A first analysis of the atmospheric measurements from TANSO/GOSAT

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau em Mestre em Engenharia Física

Variação dos parâmetros paleomagnéticos em perfis dos Tufos de Condeixa (Baixo Mondego, Portugal)

Paleomagnetic parameters measured in 4 outcrops of the Tufos de Condeixa are presented. The directional signatures allow the admission of the Reversed Polarity Criptchron C1n-1 in the Condeixa-a-Velha outcrop. It was possible to discuss the magnetic properties, origin of the magnetic grains and paleoclimatic variation relationships. The downward of the initial magnetization and magnetic susceplibility values in the Condeixa-a-Velha outcrop coincide with the Cln-1 (504-493 Ka) occurrence and may represent a paleoclimatic variation. The Tufos de Condeixa were formed during the Ionian-Tarentian(?).

High-resolution correlation of coastal cliff sections in the Lagos- Portimao Formation (Lower - Middle Miocene, central Algarve, Portugal)

This paper describes a high-resolution stratigraphic correlation scheme for the early to middle Miocene Lagos-Portimão Formation of central Algarve, southern Portugal. The Lagos Portimão-Formation of central Algarve is a 60 m thick package of horizontally bedded siliciclastics and carbonates. The bryozoan and mollusc dominated biofacies is typical of a shallow marine, warm-temperate climatic environment. We define four stratigraphic marker beds based on biofacies, lithology, and gamma-ray signatures. Marker bed 1 is a reddish shell bed composed predominantly of bivalve shells in various stages of fragmentation. Marker bed 2 is a fossiliferous sandstone / sandy rudstone characterized by bryozoan masses. Marker bed 3 is also a fossiliferous sandstone with abundant larger foraminifers and foliate bryozoans. Marker bed 4 is composed of three distinct layers; two fossiliferous sandstones with an intercalated shell bed. The upper sandstone unit displays thickets of the bryozoan Celleporaria palmate associated with the coral Culizia parasitica. This stratigraphic framework allows to correlate isolated outcrops within the stratigraphic context of the Lagos-Portimão Formation and to establish high resolution chronostratigraphic Sr-isotopic dating.

Biological predictors of survival in limphoma and mechanisms underlying follicular lymphoma transformaion into diffuse large B cell lymphoma (vol I e II)

RESUMO: Os biomarcadores tumorais permitem identificar os doentes com maior risco de recorrência da doença, predizer a resposta tumoral à terapêutica e, finalmente, definir candidatos a novos alvos terapêuticos. Novos biomarcadores são especialmente necessários na abordagem clínica dos linfomas. Actualmente, esses tumores são diagnosticados através de uma combinação de características morfológicas, fenotípicas e moleculares, mas o prognóstico e o planeamento terapêutico estão quase exclusivamente dependentes de características clínicas. Estes factores clínicos são, na maioria dos linfomas, insuficientes numa proporção significativa dos doentes, em particular, aqueles com pior prognóstico. O linfoma folicular (LF) é, globalmente, o segundo subtipo mais comum de linfoma. É tipicamente uma doença indolente com uma sobrevida média entre os 8 e 12 anos, mas é geralmente fatal quando se transforma num linfoma agressivo de alto grau, habitualmente o linfoma difuso de grandes células B (LDGCB). Morfologicamente e funcionalmente, as células do LF recapitulam as células normais do centro germinativo na sua dependência de sobrevivência do microambiente não-tumoral, especialmente das células do sistema imunológico. Biomarcadores preditivos de transformação não existem pelo que um melhor conhecimento da biologia intrínseca de progressão do LF poderá revelar novos candidatos. Nesta tese descrevo duas abordagens distintas para a descoberta de novos biomarcadores. A primeira, o estudo da expressão global de genes ('genomics') obtidos por técnicas de alto rendimento que analisam todo o genoma humano sequenciado, permitindo identificar novas anomalias genéticas que possam representar mecanismos biológicos importantes de transformação. São descritos novos genes e alterações genómicas associados à transformação do LF, sendo especialmente relevantes as relacionadas com os eventos iniciais de transformação em LDGCB. A segunda, baseou-se em várias hipóteses centradas no microambiente do LF, rico em vários tipos de células nãomalignas. Os estudos imunoarquitectural de macrófagos, células T regulatórias e densidade de microvasos efectuado em biopsias de diagnóstico de doentes com LF tratados uniformemente correlacionaram-se significativamente, e independentemente dos critérios clínicos, com a evolução clínica e, mais importante, com o risco de transformação em LDGCB. Nesta tese, foram preferencialmente utilizadas (e optimizadas) técnicas que permitam o uso de amostras fixadas em parafina e formalina (FFPET). Estas são facilmente acessíveis a partir das biopsias de diagnóstico de rotina presentes nos arquivos de todos os departamentos de patologia, facilitando uma transição rápida dos novos marcadores para a prática clínica. Embora o FL fosse o tema principal da tese, os novos achados permitiram estender facilmente hipóteses semelhantes a outros subtipos de linfoma. Assim, são propostos e validados vários biomarcadores promissores e relacionados com o microambiente não tumoral, sobretudo dependentes das células do sistema imunológico, como contribuintes importantes para a biologia dos linfomas. Estes sugerem novas opções para a abordagem clínica destas doenças e, eventualmente, novos alvos terapêuticos.------------- ABSTRACT: Cancer biomarkers provide an opportunity to identify those patients most at risk for disease recurrence, predict which tumours will respond to different therapeutic approaches and ultimately define candidate biomarkers that may serve as targets for personalized therapy. New biomarkers are especially needed in the management of lymphoid cancers. At present, these tumours are diagnosed using a combination of morphologic, phenotypic and molecular features but prognosis and overall survival are mostly dependent on clinical characteristics. In most lymphoma types, these imprecisely assess a significant proportion of patients, in particular, those with very poor outcomes. Follicular lymphoma (FL) is the second most common lymphoma subtype worldwide. It is typically an indolent disease with current median survivals in the range of 8-12 years, but is usually fatal when it transforms into an aggressive high-grade lymphoma, characteristically Diffuse Large B Cell Lymphoma (DLBCL). Morphologically and functionally it recapitulates the normal cells of the germinal center with its survival dependency on non-malignant immune and immunerelated cells. Informative markers of transformation related to the intrinsic biology of FL progression are needed. Within this thesis two separate approaches to biomarker discovery were employed. The first was to study the global expression of genes (‘genomics’) obtained using high-throughput, wholegenome-wide approaches that offered the possibility for discovery of new genetic abnormalities that might represent the important biological mechanisms of transformation. Gene signatures associated with early events of transformation were found. Another approach relied on hypothesis-driven concepts focusing upon the microenvironment, rich in several non-malignant cell types. The immunoarchitectural studies of macrophages, regulatory T cells and microvessel density on diagnostic biopsies of uniformly treated FL patients significantly predicted clinical outcome and, importantly, also informed on the risk of transformation. Techniques that enabled the use of routine formalin fixed paraffin embedded diagnostic specimens from the pathology department archives were preferentially used in this thesis with the goal of fulfilling a rapid bench-to-beside” translation for these new findings. Although FL was the main subject of the thesis the new findings and hypotheses allowed easy transition into other lymphoma types. Several promising biomarkers were proposed and validated including the implication of several non-neoplastic immune cells as important contributors to lymphoma biology, opening new options for better treatment planning and eventually new therapeutic targets and candidate therapeutics.

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.

Diversidade genética e resistência natural ao Maraviroc em estirpes do vírus da imunodeficiência humana Tipo 1 (HIV-1) em circulação em utilizadores de drogas por via endovenosa na Grande Lisboa

RESUMO: O maraviroc (MVC) é o único anti-retroviral antagonista do co-receptor CCR5 licenciado e interage com as ansas transmembranares de CCR5, induzindo uma alteração da sua conformação e impedindo a interacção com gp120. O MVC é activo apenas contra estirpes R5 de HIV-1, sendo utilizado em terapia de recurso. Neste trabalho, foi estudada a diversidade genética da região C2V3C3 do gene env de estirpes de HIV-1 de oxicodependentes por via endovenosa da Grande Lisboa, pesquisando-se também a presença de polimorfismos genéticos naturais. Foram utilizadas 52 amostras de plasma e para 35 destas foi amplificado por RT-nested PCR um produto de 565 pb. A análise filogenética revelou a seguinte distribuição de genótipos: 23 B (incluindo, provavelmente, 2 CRF14_BG), 8 A, 3 G e 1 F1. Após tradução, e por comparação com a sequência consenso B, verificou-se uma elevada frequência de polimorfismos genéticos, sendo encontradas algumas “assinaturas de aminoácidos” relativas aos subtipos não-B. Realizou-se ainda uma pesquisa de locais de N-glicosilação e a previsão da utilização de co-receptores (abordagem genotípica), com recurso às regras 11/25 e da carga líquida da ansa V3 e aos programas PSSM e geno2pheno[coreceptor]. Observou-se uma conservação genérica do número de locais de N-glicosilação e foram identificadas 5 sequências com tropismo X4 ou duplo. Por fim, com base na literatura, realizou-se uma pesquisa de polimorfismos genéticos associados a resistência ao MVC presentes na ansa V3. Foi observado um número elevado destas mutações. A presença dos padrões 11S+26V e 20F+25D+26V, num total de 3 sequências, é relevante, visto estes estarem inequivocamente associados à resistência in vivo ao MVC. Apesar de não estar ainda definido um perfil de resistência para o MVC, a presença das mutações encontradas, em indivíduos sem contacto prévio com o fármaco, trará implicações relevantes na sua gestão clínica, considerando a introdução do MVC na terapia de recurso.---------- ABSTRACT: Maraviroc (MVC) is the only CCR5 inhibitor licensed today. This drug interacts with the transmembrane helices of CCR5 co-receptor, inducing a conformation change of its extracellular loops and preventing the interaction with gp120. MVC is only active against R5 strains of HIV-1 and is currently used in salvage therapy. The genetic diversity of the env C2V3C3 region of HIV-1 strains from injecting drug users in the Greater Lisbon was studied, along with the presence of natural genetic polymorphisms. 52 plasma samples were used and the amplification by RT-nested PCR of a 565 bp-product was possible in 35 of them. The phylogenetic analysis revealed 23 sequences classified as subtype B (probably including 2 CRF14_BG), 8 A, 3 G and 1 F1. After translation, the presence of natural genetic polymorphisms was studied by comparison to a subtype B consensus. A high frequency of genetic polymorphisms was observed and significant “amino acid signatures” were found in association with non-B subtypes. A full characterization of the N-glycosylation sites was also performed and a coreceptor prediction (genotypic approach) was accomplished using the 11/25 and the V3 net charge rules and the programs PSSM and geno2pheno[coreceptor]. The number of N-glycosylation sites was generically preserved. Five sequences were defined as X4 or dual-tropic. Based on published data, a search for genetic polymorphisms, present in V3loop, associated to MVC resistance was finally undertaken. Several of such mutations were observed, being particularly interesting the presence of the patterns 11S+26V and 20F+25D+26V, in a total of 3 sequences, since these patterns have unequivocally been associated with MVC resistance in vivo. Although a resistance profile for MVC is not yet defined, the presence of these mutations in MVC-naïve populations may have significant impact in their clinical management in the future, especially considering the introduction of this drug in salvage therapy.

Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase

Dissertation presented to obtain the Ph.D degree in Biochemistry

Geospatial assessment of pest-induced forest damage through the use of UAV-bases NIR imaging and Gi-technology

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.