Adenovirus 12 E1A gene detection by polymerase chain reaction in both the normal and coeliac duodenum

A 12 amino acid sequence from the adenovirus 12 E1B protein is homologous at the protein level with a similar 12-mer derived from the wheat protein A-gliadin. It has been suggested that exposure to Ad 12 could sensitise individuals to gliadins with resultant gluten sensitive enteropathy. In this study, the polymerase chain reaction (PCR) was used to analyse duodenal biopsy tissue from patients with coeliac disease for the presence of Ad 12. The sensitivity of the assay system was at least 1 in 10(5) cells and specificity was confirmed both by probing with an internal oligonucleotide and by direct sequencing. Ad 12 sequences were detected in three of 17 patients with adult coeliac disease and in five of 16 adult controls with normal duodenal biopsies. Since exposure to the virus would be predicted to occur in infancy we also studied patients with childhood coeliac disease diagnosed at less than 1 year of age. Ad 12 was positive in three of 10 childhood coeliac patients and one of seven controls. In addition, we studied a cohort of patients who presented with a diarrhoeal illness and associated anti alpha gliadin antibodies in 1983. These patients had duodenal biopsies performed at this time. One of three patients with abnormal histology had detectable Ad 12 while two of 14 with normal findings were positive for Ad 12. Finally, the potential oncogenic nature of Ad 12 prompted examination of a group of patients with intestinal tumours. Ad 12 DNA was, however, in only two of 19 tumour samples tested. These data indicate that Ad 12 can be successfully detected using PCR on paraffin embedded tissue. Furthermore, Ad 12 was detected at a relatively high level in normal duodenum. The results do not, however, support the hypothesis that prior exposure to Ad 12 is implicated in the pathogenesis of coeliac disease.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

Molecular subtyping of primary prostate cancer reveals specific and shared target genes of different ETS rearrangements

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BACKGROUND: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

METHODS: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity Pathway Analysis.

RESULTS: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

CONCLUSION: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

Meta-analysis of Positive and Negative Symptoms Reveals Schizophrenia Modifier Genes

BACKGROUND: Evidence suggests that genetic factors may influence both schizophrenia (Scz) and its clinical presentation. In recent years, genome-wide association studies (GWAS) have demonstrated considerable success in identifying risk loci. Detection of "modifier loci" has the potential to further elucidate underlying disease processes.

METHODS: We performed GWAS of empirically derived positive and negative symptom scales in Irish cases from multiply affected pedigrees and a larger, independent case-control sample, subsequently combining these into a large Irish meta-analysis. In addition to single-SNP associations, we considered gene-based and pathway analyses to better capture convergent genetic effects, and to facilitate biological interpretation of these findings. Replication and testing of aggregate genetic effects was conducted using an independent European-American sample.

RESULTS: Though no single marker met the genome-wide significance threshold, genes and ontologies/pathways were significantly associated with negative and positive symptoms; notably, NKAIN2 and NRG1, respectively. We observed limited overlap in ontologies/pathways associated with different symptom profiles, with immune-related categories over-represented for negative symptoms, and addiction-related categories for positive symptoms. Replication analyses suggested that genes associated with clinical presentation are generalizable to non-Irish samples.

CONCLUSIONS: These findings strongly support the hypothesis that modifier loci contribute to the etiology of distinct Scz symptom profiles. The finding that previously implicated "risk loci" actually influence particular symptom dimensions has the potential to better delineate the roles of these genes in Scz etiology. Furthermore, the over-representation of distinct gene ontologies/pathways across symptom profiles suggests that the clinical heterogeneity of Scz is due in part to complex and diverse genetic factors.

Age-Related Macular Degeneration-Associated Genes in Alzheimer Disease

Objectives: Given the clinical and pathological similarities between age-relatedmacular degeneration (AMD) and Alzheimer disease (AD), to assess whether AMDassociatedsingle nucleotide polymorphisms (SNPs), including those from complementrelatedgenes, are associated with AD. 
Design: A case-control association study-typedesign. 
Setting: A UK tertiary care dementia clinic. 
Participants: 322 cognitivelynormal participants and 258 cases with a clinical diagnosis of AD.
Measurements:Polymorphisms in the following genes were studied: CFH, ARMS2, C2/CFB, C3, CFI/PLA2G12a, SERPING1, TLR3, TLR4, CRP, APOE, and TOMM40. Haplotypes were analysedfor CFH, TOMM40, and APOE. Univariate analysis was performed for each geneticchange and case-comparator status, and then correction for multiple testing performed. 
Results: The presence of an ε4 APOE allele was significantly associated with AD. Noassociation was evident between CFH SNPs or haplotypes, or other AMD-associated SNPstested, and AD. The exceptions were TOMM40 SNPs, which were associated with AD evenafter correction for multiple comparisons. The associations disappeared, however, whenentered into a regression model including APOE genotypes. 
Conclusions: The resultsfor most SNPs tested, as well as CFH haplotypes, are novel. The functional effects ofabnormal complement activity in AD’s pathogenesis may be contradictory, butmethodological reasons may underlie the lack of association—for example, geneticchanges other than SNPs being involved.

ISCR and their association with antibiotic resistance genes

A prevalência de bactérias resistentes a antibióticos em ambiente hospitalar tem vindo a tornar-se dramática e preocupante a nível mundial. Contudo, com a utilização inadequada de antibióticos em áreas tão diversas como a veterinária, a aquacultura e a agricultura, esta deixou de estar confinada ao ambiente hospitalar, sendo o ambiente um reservatório natural de microganismos resistentes a estes compostos. O conhecimento detalhado dos determinantes de resistência a antibióticos presentes nestes ambientes, sejam estes genes de resistência ou estruturas envolvidas na sua mobilização, é fundamental, não só do ponto de vista do conhecimento como para a eventual implementação de medidas de contenção da sua disseminação. Neste contexto, são necessários estudos que permitam conhecer o panorama mais realista da distribuição destes determinantes de resistência a antibióticos, quer no meio ambiente quer no ambiente clínico. Assim, constituiu objectivo principal deste trabalho contribuir para o conhecimento do panorama actual da prevalência e distribuição dos elementos ISCR, bem como de outros determinantes de resistência a antibióticos em espécies de Gram-negativo clinicamente relevantes (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii e Citrobacter freundii) recolhidas a partir de amostras de pacientes do Hospital Infante D. Pedro, EPE, Aveiro, Portugal, entre 2006 e 2008. Adicionalmente, foram também recolhidas bactérias de Gram-negativo do meio ambiente, a partir de amostras de águas e de vísceras de peixes, nas quais foram igualmente pesquisados os elementos acima referidos. À excepção dos isolados de E. coli e de Gramnegativo ambientais, todas as estirpes estudadas foram seleccionadas com base no seu perfil de multirresistência a antibióticos. Os resultados mostraram que em todas as espécies recolhidas no ambiente hospitalar foi detectada a presença de elementos ISCR, do tipo ISCR1 ou ISCR2. O elemento ISCR1, foi encontrado em isolados de E. coli, K. pneumoniae e C. freundii e o elemento ISCR2 em isolados de A. baumannii. Não foi detectada a presença de ISCRs nos isolados de Gram-negativo ambientais, o que sugere que a ocorrência dos mesmos é fortemente influenciada pela pressão selectiva exercida pelo ambiente em que os microrganismos se encontram. Genes qnr e integrões de classe 1 foram os determinantes de resistência mais frequentemente encontrados associados aos elementos ISCR1. Os vários determinantes de resistência foram encontrados em diferentes contextos genéticos e localizados em estruturas móveis, nomeadamente em plasmídeos. O elemento ISCR2 presente em isolados de A. baumannii encontra-se associado ao gene sul2 em todos os isolados, dentro de um mesmo contexto genético e com uma localização cromossomal. Contudo, o contexto genético encontrado nestes isolados é novo não tendo sido descrito até à data em outros microrganismos. O presente estudo constitui a primeira descrição de elementos ISCR intrinsecamente ligados a genes de resistência a antibióticos, em Portugal. Uma vez que estes elementos parecem ser responsáveis pela mobilização de um grande número de genes de resistência a antibióticos, a sua elevada incidência entre estirpes resistentes e multirresistentes, bem como a sua associação com genes de resistência é preocupante e requer vigilância.

Sistema computacional para o estudo da estrutura primária e redesenho de genes

Um dos maiores avanços científicos do século XX foi o desenvolvimento de tecnologia que permite a sequenciação de genomas em larga escala. Contudo, a informação produzida pela sequenciação não explica por si só a sua estrutura primária, evolução e seu funcionamento. Para esse fim novas áreas como a biologia molecular, a genética e a bioinformática são usadas para estudar as diversas propriedades e funcionamento dos genomas. Com este trabalho estamos particularmente interessados em perceber detalhadamente a descodificação do genoma efectuada no ribossoma e extrair as regras gerais através da análise da estrutura primária do genoma, nomeadamente o contexto de codões e a distribuição dos codões. Estas regras estão pouco estudadas e entendidas, não se sabendo se poderão ser obtidas através de estatística e ferramentas bioinfomáticas. Os métodos tradicionais para estudar a distribuição dos codões no genoma e seu contexto não providenciam as ferramentas necessárias para estudar estas propriedades à escala genómica. As tabelas de contagens com as distribuições de codões, assim como métricas absolutas, estão actualmente disponíveis em bases de dados. Diversas aplicações para caracterizar as sequências genéticas estão também disponíveis. No entanto, outros tipos de abordagens a nível estatístico e outros métodos de visualização de informação estavam claramente em falta. No presente trabalho foram desenvolvidos métodos matemáticos e computacionais para a análise do contexto de codões e também para identificar zonas onde as repetições de codões ocorrem. Novas formas de visualização de informação foram também desenvolvidas para permitir a interpretação da informação obtida. As ferramentas estatísticas inseridas no modelo, como o clustering, análise residual, índices de adaptação dos codões revelaram-se importantes para caracterizar as sequências codificantes de alguns genomas. O objectivo final é que a informação obtida permita identificar as regras gerais que governam o contexto de codões em qualquer genoma.

From genes to population: effects of toxicants on Enchytraeus albidus

Nowadays, a systems biology approach is both a challenge as well as believed to be the ideal form of understanding the organisms’ mechanisms of response. Responses at different levels of biological organization should be integrated to better understand the mechanisms, and hence predict the effects of stress agents, usable in broader contexts. The main aim of this thesis was to evaluate the underlying mechanisms of Enchytraeus albidus responses to chemical stressors. Therefore, there was a large investment on the gene library enrichment for this species, as explained ahead. Overall, effects of chemicals from two different groups (metals and pesticides) were assessed at different levels of biological organization: from genes and biochemical biomarkers to population endpoints. Selected chemicals were: 1) the metals cadmium and zinc; 2) the insecticide dimethoate, the herbicide atrazine and the fungicide carbendazim. At the gene and sub-cellular level, the effects of time and dosage were also adressed. Traditional ecotoxicological tests - survival, reproduction and avoidance behavior - indicated that pesticides were more toxic than metals. Avoidance behaviour is extremely important from an ecological point of view, but not recommended to use for risk assessment purposes. The oxidative stress related experiment showed that metals induced significant effects on several antioxidant enzyme activities and substrate levels, as well as oxidative damage on the membrane cells. To increase the potential of our molecular tool to assess transcriptional responses, the existing cDNA library was enriched with metal and pesticide responding genes, using Suppression Subtractive Hybridization (SSH). With the sequencing information obtained, an improved Agilent custom oligonucleotide microarray was developed and an EST database, including all existing molecular data on E. albidus, was made publicly available as an interactive tool to access information. With this microarray tool, most interesting and novel information on the mechanisms of chemical toxicity was obtained, with the identification of common and specific key pathways affected by each compound. The obtained results allowed the identification of mechanisms of action for the tested compounds in E. albidus, some of which are in line with the ones known for mammals, suggesting across species conserved modes of action and underlining the usefulness of this soil invertebrate as a model species. In general, biochemical and molecular responses were influenced by time of exposure and chemical dosage and these allowed to see the evolution of events. Cellular energy allocation results confirmed the gene expression evidences of an increased energetic expenditure, which can partially explain the decrease on the reproductive output, verified at a later stage. Correlations found throughout this thesis between effects at the different levels of biological organization have further improved our knowledge on the toxicity of metals and pesticides in this species.

Study of Perkinsus olseni infection mechanisms: identification and regulation of parasite genes differentially expressed in response to host and environmental stress

Tese dout., Biologia, Universidade do Algarve, 2008

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Role of the different actin genes during extrusion of capping protein mutant cells in the Drosophila wing blade epithelium

Dissertação mest., Biotecnologia, Universidade do Algarve, 2008

Identification of reliable reference genes for quantitative gene expression in the Mozambique tilapia, Oreochromis mossambicus

Dissertação de mest., Biologia Marinha (Aquacultura), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010