A let-7 microRNA-binding site polymorphism in 3'-untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy

<p>PURPOSE: recent studies have found that KRAS mutations predict resistance to monoclonal antibodies targeting the epidermal growth factor receptor in metastatic colorectal cancer (mCRC). A polymorphism in a let-7 microRNA complementary site (lcs6) in the KRAS 3' untranslated region (UTR) is associated with an increased cancer risk in non-small-cell lung cancer and reduced overall survival (OS) in oral cancers. We tested the hypothesis whether this polymorphism may be associated with clinical outcome in KRAS wild-type (KRASwt) mCRC patients treated with cetuximab monotherapy.</p><p>PATIENTS AND METHODS: the presence of KRAS let-7 lcs6 polymorphism was evaluated in 130 mCRC patients who were enrolled in a phase II study of cetuximab monotherapy (IMCL-0144). Genomic DNA was extracted from dissected formalin-fixed paraffin-embedded tumor tissue, KRAS mutation status and polymorphism were assessed using direct sequencing and PCR restriction fragment length polymorphism technique.</p><p>RESULTS: KRAS let-7 lcs6 polymorphism was found to be related to object response rate (ORR) in mCRC patients whose tumors had KRASwt. The 12 KRASwt patients harboring at least a variant G allele (TG or GG) had a 42% ORR compared with a 9% ORR in 55 KRASwt patients with let-7 lcs6 TT genotype (P = 0.02, Fisher's exact test). KRASwt patients with TG/GG genotypes had trend of longer median progression-free survival (3.9 versus 1.3 months) and OS (10.7 versus 6.4 months) compared to those with TT genotypes.</p><p>CONCLUSIONS: these results are the first to indicate that the KRAS 3'UTR polymorphism may predict for cetuximab responsiveness in KRASwt mCRC patients, which warrants validation in other clinical trials.</p>

Environmental selection on transcriptome-derived SNPs in a high gene flow marine fish, the Atlantic herring (Clupea harengus)

<p>High gene flow is considered the norm for most marine organisms and is expected to limit their ability to adapt to local environments. Few studies have directly compared the patterns of differentiation at neutral and selected gene loci in marine organisms. We analysed a transcriptome-derived panel of 281 SNPs in Atlantic herring (Clupea harengus), a highly migratory small pelagic fish, for elucidating neutral and selected genetic variation among populations and to identify candidate genes for environmental adaptation. We analysed 607 individuals from 18 spawning locations in the northeast Atlantic, including two temperature clines (5-12 ��C) and two salinity clines (5-35���). By combining genome scan and landscape genetic analyses, four genetically distinct groups of herring were identified: Baltic Sea, Baltic-North Sea transition area, North Sea/British Isles and North Atlantic; notably, samples exhibited divergent clustering patterns for neutral and selected loci. We found statistically strong evidence for divergent selection at 16 outlier loci on a global scale, and significant correlations with temperature and salinity at nine loci. On regional scales, we identified two outlier loci with parallel patterns across temperature clines and five loci associated with temperature in the North Sea/North Atlantic. Likewise, we found seven replicated outliers, of which five were significantly associated with low salinity across both salinity clines. Our results reveal a complex pattern of varying spatial genetic variation among outlier loci, likely reflecting adaptations to local environments. In addition to disclosing the fine scale of local adaptation in a highly vagile species, our data emphasize the need to preserve functionally important biodiversity.</p>

Gene-associated markers can assign origin in a weakly structured fish, Atlantic herring

<p>Regulations on the exploitation of populations of commercially important fish species and the ensuing consumer interest in sustainable products have increased the need to accurately identify the population of origin of fish and fish products. Although genomics-based tools have proven highly useful, there are relatively few examples in marine fish displaying accurate origin assignment. We synthesize data for 156 single-nucleotide polymorphisms typed in 1039 herring, Clupea harengus L., spanning the Northeast Atlantic to develop a tool that allows assignment of individual herring to their regional origin. We show the method's suitability to address specific biological questions, as well as management applications. We analyse temporally replicated collections from two areas, the Skagerrak (n = 81, 84, 66) and the western Baltic (n = 52, 52). Both areas harbour heavily fished mixed-origin stocks, complicating management issues. We report novel genetic evidence that herring from the Baltic Sea contribute to catches in the North Sea, and find support that western Baltic feeding aggregations mainly constitute herring from the western Baltic with contributions from the Eastern Baltic. Our study describes a general approach and outlines a database allowing individual assignment and traceability of herring across a large part of its East Atlantic distribution.</p>

Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility

<p>Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p &lt; 0.01). Merging gene expression and DNA methylation data revealed CHN2 as consistently hypermethylated and downregulated in this disease (Spearman -0.71, p &lt; 0.001). Mutations in TP53 which were found in more than half of the cohort (15/28) and Kazald1 hypomethylation were both were indicative of poor survival (p = 0.03, HR = 3.2 and p = 0.01, HR = 4.9 respectively). By integrating high-throughput mutational, gene expression and DNA methylation data, this study reveals for the first time the distinct molecular profile of small bowel adenocarcinoma and highlights potential clinically exploitable markers.</p>

Sequence and expression of complement factor H gene cluster variants and their roles in age-related macular degeneration risk

Purpose: To investigate how potentially functional genetic variants are coinherited on each of four common complement factor H (CFH) and CFH-related gene haplotypes and to measure expression of these genes in eye and liver tissues.<br/><br/>Methods: We sequenced the CFH region in four individuals (one homozygote for each of four common CFH region haplotypes) to identify all genetic variants. We studied associations between the haplotypes and AMD phenotypes in 2157 cases and 1150 controls. We examined RNA-seq profiles in macular and peripheral retina and retinal pigment epithelium/choroid/sclera (RCS) from eight eye donors and three liver samples.<br/><br/>Results: The haplotypic coinheritance of potentially functional variants (including missense variants, novel splice sites, and the CFHR3���CFHR1 deletion) was described for the four common haplotypes. Expression of the short and long CFH transcripts differed markedly between the retina and liver. We found no expression of any of the five CFH-related genes in the retina or RCS, in contrast to the liver, which is the main source of the circulating proteins.<br/><br/>Conclusions: We identified all genetic variants on common CFH region haplotypes and described their coinheritance. Understanding their functional effects will be key to developing and stratifying AMD therapies. The small scale of our expression study prevented us from investigating the relationships between CFH region haplotypes and their expression, and it will take time and collaboration to develop epidemiologic-scale studies. However, the striking difference between systemic and ocular expression of complement regulators shown in this study suggests important implications for the development of intraocular and systemic treatments.

QUADrATiC: scalable gene expression connectivity mapping for repurposing FDA-approved therapeutics

Background: Gene expression connectivity mapping has proven to be a powerful and flexible tool for research. Its application has been shown in a broad range of research topics, most commonly as a means of identifying potential small molecule compounds, which may be further investigated as candidates for repurposing to treat diseases. The public release of voluminous data from the Library of Integrated Cellular Signatures (LINCS) programme further enhanced the utilities and potentials of gene expression connectivity mapping in biomedicine. Results: We describe QUADrATiC (http://go.qub.ac.uk/QUADrATiC), a user-friendly tool for the exploration of gene expression connectivity on the subset of the LINCS data set corresponding to FDA-approved small molecule compounds. It enables the identification of compounds for repurposing therapeutic potentials. The software is designed to cope with the increased volume of data over existing tools, by taking advantage of multicore computing architectures to provide a scalable solution, which may be installed and operated on a range of computers, from laptops to servers. This scalability is provided by the use of the modern concurrent programming paradigm provided by the Akka framework. The QUADrATiC Graphical User Interface (GUI) has been developed using advanced Javascript frameworks, providing novel visualization capabilities for further analysis of connections. There is also a web services interface, allowing integration with other programs or scripts.Conclusions: QUADrATiC has been shown to provide an improvement over existing connectivity map software, in terms of scope (based on the LINCS data set), applicability (using FDA-approved compounds), usability and speed. It offers potential to biological researchers to analyze transcriptional data and generate potential therapeutics for focussed study in the lab. QUADrATiC represents a step change in the process of investigating gene expression connectivity and provides more biologically-relevant results than previous alternative solutions.

Tributyltin (TBT) resistance in Aeromonas molluscorum Av27

O tributilestanho (TBT) �� considerado um dos xenobi��ticos mais t��xicos, produzidos e deliberadamente introduzidos no meio ambiente pelo Homem. Tem sido usado numa variedade de processos industriais e subsequentemente descarregado no meio ambiente. O tempo de meia-vida do TBT em ��guas marinhas �� de v��rias semanas, mas em condi����es de an��xia nos sedimentos, pode ser de v��rios anos, devido �� sua degrada����o mais lenta. Embora o TBT tenha sido descrito como sendo t��xico para eucariotas e procariotas, muitas bact��rias podem ser resistentes a este composto. O presente trabalho teve como objetivo principal elucidar o mecanismo de resist��ncia ao TBT em bact��rias. Para al��m disso, pretendeu-se desenvolver um biorep��rter para detectar TBT no ambiente. Para atingir estes objetivos foram delineadas v��rias tarefas cujos principais resultados obtidos se apresentam a seguir. V��rias bact��rias resistentes ao TBT foram isoladas de sedimento e ��gua do Porto de Pesca Long��nqua (PPL) na Ria de Aveiro, Portugal. Entre estas, Aeromonas molluscorum Av27 foi selecionada devido �� sua elevada resist��ncia a este composto (concentra����es at�� 3 mM), �� sua capacidade de degradar o TBT em compostos menos t��xicos (dibutilestanho, DBT e monobutilestanho, MBT) e tamb��m por usar o TBT como fonte de carbono. A. molluscorum Av27 foi caracterizada genotipica e fenotipicamente. Os fatores de virul��ncia estudados mostraram que esta estirpe i) possui atividade lipol��tica; ii) n��o �� citot��xica para c��lulas de mam��feros, nomeadamente para c��lulas Vero; iii) n��o possui integr��es de classe I e II e iv) possui cinco plasm��deos com aproximadamente 4 kb, 7 kb, 10 kb, 100 kb e mais de 100 kb. Estes resultados mostraram que a estirpe Av27 n��o �� t��xica, aumentando assim o interesse nesta bact��ria para futuras aplica����es, nomeadamente na bioremedia����o. Os testes de toxicidade ao TBT mostraram que este composto tem um impacto negativo no crescimento desta estirpe, bem como, na densidade, no tamanho e na atividade metab��lica das c��lulas e �� respons��vel pela forma����o de agregados celulares. Assim, o TBT mostrou ser bastante t��xico para as bact��rias interferindo com a atividade celular geral. O gene Av27-sugE, que codifica a prote��na SugE pertencente �� fam��lia das ���small multidrug resistance proteins��� (SMR), foi identificado como estando envolvido na resist��ncia ao TBT nesta estirpe. Este gene mostrou ser sobreexpresso quando as c��lulas crescem na presen��a de TBT. O promotor do gene Av27-sugE foi utilizado para construir um bioreporter para detetar TBT, contendo o gene da luciferase do pirilampo como gene rep��rter. O biorep��rter obtido re��ne as caracter��sticas mais importantes de um bom biorep��rter: sensibilidade (intervalo de limite de detec����o de 1-1000 nM), rapidez (3 h s��o suficientes para a dete����o de sinal) e, possivelmente, n��o �� invasivo (pois foi constru��do numa bact��ria ambiental). Usando sedimento recolhido no Porto de Pesca Long��nqua da Ria de Aveiro, foi preparada uma experi��ncia de microcosmos com o intuito de avaliar a capacidade de Av27 para bioremediar o TBT, isoladamente ou em associa����o com a comunidade bacteriana ind��gena. A an��lise das amostras de microcosmos por PCR-DGGE e de bibliotecas de 16S rDNA revelaram que a comunidade bacteriana �� relativamente est��vel ao longo do tempo, mesmo quando Av27 �� inoculada no sedimento. Para al��m disso, o sedimento estuarino demonstrou ser dominado por bact��rias pertencentes ao filo Proteobacteria (sendo mais abundante as Delta e Gammaproteobacteria) e Bacteroidetes. Ainda, cerca de 13% dos clones bacterianos n��o revelaram nenhuma semelhan��a com qualquer dos filos j�� definidos e quase 100% afiliou com bact��rias n��o cultiv��veis do sedimento. No momento da conclus��o desta tese, os resultados da an��lise qu��mica de compostos organoest��nicos n��o estavam dispon��veis, e por essa raz��o n��o foi poss��vel tirar quaisquer conclus��es sobre a capacidade desta bact��ria remediar o TBT em sedimentos. Esses resultados ir��o ajudar a esclarecer o papel de A. molluscorum Av27 na remedia����o de TBT. Recentemente, a capacidade da estirpe Av27 remediar solo contaminado com TBT foi confirmada em bioensaios realizados com plantas, Brassica rapa e Triticum aestivum (Silva 2011a), e tamb��m com invertebrados Porcellionides pruinosus (Silva 2011B). Assim, poder-se-�� esperar que a bioremedia����o do sedimento na experi��ncia de microcosmos tamb��m tenha ocorrido. No entanto, s�� a an��lise qu��mica dos compostos organost��nicos dever�� ser conclusiva. Devido �� dificuldade em realizar a an��lise anal��tica de organoest��nicos, um m��todo de bioensaio f��cil, r��pido e barato foi adaptado para avaliar a toxicidade do TBT em laborat��rio, antes de se proceder �� an��lise qu��mica das amostras. O m��todo provou a sua utilidade, embora tenha mostrado pouca sensibilidade quando se usam concentra����es de TBT baixas. Em geral, os resultados obtidos contribu��ram para um melhor entendimento do mecanismo de resist��ncia ao TBT em bact��rias e mostraram o potencial biotecnol��gico de A. molluscorum Av27, nomeadamente, no que refere �� sua poss��vel aplica����o na descontamina����o de TBT no ambiente e tamb��m no desenvolvimento de biorep��rteres.

Estudo da variabilidade gen��tica de marcadores moleculares ligados ao gene da MGP e sua aplica����o para paternidades em Sparus aurata

Disserta����o de Mestrado, Biologia Marinha, Especializa����o em Biotecnologia Marinha, Faculdade de Ci��ncias do Mar e do Ambiente, Universidade do Algarve, 2008

Polymorphisms at the five exons of gowth hormone gene in the Algarvia Goat. Possible association with milk traits

The present preliminary study attempts to establish associations between milk production traits and genetic polymorphisms at the GH gene in the Algarvia goat. The DNA of 108 goats of the indigenous Portuguese Algarvia breed was evaluated.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

Cloning of the Bone Gla Protein gene from the teleost fish Sparus aurata (gilthead seabream). Molecular organization, developmental appearance and evolutionary implications

Tese de Doutoramento, Biologia Molecular, Faculdade de Ci��ncias do Mar e do Ambiente, Universidade do Algarve, 2001

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ci��ncias Biom��dicas, Departamento de Ci��ncias Biom��dicas e Medicina, Universidade do Algarve, 2015

The role of reciprocal regulation between TAL1 and miRNA expression in T-cell leukemia progression

Tese de doutoramento, Ci��ncias Biom��dicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2015

Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ci��ncias Biom��dicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ci��ncias, 2015