Synthesis, characterization and reactivity of new cobalt, palladium and nickel complexes bearing α-diimines and P, O ligands

This work describes the synthesis and characterization of a series of new α-diimine and P,O, β-keto and acetamide phosphines ligands, and their complexation to Ni(II), Co(II),Co(III) and Pd(II) to obtain a series of new compounds aiming to study their structural characteristics and to test their catalytic activity. All the compounds synthesized were characterized by the usual spectroscopic and spectrometric techniques: Elemental Analysis, MALDI-TOF-MS spectrometry, IR, UV-vis, 1H, 13C and 31P NMR spectroscopies. Some of the paramagnetic compounds were also characterized by EPR. For the majority of the compounds it was possible to solve their solid state structure by single crystal X-ray diffraction. Tests for olefin polymerization were performed in order to determine the catalytic activity of the Co(II) complexes. Chapter I presents a brief introduction to homogenous catalysis, highlighting the reactions catalyzed by the type of compounds described in this thesis, namely olefin polymerization and oligomerization and reactions catalyzed by the complexes bearing α-diimines and P,O type ligands. Chapter II is dedicated to the description of the synthesis of new α-diimines cobalt (II) complexes, of general formula [CoX2(α-diimine)], where X = Cl or I and the α-diimines are bis(aryl)acenaphthenequinonediimine) (Ar-BIAN) and 1,4-diaryl-2,3-dimethyl-1,4-diaza-1,3-butadiene (Ar-DAB). Structures solved by single crystal X-ray diffraction were obtained for all the described complexes. For some of the compounds, X-band EPR measurements were performed on polycrystalline samples, showing a high-spin Co(II) (S = 3/2) ion, in a distorted axial environment. EPR single crystal experiments on two of the compounds allowed us to determine the g tensor orientation in the molecular structure. In Chapter III we continue with the synthesis and characterization of more cobalt (II)complexes bearing α-diimines of general formula [CoX2(α-diimine)], with X = Cl or I and α-diimines are bis(aryl)acenaphthenequinonediimine) (Ar-BIAN) and 1,4-diaryl-2,3-dimethyl- 1,4-diaza-1,3-butadiene (Ar-DAB). The structures of three of the new compounds synthesized were determined by single crystal X-ray diffraction. A NMR paramagnetic characterization of all the compounds described is presented. Ethylene polymerization tests were done to determine the catalytic activity of several of the Co(II) complexes described in Chapter II and III and their results are shown. In Chapter IV a new rigid bidentate ligand, bis(1-naphthylimino)acenaphthene, and its complexes with Zn(II) and Pd(II), were synthesized. Both the ligand and its complexes show syn and anti isomers. Structures of the ligand and the anti isomer of the Pd(II) complex were solved by single crystal X-ray diffraction. All the compounds were characterized by elemental analysis, MALDI-TOF-MS spectrometry, and by IR, UV-vis, 1H, 13C, 1H-1H COSY, 1H-13C HSQC, 1H-13C HSQC-TOCSY and 1H-1H NOESY NMR when necessary. DFT studies showed that both conformers of [PdCl2(BIAN)] are isoenergetics and can be obtain experimentally. However, we can predict that the isomerization process is not available in square-planar complex, but is possible for the free ligand. The molecular geometry is very similar in both isomers, and only different orientations for naphthyl groups can be expected. Chapter V describes the synthesis of new P, O type ligands, β-keto phosphine, R2PCH2C(O)Ph, and acetamide phosphine R2PNHC(O)Me, as well as a series of new cobalt(III) complexes namely [(η5-C5H5)CoI2{Ph2PCH2C(O)Ph}], and [(η5- C5H5)CoI2{Ph2PNHC(O)Me}]. Treating these Co(III) compounds with an excess of Et3N, resulted in complexes η2-phosphinoenolate [(η5-C5H5)CoI{Ph2PCH…C(…O)Ph}] and η2- acetamide phosphine [(η5-C5H5)CoI{Ph2PN…C(…O)Me}]. Nickel (II) complexes were also obtained: cis-[Ni(Ph2PN…C(…O)Me)2] and cis-[Ni((i-Pr)2PN…C(…O)Me)2]. Their geometry and isomerism were discussed. Seven structures of the compounds described in this chapter were determined by single crystal X-ray diffraction. The general conclusions of this work can be found in Chapter VI.

Analysis of support for modularity in object teams based on design patterns

Dissertação de Mestrado em Engenharia Informática

Comparative analysis of time-frequency methods estimating the time-varying microstructure of sleep EEG spindles

Proceedings of the Information Technology Applications in Biomedicine, Ioannina - Epirus, Greece, October 26-28, 2006

Energy-dispersive x-ray fluorescence analysis of modern coloured glasses from Marinha Grande (Portugal)

X-Ray Spectrom. 2003; 32: 396–401

Penicillium glabrum cork colonising isolates – preliminary analysis of their genomic similari

Cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are yet to be completely understood and are considered to be involved in the so called “cork taint” of bottled wine. It is essential to identify environmental constraints which define the appearance of the colonising fungal species and to trace their origin to the forest and/or as residents in the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic biologic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was identified, followed by a high diversity of Penicillium species. Penicillium glabrum, found in all samples, was the most frequent isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork resident fungi can also contrib

Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain

J Biol Inorg Chem (2011) 16:881–888 DOI 10.1007/s00775-011-0785-8

Earth-based repair mortars: experimental analysis with different binders and natural fibers

RESTAPIA 2012 - Int. Conf. on Rammed Earth Conservation, Valencia, 21-23 June 2012

Adaptação cultural e contributo para a validação da escala petterns of activity measura - pain (POAM-P)

RESUMO: A dor lombar crónica (DLC) é uma das condições clínicas mais comuns e com elevados custos socioeconómicos no mundo ocidental. Estudos recentes indicam que os utentes com DLC apresentam diferentes padrões de atividade que influenciam os níveis de incapacidade funcional. Contudo, a evidência acerca destas associações é, ainda, limitada e inconclusiva. Em Portugal, não existe, do nosso conhecimento, nenhuma escala validada para a população portuguesa que meça estes padrões de atividade em utentes com DLC. Objetivos: Adaptar culturalmente a escala Patterns of Activity Measure – Pain (POAM-P) para a população portuguesa com dor lombar crónica inespecífica (DLCI) e contribuir para a sua validação. Metodologia: A versão original (inglesa) do POAM-P foi traduzida e adaptada para a língua portuguesa (POAM-P-VP) através de uma equipa multidisciplinar que incluiu tradutores, retrotradutores (cegos e independentes), peritos de diferentes áreas e utentes com DLCI, de acordo com as recomendações de linhas orientadoras atuais para este processo. A análise factorial e das propriedades psicométricas da POAM-P-VP contou com uma amostra de 132 utentes. A consistência interna foi analisada através do coeficiente alpha de Cronbach (α) e para a análise da fiabilidade teste-reteste recorreu-se ao coeficiente de correlação intraclasse (ICC:2,1). A análise da validade de construto convergente e discriminativa das componentes da POAM-P-VP foi conseguida através da aplicação da versão portuguesa da escala Tampa Scale of Kinesiophobia (TSK-13-VP), e recorrendo ao cálculo do coeficiente de Spearman. Todos os cálculos estatísticos foram realizados no software IBM SPSS Statistics (versão 20). Resultados: A análise factorial permitiu identificar três componentes da POAM-P-VP (evitamento, persistência excessiva e persistência consistente com a dor), sendo estruturalmente diferentes das subescalas do POAM-P original. Estas componentes apresentaram uma consistência interna boa a elevada. As componentes 1 e 2 apresentaram uma fiabilidade teste-reteste moderada a excelente, e a componente 3 uma fiabilidade teste-reteste pobre, limitando o seu poder de uso na prática clínica e em investigação. Relativamente à validade de construto, nenhuma das hipóteses estabelecidas no estudo apriori foram verificadas, não podendo aferir acerca da relação dos padrões de atividade com a cinesiofobia, medida pelo TSK-13-VP. Porém, a componente de evitamento da POAM-P-VP parece medir conteúdos partilhados com a TSK-13-VP (rs = 0.15, p<0.048). Conclusão: A adaptação e contributo para a validação da versão portuguesa da escala POAM-P constituiu um ponto de partida para a existência de um instrumento de medição de padrões de atividade de utentes portugueses com DLC, requerendo mais estudos para a sua validação. Apesar de algumas limitações, considera-se que este estudo é de grande importância para os fisioterapeutas e investigadores que buscam um maior conhecimento e efetividade das abordagens de intervenção em utentes com dor lombar crónica.-------------- ABSTRACT: Chronic low back pain (CLBP) is one of the most common clinical conditions as well as one with high economical costs within western countries. Recent studies have shown that patients with LBP present different patterns of activity which influence their levels of functional capacity. However, evidence on these associations is still limited and inconclusive. To our knowledge, there is in Portugal no valid scale for measuring these patterns of activity in CLBP patients. Purpose: Culturally adapt the Patterns of Activity Measure – Pain (POAM-P) scale to the Portuguese population with non-specific chronic low back pain (NSLBP) and contribute to its validation. Method: The original English version of POAM-P was blindly and independently translated, back translated and adapted to the Portuguese language (POAM-P-VP) by a multidisciplinary team of translators, experts from different fields, and patients with NSLBP, according to established guidelines for this process. Factorial and psychometric properties’ analysis of POAM-P-VP comprised a sample of 132 patients. The internal consistency was analyzed based on Cronbach's alpha-coefficient (α) and for test-retest reliability analysis the Intraclass Correlation Coefficient (ICC) was used. The analysis of convergent and discriminant construct validity of POAM-P-VP components was achieved through the use of the Portuguese version of the Tampa Scale of Kinesiophobia (TSK-13-VP), using the Spearman coefficient calculation. All statistical calculations were performed using IBM SPSS Statistics software (v.20). Results: The factor analysis allowed for the identification of three components of POAM-P-VP (avoidance, excessive persistence and pain-contingent persistence), structurally different from the original POAM-P subscales. These components demonstrated a good to high level of internal consistency. Components 1 and 2 demonstrated moderate to excellent test-retest reliability, whereas component 3 presented low test-retest reliability thus limiting its clinical and investigative use. With regard to construct validity, none of the previously established hypothesis was verified, therefore not making it possible to assess the relation between activity patterns and kinesiophobia, measured by TSK-13-VP. However, the avoidance component of POAM-P-VP seems to share measurable contents with TSK-13-VP (rs = 0.15, p<0.048). Conclusion: The adaptation and contribution to the validation of the Portuguese version of POAM-P scale, sets a starting point to the existence of a useful instrument for measuring activity patterns in Portuguese CLBP patients, requiring further studies towards its validation. Despite some limitations, this study is considered of high importance to physiotherapists as well as investigators in search of deeper knowledge and effective practical approaches on chronic low back pain patients.

Territorial Strategic Planning as a support instrument for Regional and Local Development: a comparative analysis between Lisbon and Barcelona Metropolitan Areas

MARQUES, B.P. (2011) "Territorial Strategic Planning as a support instrument for Regional and Local Development: a comparative analysis between Lisbon and Barcelona Metropolitan Areas", in Atas do 17.º Congresso da APDR, do 5.º Congresso de Gestão e Conservação da Natureza e do Congresso Internacional da APDR/AECR, Bragança e Zamora, pp. 1265-1272, ISBN 978-989-96353-2-6.

Analysis and treatment of a nineteenth century oil painting

This thesis focused on the study and treatment of a 19th century female portrait in oil from ECOMUSEU Municipal do Seixal, Portugal. The portrait, which depicts Isabel Maria Lourenço Affonso was in poor condition and a large strip of paint and canvas was missing (approximately 9cm by 66cm, almost 11% of the total surface area). The portrait is a companion piece to a male portrait (the relationship was established as part of this study), therefore a technical study of both paintings was considered essential to support the choices made during the treatment. The project involved three main areas: - The study of the history, condition, materials and techniques of both paintings. This allowed their comparison and understanding of their relationship; - The treatment of Isabel Maria Lourenço Affonso. The choices made and problems encountered are described. - The production of a replacement for the missing strip of paint and canvas. The practical solution developed to overcome such an unusual challenge is described along with the creative and flexible thinking required. Because not all traditional infill materials cope well on a mechanical level with thin layers over a very large surface (many are too brittle), strict criteria had to be employed to choose the appropriate material. The primary goal was to find a fill which would remain flexible and be capable of accepting surface texture, such that there would be a good visual match with the painting. Analysis and testing was carried out to evaluate the physical properties of the fill material chosen, BEVA® Gesso-P. The successful creation of the replacement strip has resulted in two publications and one presentation: Publication pending in The Picture Restorer, Leslie Carlyle, Raquel Marques, Isabel Pombo Cardoso and Sara Babo, “Creating a Textured Replacement Strip for the Missing Lower Portion of an Oil Portrait: Problem Solving and Practical Solutions”. Abstract accepted for presentation and publication, International Meeting on Retouching of Cultural Heritage (2RECH), Raquel Marques, Leslie Carlyle and Isabel Pombo Cardoso, “Textured Replacement Strip for a Missing Portion of a Portrait: Problem Solving and Practical Solutions”.

Satisfação dos utilizadores de sistemas de informação e documentação de enfermagem em suporte eletrónico no Centro Hospitalar Lisboa Norte, E.P.E.

RESUMO - Os registos de enfermagem no Centro Hospitalar Lisboa Norte, E.P.E. (CHLN) são feitos em suporte de papel ou através de sistemas de informação (SI) próprios de cada serviço, com a utilização de várias aplicações como o Alert, Picis, etc. Esta diversidade gera alguns constrangimentos em termos de fluxo de informação, em virtude da falta de interoperabilidade dos respetivos sistemas. Esta realidade pode ter impactos na área da qualidade e segurança do utente, com a possibilidade de ocorrência de erros e/ou eventos adversos. Podem ainda ser notórios na área da privacidade e confidencialidade dos dados clínicos, na tomada de decisão, na gestão clínica e financeira e na produção de informação útil para a investigação científica. No CHLN está em curso a implementação de um SI capaz de dar resposta aos registos de enfermagem, integrados num registo de saúde eletrónico focado no utente que obedece à metodologia do processo de enfermagem e utiliza a linguagem codificada da Classificação Internacional para a Prática de Enfermagem (CIPE). Com o desenvolvimento desta investigação, devidamente autorizada pelo Conselho de Administração do CHLN, pretendeu-se dar resposta à pergunta de partida:  Estarão os enfermeiros, utilizadores do Desktop de Enfermagem do CHLN, satisfeitos com esse sistema de informação? Com esse propósito, foi elaborada uma abordagem exploratória com recurso a pesquisa bibliográfica sobre os sistemas de informação de enfermagem e a sua avaliação, com base no “Modelo de Sucesso dos Sistemas de Informação de DeLone e McLean”, tendo sido desenvolvido um estudo de caso com uma abordagem quantitativa, mediante a aplicação de um inquérito por questionário aos 262 enfermeiros do CHLN, nos serviços onde já utilizavam o referido SI, entre maio e junho de 2014, com uma taxa de resposta de 84%. Os resultados da aplicação do questionário, objeto de análise estatística univariada e bivariada com recurso a procedimentos descritivos e inferenciais, visando a produção de sínteses dirigidas aos objetivos do estudo, permitiram caracterizar o nível de satisfação dos enfermeiros, enquanto utilizadores do “desktop de enfermagem”, suportados por Tecnologias de Informação e Comunicação. Na escala utilizada (de 1 a 5), o nível médio de satisfação global (2,78) foi ligeiramente inferior ao seu ponto médio (3). No entanto, a maioria dos inquiridos (81,5%) não pretende abandonar o SI que utilizam. Os resultados obtidos permitem demonstrar que a satisfação dos enfermeiros face à implementação e utilização do SIE se trata de uma estratégia bem sucedida do CHLN, ainda que haja áreas onde foram evidenciados menores níveis de satisfação, tais como a “velocidade de processamento”, o “equipamento informático” e o “apoio técnico”, que podem ser alvo de uma maior atenção e reflexão pela gestão de topo, numa estratégia de melhoria contínua da qualidade, com importantes benefícios para a governação da instituição, para os profissionais e para os utentes, no futuro.

Analysis of outcome and cancer stem cells status in patients with resectable pancreatic adenocarcinoma

RESUMO: Actualmente, a única possibilidade de cura para doentes com adenocarcinoma do pâncreas (PDAC) é a ressecção cirúrgica, no início deste estudo, perguntamo-nos se os predictores clínico-patológicos clássicos de prognostico poderiam ser validados em uma grande cohort de doentes com cancro do pâncreas ressecável e se outros predictores clínicos poderiam ter um papel na decisão de que doentes beneficiariam de ressecção cirúrgica. No capítulo 2, observamos que até 30% dos doentes morrem no primeiro ano após a ressecção cirúrgica, pelo que o nosso objectivo foi determinar factores pré-operatórios que se correlacionam com mortalidade precoce após ressecação cirúrgica com recurso a um instrumento estatisticamente validado, o Charlson-Age Comorbidity Index (CACI), determinamos que um CACI score superior a 4 foi preditivo de internamentos prolongados (p <0,001), complicações pós-operatórias (p = 0,042), e mortalidade em 1 ano pós- ressecção cirúrgica (p <0,001). Um CACI superior a 6 triplicou a mortalidade no primeiro ano pós-cirurgia e estes doentes têm menos de 50% de probabilidade de estarem vivos um ano após a cirurgia. No capítulo 3, o nosso objectivo foi identificar uma proteína de superfície que se correlacionasse estatisticamente com o prognostico de doentes com adenocarcinoma do pâncreas e permitisse a distinção de subgrupos de doentes de acordo com as suas diferenças moleculares, perguntamo-nos ainda se essa proteína poderia ser um marcador de células-estaminais. No nosso trabalho anterior observamos que as células tumorais na circulação sanguínea apresentavam genes com características bifenotípica epitelial e mesenquimal, enriquecimento para genes de células estaminais (ALDH1A1 / ALDH1A2 e KLF4), e uma super-expressão de genes da matriz extracelular (colagénios, SPARC, e DCN) normalmente identificados no estroma de PDAC. Após a avaliação dos tumores primários com RNA-ISH, muitos dos genes identificados, foram encontrados co-localizando em uma sub-população de células na região basal dos ductos pancreáticos malignos. Além disso, observamos que estas células expressam o marcador SV2A neuroendócrino, e o marcador de células estaminais ALDH1A1/2. Em comparação com tumores negativos para SV2, os doentes com tumores SV2 positivos apresentaram níveis mais baixos de CA 19-9 (69% vs. 52%, p = 0,012), tumores maiores (> 4 cm, 23% vs. 10%, p = 0,0430), menor invasão de gânglios linfáticos (69% vs. 86%, p = 0,005) e tumores mais diferenciados (69% vs. 57%, p = 0,047). A presença de SV2A foi associada com uma sobrevida livre de doença mais longa (HR: 0,49 p = 0,009) bem como melhor sobrevida global (HR: 0,54 p = 0,018). Em conjunto, esta informação aponta para dois subtipos diferentes de adenocarcinoma do pâncreas, e estes subtipos co-relacionam estatisticamente com o prognostico de doentes, sendo este subgrupo definido pela presença do clone celular SV2A / ALDH1A1/2 positivo com características neuroendócrinas. No Capítulo 4, a expressão de SV2A no cancro do pâncreas foi validado em linhas celulares primárias. Demonstramos a heterogeneidade do adenocarcinoma do pâncreas de acordo com características clonais neuroendócrinas. Ao comparar as linhas celulares expressando SV2 com linhas celulares negativas, verificamos que as linhas celulares SV2+ eram mais diferenciadas, diferindo de linhas celulares SV2 negativas no que respeita a mutação KRAS, proliferação e a resposta à quimioterapia. No capítulo 5, perguntamo-nos se o clone celular SV2 positivo poderia explicar a resistência a quimioterapia observada em doentes. Observamos um aumento absoluto de clones celulares expressando SV2A, em múltiplas linhas de evidência - doentes, linhas de células primárias e xenotransplantes. Embora, tenhamos sido capazes de demonstrar que o adenocarcinoma do pâncreas é uma doença heterogénea, consideramos que a caracterização genética destes clones celulares expressando SV2A é de elevada importância. Pretendemos colmatar esta limitação com as seguintes estratégias: Após o tratamento com quimioterapia neoadjuvante na nossa coorte, realizamos microdissecação a laser das amostras primarias em parafina, de forma a analisar mutações genéticas observadas no adenocarcinoma pancreático; em segundo lugar, pretendemos determinar consequências de knockdown da expressão de SV2A em nossas linhas celulares seguindo-se o tratamento com gemicitabina para determinação do papel funcional de SV2A; finalmente, uma vez que os nossos esforços anteriores com um promotor - repórter e SmartFlare ™ falharam, o próximo passo será realizar RNA-ISH PrimeFlow™ seguido de FACS e RNA-seq para caracterização deste clone celular. Em conjunto, conseguimos provar com várias linhas de evidência, que o adenocarcinoma pancreático é uma doença heterogénea, definido por um clone de células que expressam SV2A, com características neuroendócrinas. A presença deste clone no tecido de doentes correlaciona-se estatisticamente com o prognostico da doença, incluindo sobrevida livre de doença e sobrevida global. Juntamente com padrões de proliferação e co-expressão de ALDH1A1/2, este clone parece apresentar um comportamento de células estaminais e está associado a resistência a quimioterapia, uma vez que a sua expressão aumenta após agressão química, quer em doentes, quer em linhas de células primárias.----------------------------- ABSTRACT: Currently, the only chance of cure for patients with pancreatic adenocarcinoma is surgical resection, at the beginning of my thesis studies, we asked if the classical clinicopathologic predictors of outcome could be validated in a large cohort of patients with early stage pancreatic cancer and if other clinical predictors could have a role on deciding which patients would benefit from surgery. In chapter 2, we found that up to 30% of patients die within the first year after curative intent surgery for pancreatic adenocarcinoma. We aimed at determining pre-operative factors that would correlate with early mortality following resection for pancreatic cancer using a statistically validated tool, the Charlson-Age Comorbidity Index (CACI). We found that a CACI score greater than 4 was predictive of increased length of stay (p<0.001), post-operative complications (p=0.042), and mortality within 1-year of pancreatic resection (p<0.001). A CACI score of 6 or greater increased 3-fold the odds of death within the first year. Patients with a high CACI score have less than 50% likelihood of being alive 1 year after surgery. In chapter 3 we aimed at identifying a surface protein that correlates with patient’s outcome and distinguishes sub-groups of patients according to their molecular differences and if this protein could be a cancer stem cell marker. The most abundant class of circulating tumor cells identified in our previous work was found to have biphenotypic features of epithelial to mesenchymal transition, enrichment for stem-cell associated genes (ALDH1A1/ALDH1A2 and KLF4), and an overexpression of extracellular matrix genes (Collagens, SPARC, and DCN) normally found in the stromal microenvironment of PDAC primary tumors. Upon evaluation of matched primary tumors with RNA-ISH, many of the genes identified were found to co-localize in a sub-population of cells at the basal region of malignant pancreatic ducts. In addition, these cells expressed the neuroendocrine marker SV2A, and the stem cell marker ALDH1A1/2. Compared to SV2 negative tumors, patients with SV2 positive tumors were more likely to present with lower CA 19-9 (69% vs. 52%, p = 0.012), bigger tumors (size > 4 cm, 23% vs. 10%, p= 0.0430), less nodal involvement (69% vs. 86%, p = 0.005) and lower histologic grade (69% vs. 57%, p = 0.047). The presence of SV2A expressing cells was associated with an improved disease free survival (HR: 0.49 p=0.009) and overall survival (HR: 0.54 p=0.018) and correlated linearly with ALDH1A2. Together, this information points to two different sub-types of pancreatic adenocarcinoma, and these sub-types correlated with patients’ outcome and were defined by the presence of a SV2A/ ALDH1A1/2 expressing clone with neuroendocrine features. In Chapter 4, SV2A expression in cancer was validated in primary cell lines. We were able to demonstrate pancreatic adenocarcinoma heterogeneity according to neuroendocrine clonal features. When comparing SV2 expressing cell lines with SV2 negative cell lines, we found that SV2+ cell lines were more differentiated and differ from SV2 negative cell lines regarding KRAS mutation, proliferation and response to chemotherapy. In Chapter 5 we aimed at determining if this SV2 positive clone could explain chemoresistance observed in patients. We found an absolute increase in SV2A expressing cells, with multiple lines of evidence, in patients, primary cell lines and xenografts. Although, we have been able to show evidence that pancreatic adenocarcinoma is a heterogeneous disease, our findings warrant further investigation. To further characterize SV2A expressing clones after treatment with neoadjuvant chemotherapy in our cohort, we have performed laser capture microdissection of the paraffin embedded tissue in this study and will analyze the tissue for known genetic mutations in pancreatic adenocarcinoma; secondly, we want to know what will happen after knocking down SV2A expression in our cell lines followed by treatment with gemcitabine to determine if SV2A is functionally important; finally, since our previous efforts with a promoter – reporter and SmartFlare™ have failed, we will utilize a novel PrimeFlow™ RNA-ISH assay followed by FACS and RNA sequencing to further characterize this cellular clone. Overall our data proves, with multiple lines of evidence, that pancreatic adenocarcinoma is a heterogeneous disease, defined by a clone of SV2A expressing cells, with neuroendocrine features. The presence of this clone in patients’ tissue correlates with patient’s disease free survival and overall survival. Together with patterns of proliferation and ALDH1A1/2 co-expression, this clone seems to present a stem-cell-like behavior and is associated with chemoresistance, since it increases after chemotherapy, both in patients and primary cell lines.