Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 105 molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 105 molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

Imidazo[4,5-f]-1,10-phenanthrolines: Versatile ligands for the design of metallomesogens

2-Aryl-substituted imidazo[4,5-f]-1,10-phenanthrolines were used as building blocks for metal-containing liquid crystals (metallomesogens). Imidazo[4,5-f]-1,10-phenanthrolines are versatile ligands because they can form stable complexes with various d-block transition metals, including platinum(II) and rhenium(I), as well as with lanthanide(III) and uranyl ions and they can easily be structurally modified by a judicious choice of benzaldehyde precursor. None of the ligands designed for this study were liquid-crystalline. However, mesomorphism could be induced by their coordination to various metallic fragments. The thermal behavior of the metal complexes depended on the metal-to-ligand ratio and the substitution pattern of the coordinating ligands. Complexes with a metal-to-ligand ratio of 1:1 [ML, with M = Pt(II), Re(I)] were not liquid-crystal line. The lanthanide(III) complexes with a metal-to-ligand ratio of 1:2 [ML2 with M = Ln(III)] formed an enantiotropic cubic mesophase or were not liquid-crystalline, depending on the nature of the lanthanide(III) ion and the substitution pattern of the ligands. A 1:3 uranyl complex of the type [ML3](2+) exhibited a hexagonal columnar mesophase over a broad temperature range. Self-assembled monolayers of a europium(III) complex were investigated by scanning tunneling microscopy, which revealed that the complex formed well-ordered structures over long distances at the 1-octanoic acid-graphite interface. The rhenium(I) complexes and the europium(III) complexes with 2-thenoyl-trifluoroacetonate or dibenzoylmethanate and imidazo[4,5-f]-1,10-phenanthroline showed good luminescence properties.

Characterization of a picosecond laser generated 4.5 keV TiK-alpha source for pulsed radiography

K alpha radiation generated by interaction of an ultrashort (1 ps) laser with thin (25 mu m) Ti foils at high intensity (2x10(16) W/cm(2)) is analyzed using data from a spherical Bragg crystal imager and a single hit charge-coupled device spectrometer together with Monte Carlo simulations of K alpha brightness. Laser to K alpha and electron conversion efficiencies have been determined. We have also measured an effective crystal reflectivity of 3.75 +/- 2%. Comparison of imager data with data from the relatively broadband single hit spectrometer has revealed a reduction in crystal collection efficiency for high K alpha yield. This is attributed to a shift in the K-shell spectrum due to Ti ionization. (c) 2005 American Institute of Physics.

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

A study of HPV 1, 2 and 4 antibody prevalence in patients presenting for treatment with cutaneous warts to general practitioners in N. Ireland

Three hundred and seventy-six patients attending their general practitioner with cutaneous warts at five health centres in Northern Ireland were screened for human papilloma virus (HPV) types 1 and 2 IgM antibody using an indirect immunofluorescence test. Eight-eight (23.4%) patients were positive for HPV type 1 IgM and 156 (41.5%) for HPV type 2 IgM. HPV 1 IgM antibody was significantly more likely to be associated with plantar warts than warts elsewhere (P less than 0.0001). HPV 2 IgM was present in 45 (34.1%) patients with plantar warts and 99 (45.6%) patients with warts at other sites (P = 0.1). Evidence of multiple infection by HPV types 1 and 2 was demonstrated by the finding of HPV 1 and 2 IgM antibodies in the sera of 16 (4.3%). HPV 4 was found in only 1 out of 30 biopsies and HPV 4 IgM was undetectable in 50 randomly chosen sera.

Epitaxial ferroelectric Pb(Mg1/3Nb2/3)O3-PbTiO3 thin films on La0.7Sr0.3MnO3 bottom electrode

Epitaxial (001)-oriented 0.7Pb(Mg_0.33Nb_0.67)O₃-0.3PbTiO₃ (PMN-PT) thin films were deposited by pulsed laser deposition on vicinal SrTiO₃ (001) substrates using La_0.7Sr_0.3MnO₃ as bottom electrode. Detailed microstructural investigations of these films were carried out using X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM). Polarization-field hysteresis curves were measured at room temperature. Spontaneous polarization P s , remnant polarization P r and coercive voltage V c were found to be 25 μC/cm², 15 μC/cm² and 0.81 V, respectively. Field dependent dielectric constant measurements exhibited butterfly shaped curves, indicating the true ferroelectric nature of these films at room temperature. The dielectric constant and the dielectric loss at 100 kHz were found to be 238 and 0.14, respectively. The local piezoelectric properties of PMN-PT films were investigated by piezoelectric force microscopy and were found to exhibit a local piezoelectric coefficient of 7.8 pm/V.

Tiotropium Respimat® in cystic fibrosis: Phase 3 and Pooled phase 2/3 randomized trials

Background: Tiotropium Respimat® improved lung function in a phase 2 trial in patients with cystic fibrosis (CF). We investigated its efficacy and safety in a phase 3 trial, including a pre-specified pooled analysis of the phase 2 and 3 trials. 

Methods: 12-week, randomized, double-blind, placebo-controlled trial of tiotropium Respimat® 5. μg once daily in patients with CF (N = 463). 

Results: Co-primary efficacy endpoints showed no statistical difference between tiotropium and placebo: percent-predicted forced expiratory volume in 1s (FEV₁) area under the curve from 0-4h (AUC_0-4h) (95% CI): 1.64% (0.27,3.55; p=0.092); percent-predicted trough FEV₁ (95% CI) 1.40% (0.50,3.30; p=0.15). Adverse events were similar between groups. Pooled phase 2/3 trial results showed a treatment difference in favor of tiotropium: percent-predicted FEV₁ AUC_0-4h (95% CI): 2.62% (1.34,3.90). 

Conclusion: Tiotropium was well tolerated in patients with CF; lung function improvements compared with placebo were not statistically significant in the phase 3 trial. Clinical trials: These studies are registered with clinical trial identifier numbers NCT00737100 and NCT01179347NCT00737100NCT01179347. These studies are also registered with the EudraCT number: 2008-001156-43 and 2010-019802-17.

Os alunos de risco no 2º ciclo da Escola Básica 2, 3 Ana de Castro Osório, no ano lectivo 2004/2005: um estudo de caso

O presente estudo aborda a problemática dos alunos de risco no 2° Ciclo da E.B. 2,3 Ana de Castro Osório, no ano letivo 2004/2005. Na dimensão teórica são referenciadas algumas questões de Organização Escolar (escola reflexiva, autonomia de escola, gestores intermédios e supervisão escolar, o papel do Diretor de Turma, o Projeto Curricular de Escola, algumas linhas orientadoras da política educativa portuguesa, no período em que decorre a elaboração desta investigação) e abordados alguns aspetos gerais dos pré-adolescentes, diversos conceitos de risco, a ação de diversas entidades com competência na área da infância e juventude, o papel da família e a relação desta com a escola. Foi utilizada a metodologia do “Estudo Caso”, no decurso da qual foram analisados documentos internos da escola e elaborados instrumentos de recolha de dados (questionários aplicados aos Alunos e aos seus Diretores de Turma). No tratamento das informações de natureza quantitativa, utilizou-se o programa informático Excel, enquanto nas questões abertas procedeu-se à análise de conteúdo. Em síntese, concluiu-se que: a) um terço dos alunos que frequentava o 2° Ciclo do Ensino Básico, foi sinalizado como “aluno de risco”. Cerca de 75% era do 5° Ano de Escolaridade. Os fatores de risco que mais se destacam são: assiduidade irregular, desmotivação/desinteresse/apatia, défice de atenção e concentração, indisciplina e retenções sucessivas no seu percurso escolar. A família é pouco participativa. b) a resposta curricular dá-se essencialmente dentro da sala de aula; c) a resposta organizacional assenta essencialmente nalgumas medidas previstas nos documentos legislativos. Na última parte do trabalho deixamos expressas as conclusões do estudo mais pormenorizadas, bem como algumas recomendações/sugestões que eventualmente, poderão orientar posteriores investigações. /ABSTRACT - The risk students on a 2nd Cycle of classroom of the E.B. 2, 3 Ana de Castro Osório, during academic year of 2004/2005: A Case study The present study attempts to assess risk students on a 2nd Cycle (Sth and 6th grades) classroom of the “E.B. 2, 3 Ana de Castro Osório” Public School during the academic year of 2004/2005. On a theoretical scale, are assessed some questions about the Classroom Organization (reflexive school, school autonomy, intermediate directors and school supervision, the role of the Class Tutor, the School Curricular Project, some guidelines about the Portuguese education policy during the period when this study took place), and was also assessed some general aspects of preadolescents, risk concepts, and actions taken by various organizations specialized in the areas of childhood and youth, the role of the family and the interrelation between the family and the school. It was used a “Case Study” methodology during which was assessed school documents of this particularly school, and data collecting instruments (Student-applied and Class Tutor-applied questionnaires). On dealing with quantitative data, it was used the Excel application, while for the open questions was assessed by its contents. In brief, it was concluded that: a) a third of students that attended the 2nd Cycle Basic Education schools revealed to be “risk students”. Around 75% of them were 5th grade students. Among the most important risk factors are: irregular attendance, unmotivated/aloofness /apathy, lack of attention and concentration, indiscipline and successive retentions during their education pathway. The family is scarcely participative. b) The curricular response is normally given within the classroom; c) the organizational response lies mostly on some measures included in government documents. On the last part of the paper, we focus on more detailed conclusions, as well as give recommendations/suggestions which may eventually be of guidance to future studies.

CuxSnSx+1 (x = 2, 3) thin films grown by sulfurization of metallic precursors deposited by dc magnetron sputtering

We report the results of the growth of Cu-Sn-S ternary chalcogenide compounds by sulfurization of dc magnetron sputtered metallic precursors. Tetragonal Cu2SnS3 forms for a maximum sulfurization temperature of 350 ºC. Cubic Cu2SnS3 is obtained at sulfurization temperatures above 400 ºC. These results are supported by XRD analysis and Raman spectroscopy measurements. The latter analysis shows peaks at 336 cm-1, 351 cm-1 for tetragonal Cu2SnS3, and 303 cm-1, 355 cm-1 for cubic Cu2SnS3. Optical analysis shows that this phase change lowers the band gap from 1.35 eV to 0.98 eV. At higher sulfurization temperatures increased loss of Sn is expected in the sulphide form. As a consequence, higher Cu content ternary compounds like Cu3SnS4 grow. In these conditions, XRD and Raman analysis only detected orthorhombic (Pmn21) phase (petrukite). This compound has Raman peaks at 318 cm-1, 348 cm-1 and 295 cm-1. For a sulfurization temperature of 450 ºC the samples present a multi-phase structure mainly composed by cubic Cu2SnS3 and orthorhombic (Pmn21) Cu3SnS4. For higher temperatures, the samples are single phase and constituted by orthorhombic (Pmn21) Cu3SnS4. Transmittance and reflectance measurements were used to estimate a band gap of 1.60 eV. For comparison we also include the results for Cu2ZnSnS4 obtained using similar growth conditions.

The role of α2,3- and α2,6-sialylation

RESUMO: Os glicoconjugados que decoram a superfície celular e os lípidos e proteínas secretados ocupam o ponto de encontro onde normalmente ocorrem interacções críticas homólogas (hospedeiro-hospedeiro) e heterólogas (hospedeiro-patogénio). Apesar de ser largamente aceite que os glicanos são parte integrante do processo de imunidade, continua a não ser claro qual o papel que os glicanos, em toda a sua diversidade, tomam no quadro geral da imunidade. Os glicanos, que são frequentemente terminados por resíduos de ácido siálico, podem ser alterados por factores externos, tais como patogénios, ou por acontecimentos fisiológicos celulares específicos. Normalmente em posição terminal, as glico-estruturas que contêm ácido siálico assumem um papel fundamental numa quantidade substancial de receptores imunes envolvidos na adesividade e tráfico celular, tal como as Selectinas e as Siglecs, das quais se sabe apresentarem uma relevante função imune. À altura do início desta tese, era sabido que os ácidos siálicos expressos à superfície das células poderiam modular mecanismos importantes nas respostas imunes adaptativas. Considerando a posição de charneira que as células dendríticas (DCs) ocupam na transição da resposta imune inata para a adaptativa, antecipámos que os ácidos siálicos poderiam também modular mecanismos relevantes nas DCs humanas. As DCs têm uma função muito relevante na verificação e captura antigénica, migração para os gânglios linfáticos e apresentação antigénica aos linfócitos, uma sequência de funções que conduz, em ultima instância, à indução da resposta inata adaptativa. Considerando estas premissas, a nossa hipótese principal foi que os ácidos siálicos podem influenciar funções relevantes das DCs, tais como captura de antigénios, maturação, migração para os gânglios linfáticos e apresentação antigénica às células Para testar esta hipótese, dividimos o trabalho em quatro partes: 1) Analisámos os glicanos sialilados de superfície, expressos durante a diferenciação de monócitos humanos em DCs (moDCs). Os nossos dados mostraram que a expressão dos glicanos com ligações em O (O-glicanos) e sialilados em α2,3, assim como glicanos com ligações em N (N-glicanos) sialilados em α2,6 e α2,3 aumentou durante o processo de diferenciação das moDCs. Contribuindo para esta nova configuração glicosídica, três sialiltransferases (STs) poderão estar envolvidas: a ST6Gal-1 correlaciona-se com a expressão aumentada de N-glicanos sialilados em α2,6; a ST3Gal-1 contribui para a sialilação em α2,3 de O-glicanos, em especial de antigénios T; e a ST3Gal-4 poderá ser responsável pelo aumento de N-glicanos sialilados em α2,3. Após estímulo e consequente maturação das moDCs, ambos os níveis de expressão génica de ST6Gal-1 e ST3Gal-4 são negativamente modificados sendo, também, que a expressão de ST3Gal-1 varia consoante o estímulo. 2) Estudámos posteriormente as consequências da modulação dos ácidos siálicos de superfície nas funções das DCs. Observámos que a remoção dos ácidos siálicos de superfície diminui significativamente a capacidade de macropinocitose e endocitose mediada por receptores nas moDCs. Em contrapartida, o tratamento com sialidase aumentou significativamente a capacidade das moDCs para fagocitar Escherichia coli. Determinou-se também que este mecanismo requer a existência de ácido siálico presente nas E. coli indicando um mecanismo de interacção hospedeiro-patogénio dependente de ácido siálico em ambas as partes envolvidas. As moDCs tratadas com sialidase também apresentam um nível superior de expressão de moléculas de MHC e moléculas co-estimulatórias, sugerindo um fenótipo celular mais maduro. Recorrendo ao modelo de ratinho, utilizaram-se DCs derivadas de células da medula (BMDCs) de ratinhos deficientes em ST3Gal-1 e ST6Gal-1. Estes ensaios revelaram que quer a endocitose quer a maturação são influenciadas por modificações 37 nos glicanos sialilados em α2,3 ou α2,6. A detecção e quantificação de proteínas Nglicosiladas e sialiladas em α2,6 apontou para um potencial envolvimento de integrinas β2 nestes mecanismos. 3) O efeito da sialilação em α2,6 na migração das DCs para os gânglios linfáticos foi também analisado. Observámos que BMDCs deficientes para ST6Gal-1 apresentam uma redução de cerca de 50% nos níveis de migração das DCs para os gânglios linfáticos, tal como aferido em ensaios de inflamação in situ e estudos de transferência adoptiva de células. Uma redução dos níveis deste tipo de migração foi também observada quando BMDCs nativas foram transferidas para ratinhos receptores deficientes em ST6Gal-1. São, contudo, necessários mais ensaios de forma a identificar as moléculas envolvidas neste processo. 4) Por último, analisámos o impacto da sialilação na estimulação antigénica das DCs às células T. Assim, concluiu-se que moDCs tratadas com sialidase apresentam um nível de expressão superior de IL-12, TNF-ɑ, IL-6 e IL-10, e activação do factor de transcrição nuclear kappa B (NF-κB). As DCs tratadas com sialidase induziram uma maior proliferação nas células T, com expressão correspondente de interferão-γ. Este dado sugere que a remoção de ácidos siálicos de superfície contribui para o desenvolvimento de uma resposta pro-inflamatória do tipo 1 por células T auxiliares (resposta Th1). Considerando estes dados no seu todo, concluímos que o ácido siálico tem um papel marcante nas funções imunes das DCs. Alterações à concentração de ácido siálico à superfície das células podem alterar a endocitose/fagocitose, maturação, migração para os tecidos e gânglios linfáticos e capacidade estimulatória para com as células T. Complementando estes dados, as ligações glicosídicas de ácidos siálicos criados por ST6Gal-1 e ST3Gal-1 são funcionalmente relevantes. A modulação programada da sialilação do glicocálice, mediada por sialidases individuais ou sialiltransferases é uma possibilidade aceitável para a melhoria da fagocitose por DCs e da sua potência imunológica. Este facto tem um significado particular para imunoterapias baseadas em DCs, podendo provar-se decisivo para a sua eficiência e aplicabilidade num futuro muito próximo.-------------------------------ABSTRACT: Glycans decorating cell surface and secreted proteins and lipids occupy the junction where critical host–host and host-pathogen interactions occur. In spite of the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their variety and variability contribute to the overall immune response remains poorly defined. Glycans, frequently terminated by sialic acid residues, may be modified by external factors such as pathogens or upon specific physiological cellular events. The terminal, privileged positions of sialic acid-modified structures makes them key, fundamental determinants for a number of immune receptors with known involvement in cellular adhesiveness and cell trafficking, such as Selectins and Siglecs, with known relevant immune functions. At the time this thesis was initiated, it was established that sialic acids expressed at cell surface could modulate important mechanisms of the adaptive immune responses. Given the key role of dendritic cells (DCs) in the transition from innate to the adaptive immune responses, we anticipated that sialic acids could also modulate important mechanisms of human DCs. DCs have a relevant role in antigen screening and uptake, migration to lymph nodes and antigen presentation to lymphocytes, ultimately triggering the adaptive immune response. Therefore, our primary hypothesis was that sialic acids may modulate DC functions, such as antigen uptake, maturation, homing to lymph nodes and antigen presentation to T cells. To test this hypothesis, we divided our work in four parts. 1) Surface sialylated glycans expressed during differentiation from human monocytes to DCs (moDCs) were analyzed. Our data showed that α2,3-sialylated O-glycans and α2,6- and α2,3-sialylated N-glycans expression increased during moDC differentiation. Three main sialyltransferases (STs) are committed with this new glycan configuration: ST6Gal- 1 correlates with the increased expression of α2,6-sialylated N-glycans; ST3Gal-1 32 contributes for the α2,3-sialylation of O-glycans, especially T antigens; and ST3Gal-4 may contribute for the increased α2,3-sialylated N-glycans. Upon moDC maturation, ST6Gal-1 and ST3Gal-4 are downregulated and ST3Gal-1 is altered in a stimulus dependent manner. 2) We subsequently analyzed the consequences of the modulation of cell surface sialic acids in DC functions. We observed that removing surface sialic acid by sialidase significantly decreased the capacity of moDCs to micropinocytose and receptormediated endocytose. In contrast, treatment with a sialidase significantly improved the capacity of moDCs to phagocytose Escherichia coli. The improved phagocytosis mechanism required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. Sialidase-treated moDCs have increased expression of MHC and co-stimulatory molecules, suggesting a more mature phenotype. Experiments using mouse bone marrow-derived DCs (BMDCs) from ST3Gal-1-/- and ST6Gal-1-/- strains indicated that endocytosis and maturation are influenced by changes in either α2,3 or α2,6-sialylated glycans. The analysis of α2,6-sialylated, N-glycosylated proteins, strongly suggested the potential involvement of β2 integrins, underlying these mechanisms. 3) The effect of α2,6-sialylation in DC homing to lymph nodes was also analyzed. We observed that BMDCs deficient for ST6Gal-1 have an almost 50% reduction in DC homing, as assayed by in situ inflammation and adoptive transfer studies. A reduction in DC homing was also observed when wild type BMDCs were transferred into ST6Gal-1-/- recipient mice. Further investigations are necessary to identify the molecules involved in this process. 4) Finally, we also analyzed the impact of sialylation on DCs ability to prime T cells. Sialidase-treated moDCs show increased gene expression of IL-12, TNF-α, IL-6 and IL- 10 cytokines, and activation of the transcription factor nuclear factor-κB. Sialidase33 treated DCs induced a higher proliferative response of T cells with concomitant higher expression of interferon-γ, suggesting that the clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. Together, our data strongly support sialic acid’s relevance in DC immune functions. Alterations of cell surface sialic acid content can alter the endocytosis/phagocytosis, maturation, migration/homing and the ability for T cell priming in human DCs. Moreover, sialic acid linkages created by ST6Gal-1 and ST3Gal-1 are functionally relevant. The engineering of cell surface sialylation, mediated by individual sialidases or sialyltransferases is a likely possibility to fine tune DC phagocytosis and immunological potency, with particular significance to DC-based therapies.

De mãos dadas com a escola:a participação dos encarregados de educação do 2.º ciclo da Escola EB 2,3 Barbosa du Bocage

Dissertação de Mestrado em Ciências Sociais na especialidade de Sociologia