Identification and molecular analysis of cable pilus biosynthesis genes in Burkholderia cepacia

Burkholderia cepacia is an opportunistic respiratory pathogen in cystic fibrosis patients. One highly transmissible and virulent clone belonging to genomovar IIIa expresses pili with unique cable morphology, which enable the bacterium to bind cytokeratin 13 in epithelial cells. The cblA gene, encoding the major pilin subunit, is often used as a DNA marker to identify potentially virulent isolates. The authors have now cloned and sequenced four additional genes, cblB, cblC, cblD and cblS, in the pilus gene cluster. This work shows that the products of the first four genes of the cbl operon, cblA, cblB, cblC and cblD, are sufficient for pilus assembly on the bacterial surface. Deletion of cblB abrogated pilus assembly and compromised the stability of the CblA protein in the periplasm. In contrast, deletion of cblD resulted in no pili, but there was no effect on expression and stability of the CblA protein subunit. These results, together with protein sequence homologies, predicted structural analyses, and the presence of typical amino acid motifs, are consistent with the assignment of functional roles for CblB as a chaperone that stabilizes the major pilin subunit in the periplasm, and CblD as the initiator of pilus biogenesis. It is also shown that expression of Cbl pili in Escherichia coli is not sufficient to mediate the binding of bacteria to the epithelial cell receptor cytokeratin 13, and that B. cepacia still binds to cytokeratin 13 in the absence of Cbl pili, suggesting that additional bacterial components are required for effective binding.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Ser6 in the maize actin-depolymerizing factor, ZmADF3, is phosphorylated by a calcium-stimulated protein kinase and is essential for the control of functional activity

Maize actin-depolymerizing factor, ZmADF, binds both G- and F-actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal-responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium-stimulated protein kinase present in a 40-70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3-4) does not bind G- or F-actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.

Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

Identification of lactic acid bacteria strains modulating incretin hormone secretion and gene expression in enteroendocrine cells

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released from intestinal enteroendocrine (EE) cells and have well-established glucose-lowering actions. Lactic acid bacteria (LAB) colonise the human intestine, but it is unknown whether LAB and EE cells interact. Acute co-culture of LAB with EE cells showed that certain LAB strains elicit GLP-1 and GIP secretion (13-194-fold) and upregulate their gene expression. LAB-induced incretin hormone secretion did not appear to involve nutrient mechanisms, nor was there any evidence of cytolysis. Instead PCR array studies implicated signalling agents of the toll-like receptor system, e.g. adaptor protein MyD88 was decreased 23-fold and cell surface antigen CD14 was increased 17-fold. Mechanistic studies found that blockade of MyD88 triggered significant GLP-1 secretion. Furthermore, blocking of CD14 completely attenuated LAB-induced secretion. A recent clinical trial clearly shows that LAB have potential for alleviating type 2 diabetes, and further characterisation of this bioactivity is warranted.

Identification of novel Factor XI mutations in a patient requiring dental extractions

Factor XI is a serine protease that participates in the intrinsic pathway of blood coagulation. Patients deficient in factor XI exhibit varying degrees of post operative bleeding following invasive surgical procedures such as dental extractions. Objectives: The aim of the study was to identify the specific mutations in a patient from a family with known factor XI deficiency. Methods: Samples were obtained from the patient, his mother and his father and subjected to DNA sequencing. Each protein coding exon 2-15 of the factor XI gene was amplified by polymerase chain reaction (PCR) followed by bidirectional sequencing utilizing di-deoxy chain termination chemistry. Results: The patient had a factor XI level of 20% of normal. Initial sequencing of factor XI from the patient identified a point mutation (646G>A) and a putative splice site mutation (1567+4A>T) in intron 13. These are novel previously unreported mutations. DNA sequence analysis of the mother revealed the 1567+4A>T mutation and the father exhibited the 646G>A mutation. As a consequence the treatment proceeded without serious bleeding complication and required administration only of transexamic acid though factor XI was available as haemostatic cover. Conclusion: The two mutations identified in this family are novel; further laboratory investigation of the functional consequences of those mutations is currently underway. Although factor XI deficiency is rare in the Northern Irish population this study highlights the techniques available to sequence and analyse this and similar haematological disorders.

Evolution and functional relevance of Ataxin-3 paralogues

O gene ataxin-3 (ATXN3; 14q32.1) codifica uma proteína expressa ubiquamente, envolvida na via ubiquitina-proteassoma e na repressão da transcrição. Grande relevância tem sido dada ao gene ATXN3 após a identificação de uma expansão (CAG)n na sua região codificante, responsável pela ataxia mais comum em todo o mundo, SCA3 ou doença de Machado-Joseph (DMJ). A DMJ é uma doença neurodegenerativa, autossómica dominante, de início tardio. O tamanho do alelo expandido explica apenas uma parte do pleomorfismo da doença, evidenciando a importância do estudo de outros modificadores. Em doenças de poliglutaminas (poliQ), a toxicidade é causada por um ganho de função da proteína expandida; no entanto, a proteína normal parece ser, também, um dos agentes modificadores da patogénese. O gene ATXN3 possui dois parálogos humanos gerados por retrotransposição: ataxin-3 like (ATXN3L) no cromossoma X, e LOC100132280, ainda não caracterizado, no cromossoma 8. Estudos in vitro evidenciaram a capacidade da ATXN3L para clivar cadeias de ubiquitina, sendo o seu domínio proteolítico mais eficiente do que o domínio da ATXN3 parental. O objetivo deste estudo foi explorar a origem e a evolução das retrocópias ATXN3L e LOC100132280 (aqui denominadas ATXN3L1 e ATXN3L2), assim como testar a relevância funcional de ambas através de abordagens evolutivas e funcionais. Deste modo, para estudar a divergência evolutiva dos páralogos do gene ATXN3: 1) analisaram-se as suas filogenias e estimou-se a data de origem dos eventos de retrotransposição; 2) avaliaram-se as pressões seletivas a que têm sido sujeitos os três parálogos, ao longo da evolução dos primatas; e 3) explorou-se a evolução das repetições CAG, localizadas em três contextos genómicos diferentes, provavelmente sujeitos a diferentes pressões seletivas. Finalmente, para o retrogene que conserva uma open reading frame (ORF) intacta, ATXN3L1, analisou-se, in silico, a conservação dos locais e domínios proteicos da putativa proteína. Ademais, para este retrogene, foi estudado o padrão de expressão de mRNA, através da realização de PCR de Transcriptase Reversa, em 16 tecidos humanos. Os resultados obtidos sugerem que dois eventos independentes de retrotransposição estiveram na origem dos retrogenes ATXN3L1 e ATXN3L2, tendo o primeiro ocorrido há cerca de 63 milhões de anos (Ma) e o segundo após a divisão Platirrínios-Catarrínios, há cerca de 35 Ma. Adicionalmente, outras retrocópias foram encontradas em primatas e outros mamíferos, correspondendo, no entanto, a eventos mais recentes e independentes de retrotransposição. A abordagem evolutiva mostrou a existência de algumas constrições selectivas associadas à evolução do gene ATXN3L1, à semelhança do que acontece com ATXN3. Por outro lado, ATXN3L2 adquiriu codões stop prematuros que, muito provavelmente, o tornaram num pseudogene processado. Os resultados da análise de expressão mostraram que o gene ATXN3L1 é transcrito, pelo menos, em testículo humano; no entanto, a optimização final da amplificação específica dos transcriptos ATXN3L1 permitirá confirmar se a expressão se estende a outros tecidos. Relativamente ao mecanismo de mutação inerente à repetição CAG, os dois parálogos mostraram diferentes padrões de evolução: a retrocópia ATXN3L1 é altamente interrompida e pouco polimórfica, enquanto a ATXN3L2 apresenta tratos puros de (CAG)n em algumas espécies e tratos hexanucleotídicos de CGGCAG no homem e no chimpanzé. A recente aquisição da repetição CGGCAG pode ter resultado de uma mutação inicial de CAG para CGG, seguida de instabilidade que proporcionou a expansão dos hexanucleótidos.Estudos futuros poderão ser realizados no sentido de confirmar o padrão de expressão do gene ATXN3L1 e de detetar proteína endógena in vivo. Adicionalmente, a caracterização da proteina ataxina-3 like 1 e dos seus interatores moleculares poderá povidenciar informação acerca da sua relevância no estado normal e patológico.

Identification of protein complexes in Alzheimer's disease

A Doença de Alzheimer (AD) é a maior doença neurodegenerativa a nível mundial, e a principal causa de demência na população idosa. O processamento da proteína precursora de amilóide (APP) pelas β- e g- secretases origina o peptídeo Aβ, que agrega em oligómeros neurotóxicos e em placas senis. Estes são eventos-chave na patogénese da DA que levam à rutura da neurotransmissão sináptica, morte neuronal e inflamação neuronal do hipocampo e córtex cerebral, causando perda de memória disfunção cognitiva geral. Apesar dos grandes avanços no conhecimento do papel do processamento da APP na DA, a sua função fisiológica ainda não foi totalmente elucidada. Os mapas de interações proteína-proteína (PPI) humanos têm desempenhado um papel importante na investigação biomédica, em particular no estudo de vias de sinalização e de doenças humanas. O método dois-híbrido em levedura (YTH) consiste numa plataforma para a produção rápida de redes de PPI em larga-escala. Neste trabalho foram realizados vários rastreios YTH com o objetivo de identificar proteínas específicas de cérebro humano que interagissem com a APP, ou com o seu domínio intracelular (AICD), tanto o tipo selvagem como com os mutantes Y687F, que mimetizam o estado desfosforilado do resíduo Tyr-687. De facto, a endocitose da APP e a produção de Aβ estão dependentes do estado de fosforilação da Tyr-687. Os rastreios YTH permitiram assim obter de redes proteínas que interagem com a APP, utilizando como “isco” a APP, APPY687F e AICDY687F. Os clones positivos foram isolados e identificados através de sequenciação do cDNA. A maior parte dos clones identificados, 118, correspondia a sequências que codificam para proteínas conhecidas, resultando em 31 proteínas distintas. A análise de proteómica funcional das proteínas identificadas neste estudo e em dois projetos anteriores (AICDY687E, que mimetiza a fosforilação, e AICD tipo selvagem), permitiram avaliar a relevância da fosforilação da Tyr-687. Três clones provenientes do rastreio YTH com a APPY687F foram identificados como um novo transcrito da proteína Fe65, resultante de splicing alternativo, a Fe65E3a (GenBank Accession: EF103274), que codifica para a isoforma p60Fe65. A p60Fe65 está enriquecida no cérebro e os seus níveis aumentam durante a diferenciação neuronal de células PC12, evidenciando o potencial papel que poderá desempenhar na patologia da DA. A RanBP9 é uma proteína nuclear e citoplasmática envolvida em diversas vias de sinalização celulares. Neste trabalho caracterizou-se a nova interação entre a RanBP9 e o AICD, que pode ser regulada pela fosforilação da Tyr-687. Adicionalmente, foi identificada uma nova interação entre a RanBP9 e a acetiltransferase de histonas Tip60. Demonstrou-se ainda que a RanBP9 tem um efeito de regulação inibitório na transcrição mediada por AICD, através da interação com a Tip60, afastando o AICD dos locais de transcrição ativos. O estudo do interactoma da APP/AICD, modelado pela fosforilação da Tyr-687, revela que a APP poderá estar envolvida em novas vias celulares, contribuindo não só para o conhecimento do papel fisiológico da APP, como também auxilia a revelar as vias que levam à agregação de Aβ e neurodegeneração. A potencial relevância deste trabalho relaciona-se com a descoberta de algumas interações proteicas/vias de sinalização que podem que podem ser relevantes para o desenvolvimento de novas estratégias terapêuticas na DA.

Identification and study of novel genes expressed in the heart precursor cells

Understanding heart development on a molecular level is a requirement for uncovering the causes of congenital heart diseases. Several genes have been implicated as critical for heart development. However, the inducers of these genes as well as their targets and pathways, remain largely unknown. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip® system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Five genes were selected for further characterization by whole mount in situ hybridization leading to the validation of their expression in the HPC. From those, Adtk1 and Ccbe1 were selected for functional analysis. Regarding to ccbe1, a more detailed WISH analysis was performed and showed that Ccbe1 is expressed specifically on the cardiac progenitors regions at HH4, more specifically in primary heart field and at later stages is present in the second heart field. Further functional analyses by knockdown and overexpression revealed an important role for Ccbe1 in early heart tube formation. Moreover, the results presented in this thesis suggested that Ccbe1 is a key gene during heart development and might be limited to multipotent and highly proliferative progenitors and downregulated upon cellular commitment into more specific cardiac phenotypes. Other of the genes identified, Adtk1 was also subjected to further functional studies. Knockdown of Adtk1 using morpholino oligonucleotides suggested that it might be necessary for the migration and fusion of the heart tube as well as for neural tube closure.

Identification of novel molecular determinants of tissue mineralization in fish

The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.

Cystathionine beta-synthase variants : identification, characterization and modulation by thiol compounds

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Ankylosing spondylitis: genomic and functional characterization of candidate genes and their repercussion in clinical practice

RESUMO: Introdução: A espondilite anquilosante (EA) é uma doença inflamatória crónica caracterizada pela inflamação das articulações sacroilíacas e da coluna. A anquilose progressiva motiva uma deterioração gradual da função física e da qualidade de vida. O diagnóstico e o tratamento precoces podem contribuir para um melhor prognóstico. Neste contexto, a identificação de biomarcadores, assume-se como sendo muito útil para a prática clínica e representa hoje um grande desafio para a comunidade científica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar possíveis associações entre genes, MHC e não-MHC, com a suscetibilidade e as características fenotípicas da EA; 3 - identificar genes candidatos associados a EA através da tecnologia de microarray. Material e Métodos: Foram recrutados doentes com EA, de acordo com os critérios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demográficos, clínicos e radiológicos e colhidas amostras de sangue periférico. Selecionaram-se de forma aleatória, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os haplótipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes alélicas dos genes IL23R, ERAP1 e ANKH foram estudadas através de ensaios de discriminação alélica TaqMan. A análise de associação foi realizada utilizando testes da Cochrane-Armitage e de regressão linear, tal como implementado pelo PLINK, para variáveis qualitativas e quantitativas, respetivamente. O estudo de expressão génica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram incluídos 369 doentes (62,3% do sexo masculino, com idade média de 45,4 ± 13,2 anos, duração média da doença de 11,4 ± 10,5 anos). No momento da avaliação, 49,9% tinham doença axial, 2,4% periférica, 40,9% mista e 7,1% entesopática. A uveíte anterior aguda (33,6%) foi a manifestação extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os haplótipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir proteção em termos de atividade, repercussão funcional e radiológica da doença. Três variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associação com a doença, confirmando a associação destes genes com a EA na população Portuguesa. O mesmo não se verificou com as variantes estudadas do ANKH. Não se verificou associação entre as variantes génicas não-MHC e as manifestações clínicas da EA. Foi identificado um perfil de expressão génica para a EA, tendo sido validados catorze genes - alguns têm um papel bem documentado em termos de inflamação, outros no metabolismo da cartilagem e do osso. Conclusões: Foi estabelecido um perfil demográfico e clínico dos doentes com EA em Portugal. A identificação de variantes génicas e de um perfil de expressão contribuem para uma melhor compreensão da sua fisiopatologia e podem ser úteis para estabelecer modelos com relevância em termos de diagnóstico, prognóstico e orientação terapêutica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4±13.2 years; mean disease duration 11.4±10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.

Evolutionary dynamics of Chlamydia trachomatis genome and identification of molecular patterns of hypothetical protein coding genes

The obligate intracellular bacterium Chlamydia trachomatis is a human pathogen of major public health significance. Strains can be classified into 15 main serovars (A to L3) that preferentially cause ocular infections (A-C), genital infections (D-K) or lymphogranuloma venereum (LGV) (L1-L3), but the molecular basis behind their distinct tropism, ecological success and pathogenicity is not welldefined. Most chlamydial research demands culture in eukaryotic cell lines, but it is not known if stains become laboratory adapted. By essentially using genomics and transcriptomics, we aimed to investigate the evolutionary patterns underlying the adaptation of C. trachomatis to the different human tissues, given emphasis to the identification of molecular patterns of genes encoding hypothetical proteins, and to understand the adaptive process behind the C. trachomatis in vivo to in vitro transition. Our results highlight a positive selection-driven evolution of C. trachomatis towards nichespecific adaptation, essentially targeting host-interacting proteins, namely effectors and inclusion membrane proteins, where some of them also displayed niche-specific expression patterns. We also identified potential "ocular-specific" pseudogenes, and pointed out the major gene targets of adaptive mutations associated with LGV infections. We further observed that the in vivo-derived genetic makeup of C. trachomatis is not significantly compromised by its long-term laboratory propagation. In opposition, its introduction in vitro has the potential to affect the phenotype, likely yielding virulence attenuation. In fact, we observed a "genital-specific" rampant inactivation of the virulence gene CT135, which may impact the interpretation of data derived from studies requiring culture. Globally, the findings presented in this Ph.D. thesis contribute for the understanding of C.trachomatis adaptive evolution and provides new insights into the biological role of C. trachomatishypothetical proteins. They also launch research questions for future functional studies aiming toclarify the determinants of tissue tropism, virulence or pathogenic dissimilarities among C. trachomatisstrains.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.