First Comprehensive In Silico

GalNAc-T1, a key candidate of GalNac-transferases genes family that is involved in mucin-type O-linked glycosylation pathway, is expressed in most biological tissues and cell types. Despite the reported association of GalNAc-T1 gene mutations with human disease susceptibility, the comprehensive computational analysis of coding, noncoding and regulatory SNPs, and their functional impacts on protein level, still remains unknown. Therefore, sequence- and structure-based computational tools were employed to screen the entire listed coding SNPs of GalNAc-T1 gene in order to identify and characterize them. Our concordant in silico analysis by SIFT, PolyPhen-2, PANTHER-cSNP, and SNPeffect tools, identified the potential nsSNPs (S143P, G258V, and Y414D variants) from 18 nsSNPs of GalNAc-T1. Additionally, 2 regulatory SNPs (rs72964406 and #x26; rs34304568) were also identified in GalNAc-T1 by using FastSNP tool. Using multiple computational approaches, we have systematically classified the functional mutations in regulatory and coding regions that can modify expression and function of GalNAc-T1 enzyme. These genetic variants can further assist in better understanding the wide range of disease susceptibility associated with the mucin-based cell signalling and pathogenic binding, and may help to develop novel therapeutic elements for associated diseases

Mining Genomic Variants And Causal Pathways Linking Hdl And Triglycerides To Coronary Disease

Blood lipids are important biomarkers of risk of coronary heart disease (CHD), the leading cause of death in the world. Myriad data support a causal role of low-density lipoprotein cholesterol (LDL-C) in increasing risk of CHD. Long-standing epidemiology suggests that high-density lipoprotein cholesterol (HDL-C) may protect from disease while high triglycerides (TGs) increase CHD risk. However, the causality of HDL-C and TG to CHD remains controversial. New genetic methodologies have allowed a better look into causal pathways underlying relationships between these traits and disease. Using a combination of approaches for interrogating rare genetic variation in humans, we investigated how HDL and TG may relate to CHD. First, through sequencing and exome-wide genotyping of subjects with extremely high HDL-C, we identified the first homozygote for a loss-of-function (LOF) variant in SCARB1, which encodes scavenger receptor class BI (SR-BI), a hepatic receptor for HDL-C. Despite markedly elevated HDL-C, carriers of this variant had an increased risk of CHD. These findings suggest that HDL functionality in driving cholesterol removal through SR-BI (the reverse cholesterol transport hypothesis) is protective from CHD in humans. Next, we functionally examined one of the first novel loci from genome-wide association studies (GWAS) for HDL-C, GALNT2. Through discovery of humans with genetic GALNT2 LOF and additional studies in rodents and nonhuman primates, we showed that GALNT2 LOF lowers HDL-C across mammals. We also identify one physiological mechanism linking GALNT2 to HDL-C through its enzymatic function. Thirdly, we studied the mechanism of protection of the APOC3 A43T variant recently reported to lower TGs and CHD risk from exome sequencing. Studies in human carriers and animal models suggest that A43T accelerates renal clearance of circulating ApoC-III, thus hindering its function in delaying TG-rich lipoprotein turnover. These data establish ApoC-III clearance mechanisms as potential therapeutic targets for TG lowering. Finally, we adapted a targeted sequencing approach to increase discovery of causal rare coding and noncoding variants at candidate loci influencing HDL-C and TGs. Collectively, this work provides a sampling of approaches for leveraging the spectrum of genomic methods to identify clinically relevant variants impacting HDL, TG and CHD risk

Glycoproteomic research using mass spectrometry

The development of problem-specific mass spectrometric (MS) glycoproteomic strategies has allowed the discovery of previously unknown protein glycosylation in both eukaryotic and prokaryotic organisms. The research in this thesis focuses on the identification and structural characterisation of novel glycan structures in ADAMTS13 and Clostridium difficile. ADAMTS13 is a large multi-domain protein which regulates thrombogenesis by cleavage of the adhesive blood glycoprotein, VWF, so generating smaller less thrombogenic fragments. Glycoproteomic strategies were employed to investigate a secretion-enhancing mutant by comparing the O-glycome of wild type (WT) with synonymous substitution, P118P, and a non-synonymous control, P118F. Identical post-translational modifications (PTMs) but several novel PTMs were discovered in ADAMTS13 including TSR1 O-glycosylation, C-mannosylation of W387 and DiSialyl Core-1 O-glycosylation of S1170. Clostridium difficile is one of the main organisms responsible for morbidity in hospitalised patients, and is the etiological agent of antibiotic-associated diarrhoea and pseudomembranous colitis. The C.difficile cell wall is surrounded by an S-layer composed of two proteins, high molecular weight (HMW) and low molecular weight (LMW) SLPs. 12 slpA gene cassettes have been recently described and cassette-11 carries an insert containing 19 ORFs. Combining different biochemical and ES- and MALDI-MS approaches, including the ETD technique, with genetic experiments, it was demonstrated that LMW SLP in strain Ox247 is glycosylated with a surprisingly large linear pentose-branched oligosaccharide of more than forty sugar residues, and a collaborative NMR study suggests a Phospho- and Acetyl- substituted non-reducing terminal rhamnose. Analysing different C.difficile hypervirulent strains, novel flagellar sulphonated peptidylamido-glycan structures not previously observed in sugar or amino acid chemistry were identified. High resolution mass measurement and negative-ion nanospray MS/MS of cone-voltage-induced fragment ions were crucial in allowing the discovery of a unique terminal Taurine (aminoethyl-sulphonic acid) peptidylamidoglycan unit which could provide a novel strategy to escape the immune system, by the C.difficile becoming more virulent.Open Acces

Gastric Carcinoma

Gastric cancer is one of the most common tumors worldwide. It has a heterogeneous milieu, where the genetic background, tumor immunology, oxidative stress, and microbial infections are key players in the multiple stages of tumorigenesis. These diverse factors are linked to the prognosis of the gastric cancer and the survival of gastric cancer patients. This book is appropriate for scientists and students in the field of oncology, gastroenterology, molecular biology, immunology, cell biology, biology, biochemistry, and pathology. This authoritative text carefully explains the fundamentals, providing a general overview of the principles followed by more detailed explanations of these recent topics efficiently. The topics presented herein contain the most recent knowledge in gastric cancer concerning the oncogenic signaling, genetic instability, the epigenetic aspect, molecular features and their clinical implications, miRNAs, integrin and E-cadherin, carbohydrate-associated-transferases, free radicals, immune cell responses, mucins, Helicobacter-pylori, neoadjuvant and adjuvant therapy, prophylactic strategy for peritoneal recurrence, and hepatic metastasis

Adaptability of metabolic networks in evolution and disease

There are 114.101 small molecule metabolites currently annotated in the Human Metabolome Database, which are highly connected amongst each other, with a few metabolites exhibiting an estimated number of more than 103 connections. Redundancy and plasticity are essential features of metabolic networks enabling cells to respond to fluctuating environments, presence of toxic molecules, or genetic perturbations like mutations. These system-level properties are inevitably linked to all aspects of biological systems ensuring cell viability by enabling processes like adaption and differentiation. To this end, the ability to interrogate molecular changes at omics level has opened new opportunities to study the cell at its different layers from the epigenome and transcriptome to its proteome and metabolome. In this thesis, I tackled the question how redundancy and plasticity shape adaptation in metabolic networks in evolutionary and disease contexts. I utilize a multi-omics approach to study comprehensively the metabolic state of a cell and its regulation at the transcriptional and proteomic level. One of the challenges with multi-omics approaches is the integration and interpretation of multi-layered data sets. To approach this challenge, I use genome scale metabolic models as a knowledge-based scaffold to overlay omics data and thereby to enable biological interpretation beyond statistical correlation. This integrative methodology has been applied to two different projects, namely the evolutionary adaptation towards a nutrient source in yeast and the metabolic adaptations following disease progression. For the latter, I also curated a current human genome-scale metabolic model and made it more suitable for flux predictions. In the yeast case study, I investigate the metabolic network adaptations enabling yeast to grow on an alternative carbon source – glycerol. I could show that network redundancy is one of the key features of fast adaptation of the yeast metabolic network to the new nutrient environment. Genomics, transcriptomics, proteomics, metabolomics and metabolic modeling together revealed a shift of the organism’s redox-balance under glycerol consumption as a driving force of adaption, which can be linked to the causal mutation in the enzyme Kgd1. On the other hand, the limitations of metabolic network adaptation also became apparent since all evolved and adapted strains exhibited metabolic trade-offs in other environmental conditions than the adaptation niche. Either an impaired diauxic shift (as in the case of the glycerol mutant) or an increased sensitivity towards osmotic stress (caused by mutations in the HOG pathway) was coupled with efficient use of glycerol. In the second project, the molecular phenotype of regressed breast cancer cells was studied to identify what differentiates these cells from healthy breast tissue and to characterize the potential source of tumor recurrence. Using a breast cancer mouse model with inducible oncogenes, transcriptomics together with an extensive set of different types of metabolomics (targeted and untargeted metabolomics, lipidomics and fluxomics) could show that regressed cancer cells, albeit their apparently normal morphology, possess a highly altered molecular phenotype with an oncogenic memory. While in cancer redundancy and plasticity enable the adaptation towards a proliferative state, in regressed cells, on the contrary, prolonged oncogenic signaling leads to a loss of metabolic network regulation and the entering of an irreversible metabolic state. This state appears to be insensitive to adaptation mechanisms as transcripts and metabolites reciprocally enhance each other to maintain the tumor-like metabolic phenotype. In conclusion, this work demonstrates how genome scale metabolic models can help identifying functional mechanisms from complex and multi-layered omics data. Appropriate genome scale metabolic models combined with metabolite measurements have proven particularly useful in this context. The comprehensive understanding of all integrated aspects of a cell’s physiology is a challenging endeavor and the results of this thesis might stimulate further research towards this goal

New Insights into the Pathogenesis and Therapies of IgA Nephropathy

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide and a frequent cause of kidney failure. Better understanding of the pathogenesis of IgAN and the related genetic, immunological, and cellular susceptibility factors are needed to enable the development of effective disease-specific therapy. This book brings together international experts to provide clinical and experimental studies and reviews with an emphasis on early diagnosis, prognosis, disease pathogenesis, determination of disease activity, and new strategies for treatment for IgAN

Bases moleculares da galactosémia clássica : pesquisa de novas estratégias terapêuticas

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014Classic galactosemia is a potentially lethal disease of the galactose metabolism caused by a severe deficiency of GALT, the second enzyme of the Leloir pathway. This disorder is associated with mutations in the GALT gene, and displays an autosomal recessive pattern of inheritance. Although apparently asymptomatic at birth, affected infants start developing escalating symptoms after 1 to 2 weeks of galactose ingestion due to milk feeding. Upon implementation of a galactose-restricted diet, infants show rapid clinical improvement and seem to be almost miraculously cured; however, the long-term outcome is often disappointing, as most patients go on to develop severe complications notwithstanding strict compliance with the dietary therapy. Despite decades of intensive research, classic galactosemia pathophysiology is still largely unknown, resulting in a limited and poorly effective therapy. A comprehensive understanding of the molecular mechanisms underlying classic galactosemia potentially opens new therapeutic avenues, and prompted us to develop the present work. The first part of this thesis (chapter 1) comprises a general introduction with a review of the literature concerning the molecular and biochemical basis of classic galactosemia, followed by an overview of the current therapeutic approaches in genetic disorders – with a special focus on inherited metabolic disorders – and finally the objectives of the present work. Our studies initiated with the molecular characterization of all patients currently followed by the Portuguese metabolic centers (chapter 2). The rationale for this study was the previous description that GALT genotype represents a valuable prognostic tool for the outcome of galactosemic patients. Accordingly, after genotyping 42 Portuguese galactosemic patients, we searched for genotype-phenotype correlations by a 3-fold evaluation. Firstly, we employed in silico strategies to assess the structural-functional impact of previously uncharacterized mutations; secondly, we evaluated the biochemical phenotype at both the metabolite and enzymatic levels, represented by Gal-1-P values and GALT activity in erythrocytes; and finally, evaluated the resulting clinical outcome. Establishment of correlations between genotype and biochemical or clinical phenotypes, however, was poorly attained, reiterating the complexity of this disease and emphasizing the idea that other modifiers – possibly genetic, epigenetic and environmental – contribute to the pathophysiology of classic galactosemia. The update of the Portuguese GALT mutational spectrum revealed the intronic mutation c.820+13A>G (IVS8+13A>G) as the second most prevalent variation, strongly suggesting its pathogenicity, which set the basis for the study described in chapter 3. We functionally characterized the c.820+13A>G variation by ex vivo and in vivo analyses, which were in full agreement with the previous in silico predictions. Indeed, we confirmed this variation is a disease-causing mutation, whose mechanism of action involves the activation of a cryptic donor site, which, in turn, induces an aberrant splicing of the GALT pre-mRNA, thus causing a frameshift with inclusion of a premature stop codon. Structural-functional studies of the recombinant truncated GALT showed it was devoid of enzymatic activity and prone to aggregation. Finally, antisense oligonucleotides were designed to specifically recognize the mutation, and successfully restored the constitutive splicing. Molecular studies in several inherited metabolic disorders have led to the realization that only a minor part of the mutations directly disrupt functional sites of the proteins. Accordingly, in chapter 4, we described the structural and functional characterization of the most prevalent mutations in the GALT gene – p.Q188R, p.S135L, p.K285N and p.N314D – and of other five clinically relevant mutations – p.R148Q, p.G175D, p.P185S, p.R231C and p.R231H. Interestingly, the analyzed mutations did not affect the global conformational stability of the GALT enzyme; rather, most mutations, notwithstanding their impact on the enzyme functionality, increased the propensity for aggregation, which at the cellular level reflects in a decrease of the enzyme’s effective concentration. These results are in agreement with previous studies in classic galactosemia models, and suggest that GALT aggregation might be a major pathogenic mechanism underlying this disorder. Previous studies have reported a yeast galactosemia model allowing the evaluation of human GALT mutations severity, by assaying the sensitivity of transformed yeast cultures to galactose addition to the medium. Thus, in chapter 5, we developed a prokaryotic model of galactose sensitivity to evaluate the ability of the above referred human GALT mutants in alleviating the galactose-induced toxicity. This model presents the inherent advantage of being assayed in vivo, thus providing valuable insights on mutations’ impact on human GALT function. Additionally, arginine ability to ameliorate the galactose-induced toxicity was also evaluated for each human GALT mutant. The rationale for this approach was, not only the long-recognized anti-aggregation properties of arginine, but also its important therapeutic effect described in previous studies. In effect, arginine appears to exert a mutation-specific mode of action, alleviating the galactose toxicity in the p.Q188R, p.K285N, and p.G175D mutants, which suggests that might be of some benefit in classic galactosemia. Nonetheless, further studies are underway to ascertain arginine’s potential therapeutic effect in this inherited disorder of the galactose metabolism. Chapter 6 presents a general discussion and major conclusions disclosed by this work, framing them in the current state of the art, proving also some perspectives for future studies. Taken together, our results provide important insights on classic galactosemia, namely by shedding light on the underlying pathogenic molecular mechanisms, thus contributing for a better understanding of this enigmatic disorder. Finally, and importantly, these studies paved the way to the search, development and improvement of novel and alternative therapeutic strategies, so needed to overcome the overwhelming and burdensome long-term complications presented by most classic galactosemic patients.Após a sua internalização celular, a galactose é rapidamente metabolizada a glucose-1- fosfato pela acção sequencial de três enzimas: galactocinase (GALK), galactose-1-fosfato uridililtransferase (GALT) e uridina difosfogalactose 4’-epimerase (GALE). Estas enzimas permitem, respectivamente, a fosforilação da galactose em galactose-1-fosfato (GALK), a conversão de galactose-1-fosfato e uridina difosfoglucose em glucose-1-fosfato e uridina difosfogalactose (GALT), e a interconversão de uridina difosfoglucose em uridina difosfogalactose (GALE). Estas enzimas constituem a via de Leloir e, apesar de o fígado ser o principal órgão envolvido no metabolismo da galactose, encontram-se na maioria das células e dos tecidos. Uma deficiência enzimática em qualquer uma das três enzimas da via de Leloir resulta numa diminuição ou ausência de capacidade de metabolizar a galactose e, consequentemente, conduz à sua acumulação no sangue – hipergalactosémia. A Galactosémia Clássica (OMIM #230400), a forma mais comum de hipergalactosémia primária, é uma doença genética de transmissão autossómica recessiva, que afecta 1 em cada 30.000 a 60.000 nados-vivos, apresentando uma prevalência variável entre populações, nomeadamente na Irlanda e Turquia onde atinge valores de 1/23.500 - 1/23.775 e no Japão onde a sua prevalência atinge o valor mais baixo (1/1.000.000). A ausência ou diminuição de actividade da GALT é causada por mutações no gene GALT, e encontram-se descritas mais de 250 variações, reflectindo a elevada heterogeneidade alélica desta doença metabólica. Para além disso, a maioria dos doentes são heterozigóticos compostos, um dos factores determinantes da ampla variabilidade fenotípica observada. Aquando do nascimento, a criança aparenta ser assintomática. Os sintomas tornam-se evidentes após o início da ingestão de leite, e consistem inicialmente em dificuldades alimentares e de desenvolvimento, vómitos, diarreia, letargia e hipotonia, podendo evoluir para cataratas e septicémia, e eventualmente conduzir à morte. Após implementação de uma dieta restrita em galactose – o tratamento padrão – as crianças mostram notáveis melhorias em apenas 24 horas, e em apenas uma a duas semanas as disfunções hepática e renal desaparecem completamente. Esta resposta dramática à terapia dietética levou ao conceito da Galactosémia Clássica como uma doença relativamente benigna e fácil de tratar. No entanto, os doentes com Galactosémia Clássica desenvolvem complicações a longo prazo e a diversos níveis, como neurológico e psiconeurológico, crescimento e densidade óssea, e disfunção ovárica nas mulheres, os quais parecem ser independentes de um diagnóstico precoce e de uma adesão do doente à terapia. Apesar de a primeira descrição datar de 1908, a Galactosémia Clássica continua a ser um enigma, quer ao nível do conhecimento aprofundado da sua fisiopatologia, quer ao nível do desenvolvimento de alternativas terapêuticas que permitam, sobretudo, mitigar as complicações a longo prazo. O presente trabalho pretende, assim, contribuir para a elucidação dos mecanismos moleculares subjacentes à patogénese das mutações GALT prevalentes na população galactosémica Portuguesa, assim como explorar novas estratégias terapêuticas direccionadas para cada tipo específico de mutação. O primeiro capítulo da tese apresenta uma revisão geral da literatura sobre vários aspectos relacionados com a Galactosémia Clássica: as bases bioquímicas e moleculares, a fisiopatologia e as actuais abordagens terapêuticas. Em seguida, descrevem-se em pormenor os mecanismos moleculares subjacentes à maioria das mutações que originam Erros Hereditários do Metabolismo (onde se inclui a Galactosémia Clássica) e que causam perca de função, nomeadamente as mutações missense e as que afectam o splicing, mecanismos esses que condicionam as novas terapias desenhadas de acordo com o tipo de mutação. Por fim, são descritos os objectivos delineados para o presente trabalho. O trabalho experimental teve início com a caracterização molecular de todos os doentes actualmente seguidos nos centros metabólicos nacionais de Lisboa, Porto e Coimbra, sendo o objectivo final averiguar se, como sugerido na literatura, o genótipo GALT pode ter um valor prognóstico sobre a evolução fenotípica dos doentes galactosémicos (capítulo 2). Os dados obtidos permitiram estabelecer o espectro mutacional da Galactosémia Clássica em Portugal e compará-lo com o de outras populações. O estudo de 42 doentes revelou a presença de 14 substituições nucleotídicas, sendo 10 missense, 2 nonsense e 2 de splicing. Identificaram-se 16 genótipos diferentes, mas metade dos doentes são homozigóticos para p.Q188R, a mutação prevalente não só em Portugal como a nível mundial. Surpreendentemente, a segunda mutação mais frequente é uma mutação de splicing, descrita até então como benigna. Em seguida, e recorrendo a programas bioinformáticos adequados, procedeu-se à análise da potencial patogenicidade das mutações ainda não caracterizadas na literatura. Os resultados sugeriram que a maioria destas mutações missense afectará a estabilidade e a funcionalidade da proteína mutada, enquanto que a mutação de splicing mais frequente deverá induzir um mecanismo de splicing alternativo. Por fim, este estudo revelou, na maioria dos casos, a ausência de uma correlação clara entre a gravidade das mutações prevista pela análise in silico e o fenótipo bioquímico dos doentes, determinado pelos níveis eritrocitários de galactose-1-fosfato, assim como com o fenótipo clínico e a evolução desfavorável manifestada pelos doentes. No entanto, tal resultado não surpreende dado a Galactosémia Clássica, apesar de ser uma doença monogénica, não originar fenótipos claros. Este facto prende-se com a falta de informação estrutural sobre os componentes enzimáticos da via de Leloir, colocando-se a hipótese de uma alteração na GALT poder afectar toda a via metabólica. Por outro lado, os metabolitos da galactose estão envolvidos em diversas reacções fisiológicas, nomeadamente as de glicosilação que se reflectem aos mais variados níveis. Finalmente, a influência de outros genes modificadores, de alterações epigenéticas e de factores ambientais também não pode ser ignorada. Os capítulos seguintes são dedicados ao estudo dos mecanismos moleculares subjacentes às mutações prevalentes na população Portuguesa e ao desenvolvimento de novas abordagens terapêuticas. No capítulo 3 descreve-se o estudo da mutação de splicing IVS8+13A>G, até então considerada como benigna, mas que revelou ser a segunda mais frequente na nossa população galactosémica. A caracterização funcional do mecanismo patogénico foi efectuada por transfeção de um minigene contendo a sequência mutada em duas linhas diferentes de células eucarióticas. A análise dos produtos de transcrição revelou que a mutação activa um sítio críptico de splicing, causando um splicing anómalo do pré-mRNA GALT, o qual induz um frameshift com inclusão de um codão de terminação prematuro (p.D274GfsX291). Por outro lado, dado ter-se observado a presença do mensageiro mutado nas amostras biológicas dos doentes portadores desta mutação, colocou-se a hipótese de a proteína truncada ser produzida in vivo, o que levou à produção da proteína recombinante. Os estudos estruturais e funcionais subsequentes revelaram que esta proteína é propensa à formação de agregados e é destituída de actividade enzimática. Finalmente, e utilizando oligonucleótidos antisense que hibridam especificamente com o local da mutação, impedindo assim a ligação do spliceossoma e forçando a sua ligação ao sítio canónico dador de splicing, foi possível corrigir o splicing alternativo induzido pela mutação e obter o mensageiro selvagem. Estas experiências constituíram a prova de conceito sobre a aplicabilidade da terapia antisense como alternativa estratégica para a claramente insuficiente dieta restrita em galactose. ! O espectro mutacional da maioria dos erros hereditários do metabolismo é dominado por mutações do tipo missense. O capítulo 4 aborda a caracterização estrutural e funcional das quatro variantes GALT prevalentes a nível mundial, assim como outras cinco variantes com relevância clínica, nomeadamente na população galactosémica Portuguesa. Diversas metodologias foram empregues para determinação da actividade enzimática e do perfil de inactivação térmica, bem como métodos biofísicos para avaliação das estruturas secundária, terciária e quaternária das proteínas mutadas. Os resultados revelaram que as mutações pontuais não afectam nenhuma das estruturas acima mencionadas, mas sim a propensão para uma agregação precoce destas variantes. Este resultado constitui na realidade a principal novidade, dado que um estudo recente postulou que algumas mutações no gene GALT afectam o correcto folding das proteínas mutadas. Esta conclusão é extremamente relevante na medida em que, para além da diminuição da actividade uridililtransferásica, a acumulação de agregados proteicos interfere com a homeostase celular. Efectivamente, diversos estudos relataram a presença, em doentes galactosémicos, do aumento de actividade dos sistemas ligados ao stress do retículo endoplasmático e da unfolded protein response, assim como de níveis elevados de stress oxidativo, fenómenos característico de uma proteotoxicidade. Estes dados levam-nos então a colocar como potencial e nova hipótese terapêutica a utilização de moduladores da proteostase, os quais prolongam a semi-vida celular das variantes GALT, compensando assim parcialmente a diminuição de actividade enzimática e simultaneamente prevenindo a acumulação de agregados proteicos. No capítulo 5 desenvolvemos um modelo procariótico de sensibilidade à galactose que permitisse avaliar, não só o impacto causado pelas diversas mutações missense, mas também o efeito de compostos com potencial acção terapêutica. Utilizámos uma estirpe de E. coli com o gene galT endógeno deletado, de modo que toda a actividade GALT detectada é proveniente do mutante nela transformado. Os resultados, embora ainda preliminares, confirmaram a validade do modelo delineado e permitiram obter dados a dois níveis. Por um lado, replicámos in vivo os resultados previamente obtidos in vitro quanto à capacidade de aliviar a toxicidade induzida pela galactose, por comparação com a enzima selvagem. Por outro lado, e muito relevante, verificámos que o modelo é válido para testar moléculas com potencial terapêutico. Efectivamente, algumas destas enzimas mutadas revelaram-se sensíveis à arginina, um composto amplamente reconhecido como estabilizador de proteínas e anti-agregante, aliviando desse modo a toxicidade da galactose para a bactéria. A presente tese termina com o capítulo 6 no qual se apresenta uma discussão dos resultados obtidos, incluindo uma análise integrada de todo o trabalho e respectivas conclusões, bem como algumas perspectivas de trabalho futuro. Em suma, este trabalho contribuiu para um melhor conhecimento da Galactosémia Clássica em Portugal e para a elucidação dos mecanismos patogénicos subjacentes às mutações prevalentes, bem como para a descoberta e desenvolvimento de novas alternativas terapêuticas, tão necessárias para minorar as graves sequelas que a maioria dos doentes galactosémicos apresenta a longo termo.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/48259/2008, projeto PEst-OE/SAU/UI4013/2011

Bifidobacterium-host-diet interactions

Bacteria belonging to the genus Bifidobacterium are key members of the gut microbiota. They are widely distributed in the animal kingdom, with over 80 recognised species and subspecies, and a host range spanning from insects to mammals. Bifidobacterium are among the earliest colonisers of the human gastrointestinal tract and have been associated with health-promoting benefits. However, investigations of infant-associated Bifidobacterium across early-life changing dietary periods are lacking. In addition, there is limited information on the diversity and the saccharolytic properties of this important microbiota member in diverse animal hosts. Thus, in this work I sought to comprehensively explore human- and animal-associated Bifidobacterium strains using both genomic and phenotypic approaches. Whole genome sequencing (WGS) and bioinformatic analyses were employed to examine a unique collection of Bifidobacterium longum strains (n=75) isolated from nine either exclusively breast- or formula-fed infants across their first 18 months, encompassing pre-weaning, weaning and post-weaning dietary stages, as well as a novel collection of animal-associated Bifidobacterium isolates and publicly available sequences recovered from a diverse range of hosts (n=433). These genomes were analysed either in combination or as discrete subsets to determine their genomic diversity and predicted functional properties related to carbohydrate metabolism. To complement bioinformatic analyses, a subset of infant-associated B. longum isolates were characterised phenotypically using experimental approaches to determine their carbohydrate metabolism capabilities, which linked to genomic analysis. Glycan uptake analysis and proteomics resulted in the determination of the mechanisms employed by selected B. longum strains to metabolise different carbohydrates. Bacterial isolation resulted in the recovery of a substantial collection of animal-associated Bifidobacterium isolates (over 100) and the identification of potential novel species. The results of the bioinformatic analysis indicated a highly diverse “open” pan-genome and an overall very broad repertoire of carbohydrate utilisation genes that could be associated with the host diet. This work represents the largest phylogenetic and comparative genomic analysis of animal-associated Bifidobacterium isolates to date. Overall, this work enhances our current understanding of genomic and phenotypic properties of Bifidobacterium and lays the foundation for subsequent in-depth research aiming at further assessment of animal and human-associated Bifidobacterium diversity, and their functional potential for both therapeutic and industrial applications