Effect of sialic acid loss on dendritic cell maturation

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.

Reactivity of human fetal and adult immortalized hepatocytes to potentially toxic bilirubin and bile acid species

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Responses of higher plants to boron deficiency

Dissertation presented to obtain the degree of Doctorate in Biochemistry by Instituto de Tecnologia Química e Biológica of Universidade Nova de Lisboa

Efficient succinic acid production from glycerol by actinobacillus succinogenes using dimethyl sulfoxid as electron acceptor

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Fsp27/CIDEC is a CREB target gene induced during fasting and regulated by fatty acid oxidation rate

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria

Biochemistry, 2003, 42 (10), pp 3070–3080 DOI: 10.1021/bi026979d

Vitamin A Deficiency and Training to Farmers: Evidence from a Field Experiment in Mozambique

Vitamin A deficiency is a widespread public health problem in Sub-Saharan Africa. This paper analyzes the impact of a food-based intervention to fight vitamin A deficiency using orange-fleshed sweet potato (OFSP). We conducted a randomized evaluation of OFSP-related training to female farmers in Mozambique, in which the treatment group was taught basic concepts of nutrition, and OFSP-planting and cooking skills. We found encouraging evidence of changes in behavior and attitudes towards OFSP consumption and planting, and considerable increases in nutrition-related knowledge, as well as knowledge on cooking and planting OFSP.

Vitamin A deficiency and training to farmers: Evidence from a field experiment in Mozambique

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Economics from the NOVA – School of Business and Economics

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

Exploring motor neuron degeneration in ALS - prevention by glycoursodeoxycholic acid and signaling to microglia

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Facilitated CO2 separation membranes: mixing cyano and amino acid-based ionic liquids

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency

RESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de superfície celular ancoradas a GPI apresentam várias funções essenciais para a manutenção celular. A deficiência na síntese de GPI é o que caracteriza principalmente a deficiência hereditária em GPI, um grupo de doenças autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento, impondo a compactação da cromatina, associada à hipoacetilação de histonas, e consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência de hemólise intravascular e anemia, sendo que estas duas últimas características definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular. Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA variam entre células primárias hematopoiéticas normais. Adicionalmente, a configuração dos nucleossomas no promotor de PIGM é mais compacta em células B do que em células eritróides e tal está correlacionado com os níveis de expressão de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expressão de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrição de PIGM em ambas as células B e eritróide. Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC. Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com IGD associada a PIGM, uma das principais características que define a HPN. Por último, resultados preliminares mostraram que a repressão da transcrição de PIGM devida à mutação patogénica -270C>G está associada com a diminuição da frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”, sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, específico-detecido, por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

The influence of menopausal status on bone turnover and disease control in breast cancer patients with metastatic bone disease treated with chemotherapy plus zoledronic acid : an exploratory retrospective cohort study

RESUMO: Introdução: Uma meta-análise recente demonstrou que uso adjuvante de ácido zoledrónico (AZ) em mulheres pós-menopáusicas com cancro da mama precoce (CM) conduz a redução do risco de morte por CM em 17%. Investigámos o efeito do estado hormonal (pré [PrM] vs pós-menopausa tardia [PoM]) na remodelação óssea e controlo de doença em mulheres com CM e metástases ósseas (MO) tratadas com AZ e quimioterapia (QT). Métodos: Neste estudo de coorte retrospetivo, colhemos variáveis clinico-patológicas e quantificámos o telopéptido N-terminal (NTX) urinário e marcadores tumorais (MT) séricos em mulheres com CM e MO tratadas com QT e AZ. As doentes foram divididas em PrM (<45 anos) e PoM (>60 anos). Endpoints do estudo: variação do NTX, CA15.3 e CEA nos meses 3, 6 e 9, tempo até falência de QT de primeira-linha e sobrevivência. Quando apropriado foram usados os testes de Wilcoxon rank-sum, modelo de efeitos lineares mistos, teste log-rank e modelo de Cox. Resultados: Quarenta doentes foram elegíveis para análise (8 PrM e 32 PoM). Depois da introdução de AZ e QT, os níveis de NTX e MT caíram no coorte global. O perfil de resposta não diferiu entre grupos no mês 3 ou em tempos posteriores (valor-p para interação tempo-estado hormonal no mês 3=0.957). Ademais, o perfil de resposta dos MT também não diferiu entre grupos. O tempo mediano até falência de primeira-linha de QT em PrM e PoM foi de 15.2 e 17.4 meses, respetivamente. Não foi identificada diferença significativa entre grupos, quer em análise univariada quer após controlo para envolvimento visceral (p=0.399 e 0.469, respetivamente). Igualmente, não houve diferenças em termos de sobrevivência. Conclusões: Neste coorte, não foram identificadas diferenças no controlo de NTX ou MT em função do estado menopausico. Igualmente, não foi identificada diferença no tempo até falência de primeira-linha de CT ou sobrevivência.----------- ABSTRACT: Background: A recent meta-analysis showed that the adjuvant use of zoledronic acid (ZA) in postmenopausal women with early breast cancer (BC) leads to a reduction in the risk of breast cancer death by 17%. We investigated the effect of the hormonal status (pre [PrM] vs late post menopause [PoM]) on bone turnover and disease control among women with BC and boné metastases (BM) treated with ZA and chemotherapy (CT). Methods: In this retrospective cohort study, we collected clinicopathologic variables, urinary Nterminal telopeptide (NTX) and serum tumor marker levels from women with BC and BM treated with CT and ZA. Patients were divided in PrM (<45 years) and PoM (>60 years). Study endpoints were NTX, CA15.3 and CEA variation at 3, 6 and 9 months, and time to first-line CT failure and survival. We performed multilevel mixed-effects linear regression models to assess the variation of repeated measures and cox regression models for time to event outcomes. Results: Forty patients were eligible for analysis (8 PrM and 32 PoM). After introduction of ZA and CT, NTX and tumor markers declined in the overall cohort. Response profile was similar between menopausal groups at month 3 and at later time points (p-value for time-hormonal status interaction at month 3=0.957). Furthermore, tumor markers response profile was also equal between groups. Median time to first-line CT failure in PrM and PoM women was 15.2 and 17.4 months, respectively. No significant difference between groups was found, either using a univariate analysis or after controlling for visceral disease involvement (p=0.399 and 0.469, respectively). Likewise, no differences in survival were found. Conclusions: In this cohort, no differences were found in terms of NTX or tumor markers control according to menopausal status. Similarly, no difference in time to first-line CT failure or survival was found.

Unravelling the role of alpha 2,6 sialic acid on mouse dentritic cells functions

RESUMO: As células dendríticas (CDs) são fundamentais na imunomodulação e iniciação de respostas imunes adaptativas, enquanto os ácidos siálicos (Sias) são potenciais imunomoduladores. Estas células expressam níveis elevados da sialiltransferase ST6Gal-1, que transfere Sias para a posição terminal de oligossacáridos. De facto, a maturação de CDs está associada a uma diminuição da sialilação na sua superfície celular. Apesar de ter função biológica desconhecida, a forma solúvel, extracelular de ST6Gal-1 aumenta em cancros e inflamação. Ainda assim, esta foi recentemente identificada como moduladora da hematopoiese. Considerando o importante papel das CDs na iniciação de respostas anticancerígenas, uma ligação entre a sialilação extrínseca induzida por ST6Gal-1 extracelular e o seu papel na modulação de CDs deve ser identificada. Neste trabalho hipotetizou-se que a sialilação α2,6 extrínseca de CDs diminui o seu perfil de maturação mediante ativação por lipopolissacarídeo (LPS). O objetivo principal foi sialilar extrinsecamente em α2,6 CDs da medula óssea de murganhos, avaliando os seus perfis de maturação e de libertação de citocinas, após estimulação com LPS (por Citometria de Fluxo e ELISA, respetivamente). Ao contrário da hipótese, o perfil celular não foi modulado, usando várias abordagens. Por outro lado, a consequência da falta de α2,6 Sias na maturação de CDs foi avaliada analisando: 1) CDs da medula óssea de murganhos tratadas com sialidase, 2) CDs da medula óssea e 3) CDs das vias aéreas, ambas de murganhos deficientes em ST6Gal-1, comparando com a estirpe selvagem. Estes resultados sugerem que a perta total de α2,6 Sias se relaciona com o aumento da expressão do complexo de histocompatibilidade principal de classe II. Apesar de controverso, é provável existirem mecanismos inerentes à ativação por LPS, reduzindo a eficácia de ST6Gal-1 extracelular. Por outro lado, a modificação no perfil de CDs de murganhos deficientes em ST6Gal-1 poderá relacionar-se com uma predisposição para um estado inflamatório severo. Com isto, o trabalho desenvolvido abriu futuras linhas de investigação, nomeadamente explorar outros fatores envolvidos na (de)sialilação α2,6 de CDs, podendo ter impacto em imunoterapia com uso de CDs.--------------------------ABSTRACT: Dendritic cells (DCs) are vital for immunomodulation and the initiation of adaptive immune responses, whereas sialic acids (Sias) are potential immunomodulators. These cells express high levels of sialyltransferase ST6Gal-1, responsible for transferring Sias to the terminal position of oligosaccharide chains. Indeed, DCs’ maturation is associated with decreased cell surface sialylation. Although its biological significance is unknown, the soluble, extracellular form of ST6Gal-1 increases in cancers and inflammation. However, extracellular ST6Gal-1 was recently identified as modulator of hematopoiesis. Considering that DCs play a crucial role in the initiation of a productive anti-cancer immune response, a link between extrinsic sialylation by the extracellular ST6Gal-1 on DC function needs to be investigated. We hypothesize that extrinsic α2,6 sialylation of DCs diminishes their maturation features upon lipopolysaccharide (LPS) stimulation. The main goal was to extrinsically α2,6 sialylate mice bone marrow derived DCs (BMDCs) and to evaluate their maturation and cytokine profiles upon LPS stimulation (by Flow Cytometry and ELISA, respectively). Unlike the hypothesis, we observed that BMDCs’ profile is not modulated, even using several approaches. In contrast, the consequence of lacking cell surface α2,6 Sias in DC maturation was assessed by analysing: 1) sialidase treated BMDCs, 2) BMDCs from mice lacking ST6Gal-1 and 3) DCs from mice airways, comparing wild type with ST6Gal-1 knockout mice. These results suggest that overall lack in α2,6 Sias is related with increased expression of major histocompatibility class II (MHC-II). Although appearing to be controversial findings, other intracellular mechanisms might be occurring upon LPS-induced BMDC activation, probably reducing extracellular ST6Gal-1 effect. In opposite, the modification observed in DC profile of ST6Gal-1 knockout mice might be related to its predisposition to a more severe inflammatory status. With this, the developed work opened future lines of investigation, namely exploring other factors involved in α2,6 (de)sialylation of DC, which might have influence in immunotherapy using DCs.

Bioproduction of succinic acid from renewable feedstocks by Actinobacillus succinogenes 130Z

Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)