is possible to conclude that the in silico analysis was very important for the selection of the peptides to be synthesized, which showed results of in vitro assays in agreement with the computational prediction of antimicrobial activity.

Regulation of protein solubility, or the ability of proteins to remain soluble within the cell, is an important part of protein homeostasis. This is highlighted with the disruption of protein homeostasis and dysregulation of solubility being associated with various neurodegenerative diseases. Using quantitative mass spectrometry and computational analyses, we identify low solubility proteins under unstressed conditions in three eukaryotic model systems: yeast cells, human neuroblastoma cells, and mouse brain tissue. Using an internal reference, we account for protein abundance, and allow for the analysis of proteins based on their partitioning between the soluble and insoluble fractions, rather than purely on their abundance within the insoluble fraction. We identified several intrinsic traits such as length, disorder, abundance, molecular recognition features, and low complexity regions which are correlated with protein solubility. These features have been previously shown to be associated with protein-protein interactions. This suggests that, under unstressed conditions, lower solubility in proteins may be linked to functional aggregation, rather than aberrant aggregation. We then present two predictors which may be used to predict the in vivo solubility of proteins, built using the many traits examined in this work. The linear regression model is able to give estimates of protein solubility, although proteins near the threshold between low and normal solubility may be misclassified. The Support Vector Machine is able to reliably distinguish between low and high solubility proteins, but is unable to reliably distinguish low and normal solubility proteins. We have identified several traits that distinguish low solubility proteins from other proteins, as well as developed two models that are able to estimate the solubility of proteins.
Science, Faculty of
Graduate

Recent advances in DNA sequencing technologies have led to a vast plethora of vertebrate genomes being made available for bioinformatic analysis and investigation. This has presented retrovirologists with many new opportunities to study endogenous retroviruses (ERVs) - selfish genetic element (SGEs) endogenised within the genomic DNA of their hosts. Many of these ERVs exist as molecular fossils of past germline infections by their exogenous counterparts, representing approximately 8-10% of mammalian genomes. While the majority are thought to be inactive today, one particular retroviral group - HERV-K(HML-2) - has been implicated in recent activity. In this thesis, efficient, synergistic in silico techniques have been implemented, with which intensive, genome-wide retroviral screens were performed. This has culminated in the identification of 11 novel, insertionally polymorphic human ERVs (HERVs), belonging to the HERV-K(HML-2) lineage, in two high- coverage archaic hominid genomes. This thesis also identifies the oldest ERV described to date - orthologous across all placental mammals - estimated to have endogenised in the germline of an ancestral mammal, 128-140 million years ago. Three SGEs, found to be endogenised within this ancient ERV, have also been described and assigned a minimum age of 104 million years, making these the oldest, definitively dated SGEs. This thesis also presents a computer program for renaming all identified ERVs in vertebrate genomes, according to a newly designed nomenclature standard to be implemented globally, that aims to unambiguously catalogue all the ERVs identified, to date.

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

L'augmentation du nombre de génomes totalement séquencés rend de plus en plus efficace l'étude des mécanismes évolutifs à partir de la comparaison de génomes contemporains. L'un des principaux problèmes réside dans la reconstruction d'architectures de génomes ancestraux plausibles afin d'apporter des hypothèses à la fois sur l'histoire des génomes existants et sur les mécanismes de leur formation. Toutes les méthodes de reconstruction ancestrale ne convergent pas nécessairement vers les mêmes résultats mais sont toutes basées sur les trois mêmes étapes : l'identification des marqueurs communs dans les génomes contemporains, la construction de cartes comparatives des génomes, et la réconciliation de ces cartes en utilisant le critère de parcimonie maximum. La qualité importante des données à analyser nécessite l'automatisation des traitements et résoudre ces problèmes représente de formidables challenges computationnels. Affiner le modèles et outils mathématiques existants par l'ajout de contraintes biologiques fortes rend les hypothèses établies biologiquement plus réalistes. Dans cette thèse, nous proposons une nouvelle méthode permettant d'identifier des marqueurs communs pour des espèces évolutivement distantes. Ensuite, nous appliquons sur les cartes comparatives reconstituées une nouvelle méthode pour la reconstruction d'architectures ancestrales basée sur les adjacences entre les marqueurs calculés et les distances génomiques entre les génomes contemporains. Enfin, après avoir corrigé l'algorithme existant permettant de déterminer une séquence optimale de réarrangements qui se sont produits durant l'évolution des génomes existants depuis leur ancêtre commun, nous proposons un nouvel outil appelé VIRAGE qui permet la visualisation animée des scénarios de réarrangements entre les espèces
Abstract

My research activity has been supported by the bioinformatic platform of the Fondazione Cariplo NOBEL Project named “Understanding DNA damage checkpoint and repair”. The aim of this platform was to investigate the molecular mechanisms involved in the DNA Damage Response (DDR), through a Systems Biology approach. Among the DDR pathways, the focus of my research was the DNA Damage Tolerance (DDT) pathway called Post-Replication Repair (PRR) using Saccharomyces cerevisiae cells as a model system. This pathway leads to the bypass of UV-induced DNA lesions through a mechanism, which is poorly characterized at molecular level in comparison with the Nucleotide Excision Repair (NER) process, the mechanism leading to the effective repair of UV-induces DNA lesions. The data presented in this thesis led to the production of the first mathematical model of PRR in eukaryotic cells. In the work carried out in this thesis I had the difficult task to manage both in silico and in vivo aspects of a Systems Biology project. Given the complexity of the PRR pathway and the lack of critical experimental data, the attempt to build a mathematical model of PRR was an ambitious aim not free from difficulties. Before this study, the crosstalk between PRR and other DDR pathways were unknown and, indeed, the discrepancy between the in vivo data and the in silico simulations, observed under certain experimental conditions, led to additional experiments that uncovered new unpublished aspects of PRR and others that need to be done. In this way the so called “Circle of Systems Biology” applied to PRR can be considered closed and promising for the future: the limit of the model to particular experimental conditions is leading to a new batch of experiments to do and new hypothesis to test.

Blastocystis sp., est un straménopile parasite anaérobie fréquemment rencontré dans le tractus gastro-intestinal de l’homme et de divers animaux. Ce microorganisme, parfois responsable de désordres digestifs aigus, pourrait conduire à des troubles fonctionnels intestinaux tels que le syndrome de l’intestin irritable (IBS). Le génome de Blastocystis sp., qui a fait l'objet d'un projet de séquençage en collaboration avec le Génoscope d’Evry, nous a permis de caractériser le plus petit génome de straménopile séquencé à ce jour (18,8 Mpb), avec une capacité codante de 6020 gènes. L’acquisition de nombreux gènes par transferts horizontaux est une caractéristique majeure de ce génome, qui montre d’abondants réarrangements génomiques. Bien qu’évoluant en anaérobiose, Blastocystis sp. possède des organites morphologiquement proches des mitochondries, appelés mitochondrion-like organelles (MLOs). Nous avons montré que ces organites comportaient un génome circulaire de type mitochondrial de 29,27 kpb, mais dépourvu des gènes codant pour les cytochromes. Des analyses in silico nous ont permis de caractériser le protéome des MLOs (365 protéines), conduisant à l’établissement d’un modèle prédictif des voies métaboliques associées à ces organites, avec notamment une chaine respiratoire limitée aux complexes I et II. Nous avons ainsi montré que les MLOs présentent des caractères communs aux mitochondries anaérobies et aux hydrogénosomes (présence d’une PFOR et d’une hydrogénase à fer), suggérant que Blastocystis sp. comporte des mitochondries anaérobies modifiées, qui résulteraient d’une adaptation du parasite à son environnement. Par ailleurs, la prédiction du sécrétome de Blastocystis sp. révèle la présence de facteurs de virulence potentiels, pouvant être impliqués dans l’altération de l’épithélium intestinal et le contournement du système immunitaire de l’hôte
Blastocystis sp. is a highly prevalent anaerobic eukaryotic stramenopile parasite found in the intestinal tract of humans and various animals. This microorganism, sometimes associated with acute intestinal disorders, could be responsible for functional intestinal disorders such as the irritable bowel syndrom (IBS). As part of a collaborative sequencing project with the Genoscope (CEA Evry, France), we were able to caracterize the smallest stramenopile genome sequenced to date (18.8 Mbp) with a 6020 genes coding capacity. The gain of many genes through horizontal gene transfer is amajor characteristic of this genome, which shows extensive genomic rearrangements. Despite the anaerobic nature of Blastocytists sp., this eukaryote harbours nevertheless mitochondrion-like organelles (MLOs). We have shown that these organelles have a 29.27 kbp mitochondrial-type circular genome that lacks cytochrome coding genes. In silico analysis allowed us to predict the MLOs proteome (365 proteins), with the subsequent predictive model of the metabolic pathways associated with these organelles, including an electron transport chain (ETC) restricted to complex I and II. We have shown that MLOs shared common characteristics with anaerobic mitochondrion and hydrogenosomes (presence of a PFOR and an iron-hydrogenase), which could mean that Blastocystis sp. harbours modified anaerobic mitochondrion that resulted from the parasite adaptation to its anaerobic environment. In addition, Blastocytis sp. secretome prediction reveals the presence of potential virulence factors, which could be involved in the degradation of the intestinal epithelium as well as the host immune system bypass

Avec l'accroissement du nombre de génomes séquencés, l'organisation de ces données brutes et des données dérivées, l'extraction de l'information et des connaissances associées défie l'imagination. La notion de voisinage a été d'abord été introduite pour l'organisation des données dans des bases de données relationnelles. Pour extraire des informations pertinentes à partir de données massives, différents types de voisinages ont été étudiés ici. Tout d'abord, avec l'analysedes correspondances (CA) et en utilisant le regroupement supervisé ("model clustering" MBC), la proximité mutuelle des éléments formant deux entités biologiques centrales, les gènes (codant les protéines) et les acides aminés a été analysée. Nous montrons par exemple que les protéines de Psychromonas ingrahamii, bactérie psychrophile extrêmes, sont regroupées en six classes, et qu'il y a une forte opposition entre le comportement de l'asparagine (N) et des acides aminés sensibles à l'oxygène, ce que nous expliquons en terms de résistance au froid. Ensuite, nous avons analysé la répartition entre les îlots génomiques (GI) et le squelette du génome de base à partir d'une nouvelle méthode combinant composition en bases et en gènes, caractéristiques GI et de briser les synténies. L'application de cette approche à E. coli et B. subtilis a révélé que cette nouvelle méthode permet d'extraire certaines régions significative, non publiées auparavant.Enfin, pour illustrer un voisinage fin, la régulation de l'expression d'un gène et son évolution, nous avons étudié la relation entre les régions en amont du gène et la zone codante du gène thrS de façon approfondie. Nous avons constaté que ces deux régions associées à un gène, se sont comportés différemment dans l'histoire évolutive. Certaines des régions en amont porteuses de la fonction non-essentielle de régulation (qui contrôle l'expression de gène) ont évoluédifféremment de la région codante
With more and more genomes being sequenced, the organization of those raw data and the derived data, the extraction of information and knowledge from these data has become a challenge. A key concept in this field is that of the neighborhood, especially with respect to the organization of data in relational databases. To extract information from bulk data, different kinds of neighborhoods were studied and each show interesting results in current study. .Firstly, through the Correspondence Analysis (CA) and later Model Based Clustering (MBC), two kinds of neighbors i.e. the genes (proteins) and amino acids were analyzed respectively, and it was found that proteins from Psychromonas ingrahamii are clustered into six classes, and there is strong opposition between asparagine (N) and the oxygen-sensitive amino acids. Secondly, the relationship between genomic islands and core genome (i.e. two closely linked neighbors withlarge range on the chromosome) was studied by a new method combining composition, GI features and synteny break. On applying to E. coli and B. subtilis it was revealed that this new method can extract some meaningful regions not published before. Thirdly, the relationship between upstream and coding regions of thrS gene (i.e. a case for two closely linked neighbors with small range on the chromosome) was studied extensively. It was found that these two regions associated to one gene, behaved differently in the evolutionary history.. Some of the upstream regions bearing non-essential function (i.e. regulation of gene expression) evolved more slowly than the coding region

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Diversos estudos tÃm evidenciado que a principal funÃÃo da proteÃna desacopladora mitocondrial de plantas (pUCP) està relacionada a regulaÃÃo de espÃcies reativas de oxigÃnio (EROs). AnÃlises in silico sugerem a existÃncia de famÃlias multigÃnicas para a codificaÃÃo de pUCPs, porÃm novos estudos ainda sÃo necessÃrios para estabelecer o perfil de expressÃo gÃnica das pUCPs, assim como a quantidade de genes em cada espÃcie e suas relaÃÃes filogenÃticas. O presente trabalho teve como objetivo caracterizar, analisar filogeneticamente e avaliar o perfil de expressÃo da famÃlia multigÃnica da pUCP em diferentes tecidos durante o desenvolvimento da soja [Glycine max (L.) MERR.] e em condiÃÃes de estresse. Foi realizada uma anÃlise in silico no genoma da soja e de outras leguminosas disponÃveis no banco de dados WGS, revelando uma famÃlia multigÃnica codificadora da pUCP, UCP1 e 2 com nove Ãxons, UCP 3 com 2 Ãxons, e UCP 4 e 5 com apenas um Ãxon. Dentre as leguminosas analisadas a soja se destacou com o maior nÃmero de genes, 10 genes no total, sendo quatro genes GmUCP1, uma GmUCP2, uma GmUCP3, dois GmUCP4 e dois GmUCP5, alÃm da presenÃa de um splicing alternativo no gene GmUCP1b1. Primers especÃficos foram desenhados para cada membro da GmUCP a fim de analisar os perfis de expressÃo em diferentes tecidos (semente seca e embebida, flores, vagens, cotilÃdones, folhas unifolioladas e trifolioladas, raÃzes, hipocÃtilos e epicÃtilos) durante o desenvolvimento da soja. Para os ensaios em condiÃÃes de estresse foram utilizadas folhas e raÃzes de soja com treze dias apÃs a semeadura (DAS) que foram submetidas a estresse osmÃtico promovido pela aplicaÃÃo de polietileno glicol (PEG) e estresse biÃtico atravÃs de Ãcido salicÃlico (AS). O RNA total de cada amostra foi extraÃdo para a realizaÃÃo de RT-qPCR. Os valores de ct foram obtidos pelo programa realplex e analisados pelo programa GeNorm. O perfil de expressÃo gÃnica mostrou que todos os genes GmUCP foram expressos em todos os tecidos/ÃrgÃos analisados durante o desenvolvimento da soja, com exceÃÃo de alguns genes em semente seca e epicÃtilo. Os diferentes perfis de expressÃo de cada gene durante o desenvolvimento de cada tecido/ÃrgÃo sugerem que ocorra uma regulaÃÃo gÃnica espacial/temporal entre os membros da GmUCP. Os perfis de expressÃo dos genes GmUCP em soja durante as condiÃÃes de estresses foi diversificado, visto que 2 genes apresentaram expressÃo estÃvel em ambos tecidos/estresse, 7 genes apresentaram queda do perfil de expressÃo, enquanto apenas 4 genes apresentaram aumento dos nÃveis de transcritos.
Several studies have evidenced that the main function of the mitochondrial uncoupling protein in plants (pUCP) is related to reactive oxygen species (ROS) regulation. In silico analysis suggests the existence of multigenic families to pUCPs codification, however further studies are yet needed to establish the pUCPs genetic expression profile, just like the gene amount in each species and their phylogenetic relations. The current work had as objective to characterize, analyze phylogeneticly and evaluate the expression profile of the pUCP multigenic family in different tissues during the soybean development [Glycine max (L.) MERR.] and in stress conditions. It has been performed an in silico analysis on the soybean genome and on other legumes available in the database WGS, revealing a codifier multigenic family for pUCP, UCP1 and 2 with 9 exons, UCP3 with 2 exons, and UCP4 and 5 with only 1 exon. Amongst the legumes analyzed, the soybean stood out with the greater number of genes, 10 genes in total, giving four GmUCP1 genes, one GmUCP2, one GmUCP3, two GmUCP4 and two GmUCP5, along with the presence of an alternative splicing on GmUCP1b1 gene. Specific primers have been designed for each GmUCP member in order to analize the expression profiles in different tissues (dry and doused seed, flowers, pods, cotyledons, unifoliate and trifoliate leaves, roots, hypocotyl and epicotyl) during the soybean development. For the assays in stress conditions have been used soybean leaves and roots with thirteen days after sowing (DAS) which have been subjected to osmotic stress caused by the application of polyethylene glycol (PEG) and biotic stress caused by salicylic acid (SA). The total RNA from each sample has been extracted in order to perform the RT-qPCR. The ct values have been obtained through the realplex program and analyzed through the GeNorm program. The genetic expression profile has shown that all genes were expressed in every tissue/organ analyzed during the soybean development, with the exception on some genes in dry seeds and epicotyl. The different expression profiles of each gene during the development of each tissue/organ suggest that occurs a spatial/temporal gene regulation among the GmUCP members. The expression profiles of the GmUCP genes in soybean during the stress conditions have varied, once 2 genes have shown steady expression in both tissues/ stress, 7 genes have shown a drop in the expression profile, while only 4 genes have shown an increase of the transcript levels.

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The flowering is a physiological process that it is vital for plants. This physiological process has been well studied in the plant model Arabidopsis, but in sugarcane this process is not well known. The transition of the shoot apical meristem from vegetative to flowering is a critical factor for plant development. At Brazil northeastern region, the transition to flowering in sugarcane has an important effect as it may reduce up to 60% its production. This is a consequence of the sugar translocation from stalks to the shoot apical meristem which is necessary during the flowering process. Therefore, the aim of this work was to explore and analyze cDNAs previously identified using subtractive cDNA libraries. The results showed that these cDNAs showed differential expression profile in varieties of sugarcane (early x late flowering). The in silico analysis suggested that these cDNAs had homology to calmodulin, NAC transcription factor and phosphatidylinositol, a SEC14, which were described in the literature as having a role in the process of floral development. To better understand the role of the cDNA homologous to calmodulin, tobacco plants were transformed with overexpression cassettes in sense and antissense orientation. Plants overexpressing the cassette in sense orientation did not flowered, while plants overexpressing the cassette in the antissense orientation produced flowers. The data obtained in this study suggested the possible role from CAM sequence, SEC14 and NAC in the induction/floral development pathway in sugarcane, this is the first study in order to analyze these genes in the sugarcane flowering process.
A flora??o ? um processo fisiol?gico que demanda um alto gasto energ?tico, e ? vital para a planta, pois ? dela que depende sua propaga??o gen?tica. Este processo fisiol?gico tem sido bem estudado no modelo vegetal Arabidopsis, mas em cana-dea??car n?o ? muito conhecido. A transi??o do meristema apical no processo de flora??o ? um fator cr?tico no ciclo de desenvolvimento da planta. No nordeste brasileiro, a transi??o para a flora??o na cana-de-a??car tem um efeito dram?tico, pois pode reduzir em at? 60% a produ??o de a??car ou etanol. Isso porque, o florescimento da cana resulta na isoporiza??o do colmo que ? caracterizado pela transloca??o do a??car presente no colmo para os ?pices meristem?ticos com a finalidade de suprir a necessidade energ?tica durante o processo de flora??o. Portanto, o objetivo desse trabalho foi de prospectar e analisar cDNAs de cana-dea??car que possam estar associados ? flora??o utilizando ferramentas de bioinform?tica e de transgenia. Os resultados mostraram que os cDNAs em estudo apontaram um perfil de express?o diferencial em variedades de cana-de-a??car. As an?lises in silico sugeriram que os cDNAs estudados apresentam similaridade para calmodulina, fator de transcri??o NAC e fosfatidilinositol, uma SEC14, as quais foram descritas na literatura como tendo um papel no processo de desenvolvimento floral. Para entender melhor o papel do cDNA hom?logo ? calmodulina, plantas de tabaco foram transformadas com cassetes de superexpress?o nas orienta??es sense e antisense contendo o promotor 35S. Plantas superexpressando o cassete na orienta??o senso n?o floresceram, enquanto que plantas superexpressando o cassete na orienta??o antissense apresentaram o desenvolvimento reprodutivo. Os dados obtidos nesse trabalho apontam para o poss?vel papel de CAM, NAC e SEC14 na via de indu??o/desenvolvimento floral em cana-de-a??car, sendo um dos primeiros trabalhos a se estudar esses genes no processo de flora??o nesse organismo

Испитивано је хроматографско понашање (хроматографска липофилност) 29 стероидних једињења (триазола и тетразола, толуенсулфонилхидразида, диона, нитрила и динитрила) од потенцијалног биомедицинског значаја, испитивано је помоћу течне хроматографије високих перформанси на обрнутим фазама, применом две стационарне и две мобилне фазе. Липофилност, изражена преко ретенционог параметра logk, моделована је QSRR приступом. Формирани линеарни и нелинеарни модели омогућили су испитивање односа између ретенционих параметара и in silico молекулских дескриптора, који су израчунати на основу структуре испитиваних једињења. Добра предиктивна моћ формираних модела, добијених за калибрациони сет, потврђена је и применом екстерног тест сета и валидационог сета. Предиктивна моћ формираних модела потврђује могућност њиховог коришћења за предвиђање липофилности нових, структурно сличних, једињења. Примењене су и класификационе хемометријске методе (анализа главних компоненти и хијерархијска кластер анализа) како би се уочиле сличности и разлика између једињења. Поред тога, представљена је in vitro анализа антимикробног потенцијала испитиваних стероидних једињења према Staphylococcus aureus, Escherichia coli и Candida albicans. Два једињења, са епоксидном групом у положају 4,5, испољила су бактериостатски ефекат према S. aureus. Такође, приказана је докинг анализа одабраних испитиваних једињења са антипролиферативном активношћу према ћелијама андроген-рецептор негативног канцера простате (AR-нег. PC-3). На основу визуелизације оптималних положаја и анализе постојећих интеракција, идентификовано је једињење са највећим потенцијалом као инхибитор хуманог цитохрома P450 CYP17A1.
Ispitivano je hromatografsko ponašanje (hromatografska lipofilnost) 29 steroidnih jedinjenja (triazola i tetrazola, toluensulfonilhidrazida, diona, nitrila i dinitrila) od potencijalnog biomedicinskog značaja, ispitivano je pomoću tečne hromatografije visokih performansi na obrnutim fazama, primenom dve stacionarne i dve mobilne faze. Lipofilnost, izražena preko retencionog parametra logk, modelovana je QSRR pristupom. Formirani linearni i nelinearni modeli omogućili su ispitivanje odnosa između retencionih parametara i in silico molekulskih deskriptora, koji su izračunati na osnovu strukture ispitivanih jedinjenja. Dobra prediktivna moć formiranih modela, dobijenih za kalibracioni set, potvrđena je i primenom eksternog test seta i validacionog seta. Prediktivna moć formiranih modela potvrđuje mogućnost njihovog korišćenja za predviđanje lipofilnosti novih, strukturno sličnih, jedinjenja. Primenjene su i klasifikacione hemometrijske metode (analiza glavnih komponenti i hijerarhijska klaster analiza) kako bi se uočile sličnosti i razlika između jedinjenja. Pored toga, predstavljena je in vitro analiza antimikrobnog potencijala ispitivanih steroidnih jedinjenja prema Staphylococcus aureus, Escherichia coli i Candida albicans. Dva jedinjenja, sa epoksidnom grupom u položaju 4,5, ispoljila su bakteriostatski efekat prema S. aureus. Takođe, prikazana je doking analiza odabranih ispitivanih jedinjenja sa antiproliferativnom aktivnošću prema ćelijama androgen-receptor negativnog kancera prostate (AR-neg. PC-3). Na osnovu vizuelizacije optimalnih položaja i analize postojećih interakcija, identifikovano je jedinjenje sa najvećim potencijalom kao inhibitor humanog citohroma P450 CYP17A1.
Chromatographic behavior (chromatographic lipophilicity) of 29 steroid compounds (triazole and tetrazole, toluenesulfonylhydrazide, dione, dinitrile and nitrile) with potential biomedical importance was investigated by reversed-phases high-performance liquid chromatography using two stationary and two mobile phases. The lipophilicity expressed through the retention parameter logk was modeled using QSRR approach. Formed linear and non-linear models enabled the study of the relationship between the retention parameters and in silico molecular descriptors calculated from the structure of the investigated compounds. Good predictive power of the established models obtained for the calibration set was confirmed by the application of an external test set and validation set. The predictive power of the established model confirms the possibility of their use for lipophilicity prediction of new, structurally similar compounds. The classification chemometric methods (principal components analysis and hierarchical cluster analysis) were applied in order to recognize the similarities and differences between the compounds. Тhis dissertation presents the in vitro analysis of the antimicrobial potentials of the investigated steroid compounds against Staphylococcus aureus, Escherichia coli and Candida albicans. Two compounds, with epoxy group in the position 4,5, exhibited bacteriostatic effect against S. aureus. The docking analysis of selected test compounds with antiproliferative activity toward cells of androgen receptor-negative prostate cancer (AR-neg. PC-3) is showed. Based on the optimal position visualization and analysis of existing interactions a compound with the most promising potential as human cytochrome P450 CYP17A1 inhibitor is idetified.

Human cytomegalovirus (CMV) is a significant infectious agent causing disease in immunocompromised HIV-infected patients, transplant recipients, and neonates. The current antiviral therapeutic strategy against CMV is limited in its utility due to the inherent toxicity and lack of bioavailability of currently available anti-CMV agents, ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS). The development of the prodrug of GCV, valganciclovir (val-GCV), has vastly improved the bioavailability profile of GCV. However, val-GCV demonstrates limited effectiveness against tissue-invasive CMV diseases as side effects involved with traditional intravenously administered GCV such as haematologic and reproductive toxicities remain. In addition, the emergence of antiviral resistant CMV mutant strains due to prolonged treatment with currently available antivirals necessitates the development of novel anti-CMV agents with reduced toxicity and improved bioavailability. In this study, select groups of novel compounds were analysed for their potential for further development as anti-CMV agents. Three groups of compounds were identified based on two screening methods which included the computer simulated screening process of compounds known as in silico screening and the traditional method of random screening. The first group of compounds (CATi) were identified by in silico screening against the CMV DNA polymerase catalytic aspartate triad, resulting in the identification of 31 compounds with the potential for inhibitory activity against CMV. The second group of compounds (PRO-i) were identified through in silico screening against the CMV protease, identifying a total of 18 lead compounds exhibiting structural complementarity with CMV protease. The third and final group of compounds (TPEX) were identified through random screening and consisted of plant extracts purified from tropical plants. All three compounds were initially screened for cytotoxicity against human fibroblasts. Plaque reduction assays were performed using compounds with acceptable levels of cytotoxicity to determine the ability of the compounds to inhibit the replication of the laboratory antiviral sensitive CMV strain, Towne. Two of the PRO-i compounds demonstrated good antiviral activity against CMV. Eleven percent (2/18) of the PRO-i compounds inhibited CMV replication, with PRO-i-43 and PRO-i??-44 displaying mean 50% inhibitory concentrations (IC50) of 4.8 ?? 1.2 ??M and 8.04 ??M, respectively. PRO-i-43 and PRO-i-44 are thus good candidates for further development as novel antiviral agents against CMV. The majority of CATi and TPEX compounds displayed significant cytotoxicity against human fibroblasts and compounds with acceptable levels of cytotoxicities did not significantly inhibit CMV replication. However, the identification of compounds with low cytotoxicities provides a good foundation for further development of novel anti-CMV agents with superior antiviral activity. In silico screening against three-dimensional viral protein models is a useful strategy for the identification of novel antiviral agents with the potential for inhibitory activity against CMV. Structural modification to produce potent derivatives of the identified anti-CMV compounds (PRO-i-43 and PRO-i-44) is a good option for the further development of novel antiviral agents against CMV. Such further examination of the identified compounds with anti-CMV activity is required to investigate their activity against not only antiviral sensitive CMV strains but also resistant CMV strains. Further investigations will yield new insights into their target, allowing further identification of compounds with potential anti-CMV activity with pharmaceutical application.

Bioactive food-derived peptides have been increasingly studied and the multiple health benefits they provide have been acknowledged. Since the most investigated sources of bioactive peptides are eggs, meat, fish, soybean, wheat, milk, and their derivatives or byproducts. More recently, some attention is being paid to microalgae, a promising unconventional protein source. On this basis, the PhD thesis was focused on the evaluation of the potential of microalgae to generate peptides with cardiovascular-promoting effects, especially hypotensive and antidiabetic activities, by targeting two therapeutic agents – angiotensin I converting enzyme (ACE) and peptidyl-peptidase IV (DPP-IV), respectively. To achieve this objective, multidisciplinary approaches were employed, involving peptidomic techniques to profile the peptide sequences, biochemical tools to analyze the bioactivity, and emerging molecular modelling methods to predict potentially bioactive peptides as well as to explore their possible mechanism of action. Briefly, the results showed that the protein hydrolysates from spirulina, PBP and chlorella generally presented significant ACE and/or DPP-IV inhibitory activities. By comparison, peptic hydrolysate of spirulina protein showed the best in vitro inhibiting effect on ACE with IC50 value of 0.1 ± 0.04 mg/mL while the tryptic hydrolysate of PBP stands out with the lowest IC50 value of DPP-IV inhibition (0.5 – 1.0 mg/mL). Noticeably, when working on the intestinal Caco-2 cells, all the hydrolysates turned to be less bioactive than in vitro, indicating their susceptibility to metabolic degradation by intestinal cells. This is further in line with the kinetics of DPP-IV inhibition of PBP tryptic hydrolysate working on the intestinal cells, which showed the decreasing trend of its bioactivity after incubation with Caco-2 cells for 3 h. This reflects the issue of peptide bioavailability, which could be stressed in further studies. Moreover, peptides Pep2 (FLKPLGSGK), Pep7 (QIYTMGK), Pep8 (FLFVAEAIYK), and Pep10 (QHAGTKAK) were screened from hydrolysates of chlorella protein by peptidomics combined with docking and MD. The modelling results indicated that they may block the important domain of both ACE and DPP-IV and generate dynamically stable peptide-ACE/DPP-IV complexes. Based on this theoretical evidence, further study will focus on the verification of their actual bioactivity by biochemical approaches. In conclusion, the bioactivity investigation of microalgae protein hydrolysates provides new evidence that microalgae protein are great sources to produce peptides with health-promoting properties. The exclusive data of peptide characterization makes a foundation to isolate single bioactive peptides as well as offers useful structural and functional implications for food ingredient formulation or pharmacological use.

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The incidence of vulvovaginal fungal infections has increased dramatically over the last decades, therefore being the second most common cause of vulvovaginal candidiasis (VVC) placed after bacterial vaginosis diagnosed in 40% of the women with vaginal discharge. The increasing resistance of micro-oganismos pathogens to existing antimicrobial market has driven the search for new therapeutic alternatives, such as natural herbal products belonging to various families of the plant kingdom, such as Poaceae and Myrtaceae, which are presented as a viable solution due to the low cost and easy access of the population. Highlighted, the genus Cymbopogon and the Eucalyptus plants is one of the main sources of biologically active molecules, among them the monoterpenes holds a huge biological potential of human interest. However, the research of plant-derived compounds with pharmacological properties must always be attached to toxicity studies in order to show the absence of these substances harm to the human body. Given this context, it has studied the biological activity of enantiomers (R)-(+)- and (S)-(-)-citronellal against C. albicans and C. tropicalis strains derived from in vitro vulvovaginal secretions, as well as the parameters toxicological to predict the theoretical oral toxicity in silico. To achieve the antimicrobial in vitro studies; determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was used microdilution test. Also it was applied to disk diffusion technique on solid medium for comparative study of antifungal resistance/sensitivity profile used in the treatment of vulvovaginal candidiasis isolated and associated with monoterpenes studieds. The MIC's and MFC's of the (R)-(+)- and (S)-(-)-citronellal to 90% of fungal strains were respectively (R)MIC90% 16μg/mL and (R)MFC90% 32μg/mL; (S)MIC90% 64μg/mL and (S)MFC90% 128μg/mL (Strong antifungal activity against C. albicans and C. tropicalis). Were observed high resistance of C. albicans to fluconazole and itraconazole 12 (92.30%) strains. However, this resistance was reversed in 9 (75%) and 7 (58.33%) respectively, when combined with (R)-(+)-citronellal and 6 (50%) in combination with (S)-(-)-citronellal. Furthermore, it was also observed C. tropicalis high resistance to amphotericin B, itraconazole and miconazole. However, the resistance was reversed to amphotericin B and itraconazole, as a result of the synergistic effect in 2 (66.65%) of yeast. For miconazole resistance was reversed in 3 (100%) of the strains for both enantiomers. In the in silico analysis, both enantiomers have good oral bioavailability theoretically, however, a potential irritant was observed as possible toxic effect on these monoterpenes. In conclusion, these results suggest that the (R)-(+)- and (S)-(-)-citronellal have antimicrobial effect, and which also exert effect modifier activity when antifungal agents in combination. However, although presenting good oral bioavailability theoretically, the varied toxicological profile suggests the need to assess the risk-benefit of this compound in the production of a new antifungal drug, by conducting in vivo trials.
A incidência de infecções fúngicas vulvovaginais aumentou dramaticamente ao longo das últimas décadas, constituindo assim a segunda causa mais comum de candidíase vulvovaginal (CVV) após a vaginose bacteriana diagnosticada em 40% das mulheres com corrimento vaginal. O aumento da resistência dos micro-organismos patógenos aos antimicrobianos existentes no mercado tem impulsionado a busca de novas alternativas terapêuticas, como os produtos naturais a base de plantas pertencentes a várias famílias do reino vegetal, como por exemplo a Poaceae e a Myrtaceae, que se apresentam como uma solução viável devido ao baixo custo e fácil acesso da população. Em destaque, as plantas do gênero Cymbopogon e Eucalyptus constitui uma das principais fontes de moléculas biologicamente ativas, dentre elas os monoterpenos são detentores de um enorme potencial biológico de interesse humano. No entanto, as pesquisas de compostos derivados de plantas com propriedades farmacológicas devem sempre ser unida aos estudos toxicológicos, afim de se comprovar a ausência de malefícios destas substâncias ao organismo humano. Diante deste contexto, foi estudado a atividade biológica dos enantiômeros (R)-(+)- e (S)-(-)-citronelal contra cepas de C. albicans e C. tropicalis oriundas de secreções vulvovaginais in vitro, bem como os parâmetros toxicológicos para a previsão da toxicidade oral teórica in silico. Para a realização dos estudos antimicrobianos in vitro; determinação da concentração inibitória mínima (CIM) bem como da concentração fungicida mínima (CFM), utilizou-se o teste de microdiluição. Também foi aplicada a técnica de disco-difusão em meio sólido para estudo comparativo do perfil de resistência/sensibilidade a antifúngicos utilizados na terapêutica da candidíase vulvovaginal isolados e associados aos monoterpenos em estudo. As CIM’s e as CFM’s dos enantiômeros (R)-(+)- e (S)-(-)-citronelal para 90% das cepas fúngicas foram respectivamente (R)CIM90% 16μg/mL e (R)CFM90% 32 μg/mL; (S)CIM90% 64μg/mL e (S)CFM90% 128 μg/mL (forte atividade antifúngica contra C. albicans e C. tropicalis). Foram constatados alta resistência de C. albicans ao fluconazol e ao itraconazol 12 (92.30%) das cepas testadas. Mas, essa resistência foi revertida em 9 (75%) e 7 (58.33%) respectivamente, quando em associação com o (R)-(+)-citronelal e em 6 (50%) em combinação com o (S)-(-)-citronelal. Além disso, também foi observado alta resistência de C. tropicalis a anfotericina B, itraconazol e miconazol. Porém a resistência foi revertida para a anfotericina B e para o itraconazol, resultado do efeito sinérgico em 2 (66.65%) das leveduras. Para o miconazol a resistência foi revertida em 3 (100%) das cepas para ambos os enantiômeros. Na análise in silico, ambos os enantiômeros apresentaram boa biodisponibilidade oral teórica, no entanto, um potencial irritante foi observado como possível efeito tóxico para estes monoterpenos. Em conclusão, estes resultados sugerem que os enantiômeros (R)-(+)- e (S)-(-)-citronelal apresentam efeito antimicrobiano, e que também exercem efeito modificador de atividade aos agentes antifúngicos quando em combinação. No entanto, embora tenha apresentado boa biodisponibilidade oral teórica, o perfil toxicológico variado sugere a necessidade em avaliar o risco-benefício desse composto na produção de um novo medicamento antifúngico, por realização de ensaios in vivo.

Thesis (Ph.D. in Bioinformatics) -- University of Colorado, 2005.
Typescript. Includes bibliographical references (leaves 116-124). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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Uma das preocupações envolvendo a expressão de diferentes proteínas heterólogas em alimentos é a possibilidade do desenvolvimento de reações alérgicas ou antigênicas nos consumidores finais. A Food and Agriculture Organization of the United Nations juntamente com a World Health Organization definiram que uma sequência de aminoácidos seria considerada alergênica/antigênica quando apresentasse ao menos 35 % de identidade em uma janela de 80 aminoácidos ou 6 – 8 aminoácidos contíguos e idênticos quando comparada com sequências de alergénos conhecidos. Para categorizar proteínas alergênicas ou antigênicas foi construída uma plataforma (chamada Plataforma de Análise da Alergenicidade e Antigenicidade de Proteínas – PA³P – e disponível em http://lpa.saogabriel.unipampa.edu.br:8080/pa3p/index.jsp ou http://pluriserver.com.br/pa3p/) que congrega um conjunto de ferramentas específicas para tais análises. A plataforma fora construída através da linguagem de programação Java e HTML. Desta forma, foram separados grupos de proteínas alergênicas/antigênicas de não alergênicas/não antigênicas com redução dos casos de falsos positivos ou falsos negativos. As análises complementares utilizadas neste trabalho são necessárias, pois a metodologia proposta pela Food and Agriculture Organization of the United Nations juntamente com a World Health Organization é insuficiente, gerando resultados duvidosos. A plataforma construída neste trabalho obteve valores de 98 % de sensibilidade e 96 % de especificidade, comparada com 89 % e 85 %, respectivamente, dos testes utilizados isoladamente. Esta plataforma pode ser utilizada para embasar a decisão de utilizar determinada proteína na construção de um Organismo Geneticamente Modificado com finalidade nutricional.
A major drawback involving heterologous protein expression in organisms used for human consumption is the possibility of allergenic or antigenic reactions. Both Food and Agriculture Organization of the United Nations and World Health Organization determined that a potentially allergenic protein world present at least 35 % identity in a 80 amino acid window or 6 – 8 contiguous and identical amino acids when compared with known allergens. In order to assess the allergenic or antigenic potential of a given protein, the Platform for Analysis of Allergenicity and Antigenicity of Proteins (PA³P – available at http://lpa.saogabriel.unipampa.edu.br:8080/pa3p/index.jsp or http://pluriserver.com.br/pa3p/) was developed. The platform was constructed using Java and HTML programming languages. It was p ssible to discriminate allergenic/antigenic from non-allergenic/non-antigenic proteins, with reduction of false positive and false negative samples. The additional analyses used in the platform are necessary, since the insufficient FAO and WHO proposed methods, rendering doubtable results. In our tests, PA³P presented 98% sensibility and 96% specificity when compared with the web tools used independently (89 and 85%, respectively). This tool has a potential to contribute to decision making regarding the of a given protein in GMO construction with nutritional intent.

This thesis work, combining a virtual screening study for cytidine deaminase ligands with a study on the effect that genetic polymorphisms have on the enzyme functionality provide a useful base to: a) understand the mechanism of nucleoside recognition by CDA and identify novel effective inhibitors; and b) study the different properties of said inhibitors toward three distinct naturally occurring CDA variants (K27, Q27 and T70). Ultimately, this may assist the future design of novel CDA inhibitors or antitumor drugs not susceptible to deamination, with the aim to get more effective personalized drug therapies.

Chronic inflammation is closely related to the emergence of a number of cancers, including Breast cancer. Inflammation causes damage to the cell’s DNA which leads to its abnormal growth and formation of tumor mass. One of the most commonly known receptor responsible for inflammatory reactions is Toll-like receptor 4 (TLR4). It is activated majorly by bacterial LPS. Its activation further activates Cyclooxygenase enzyme that catalyzes the conversion of arachidonic acid into prostaglandins that lead to inflammation-like conditions. COX2 has also been correlated to the promotion of tumor growth. It enhances metastasis, neoplasia, lymphangiogenesis, etc., and is also related to poor prognosis in the breast cancer patients. Curcumin derived from turmeric is a proven inhibitor of COX2. In my project I have aimed to analyse and compare the inhibitory properties of other analogues of curcumin that have previously been known to inhibit COX2. The experimental layout began with screening the molecules on the basis of drug-likeness using Lipinski rule of five. The suitable ligand molecules were further subjected to other experiments, i.e., ligand docking and drug potential assessment. After all the experiments, out of the five selected Curcumin analogues, Isoeugenol (extracted from clove) was determined as the best fit molecule. The druglikeliness and drug potential assessment results further validate its use as a potential inhibitor and can further be tested for in-vivo efficacy. This drug can further be used in the 1st line therapy of locally advanced and metastatic breast cancer patients as it will inhibit COX2 that promotes metastasis of cancer cells. Isoeugenol extracted from Eugenia caryophyllus (Cloves) can further be proven as a better COX2 inhibitor than its chemical counterparts, as it is a natural compound and will therefore have significantly less side effects.

Cubatão, o maior pólo industrial da américa latina também já foi uma das cidades mais poluídas do mundo. Os 30 anos de intensa atividade industrial vêm pressionando o meio ambiente com substâncias tóxicas e afetando gravemente a saúde da população. Dentre as substâncias contaminantes mais importantes da região estão os derivados de petróleo como o benzeno, tolueno, etilbenzeno e xilenos. Conhecidos como BTEX, eles são produzidos e utilizados em larga escala e a contaminação ocorre frequentemente através de vazamentos. Nos solos, devido à sua solubilidade em água, essas substâncias podem se espalhar por longas distâncias a partir do ponto afetado contaminando locais distantes. Já foi comprovada a capacidade de micro-organismos de sobreviver e até utilizar BTEX como fonte de carbono. Os micro-organismos adaptados catabolizam os contaminantes transformando-os em substâncias menos tóxicas e até mesmo eliminando-os do ambiente, capacidade de grande interesse econômico e ambiental. Nessa linha, nossa proposta visa o estudo das comunidades microbianas de solos afetados e não afetados por BTEX. Para isso foi utilizada a metagenômica como abordagem de estudo identificando-se diferenças qualitativas e quantitativas nas estruturas microbianas de três diferentes locais do município de Cubatão, sendo um deles afetado diretamente por BTEX. Pelo método utilizado e aqui desenvolvido, foi possível identificar um panorama metabólico geral identificando-se genes relevantes e o potencial de degradação de hidrocarbonetos aromáticos de micro-organismos conhecidos e desconhecidos, revelando melhor o potencial metabólico dos solos identificados. Os resultados apresentados podem contribuir para um melhor entendimento da dinâmica in situ de uma comunidade microbiana afetada por BTEX assim como melhorar o conhecimento sobre a comunidade microbiana de um local altamente impactado como Cubatão.
Cubatão is the largest industrial site in Latin America and was in the past one of the most polluted cities in the world. 30 years of intense industrial activity has pushed environmental limits with toxic substances and has severely affected the inhabitants\' health. Among the contaminants found in the region, the petroleum derivatives benzene, toluene, ethylbenzene and xylenes are the most important. Known collectively as BTEX, they are produced and used at a large scale and contamination frequently occurs. Because it is highly soluble in water, when in soil BTEX can spread long distances from the original contamination site, thus affecting large areas. Some microorganisms are known to live in contaminated environments and use contaminants such as BTEX as a unique carbon source for energy production. They catabolize contaminants into less dangerous products or even eliminate them from environment, a feature which has great commercial and environmental interest. We therefore compared the microbial communities in soils which were affected and un-affected by BTEX contamination. To this end, we used a metagenomics approach and developed a comparison method to identify microorganisms and degradation potential of soils studied. We found qualitative and quantitative differences in microbial structures from three different sites in Cubatão County, one of which is contaminated with BTEX. We constructed a metabolic overview identifying important genes, degradation potential and microorganisms related to BTEX degradation. The results presented here could contribute to understanding the in situ dynamics of a BTEX affected microbial community as well as improving our knowledge of the microbial community of Cubatão, a highly environmentally impacted place.