Differences in the prices of physical ETF’s and synthetic ETF’s

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

Sudden sensorineural hearing loss : evaluation of co-morbidities and potential clinical associations

RESUMO: A surdez súbita (SS) caracteriza-se por uma perda abrupta de audição, mais frequentemente unilateral e associada a sensação de preenchimento aural, acufenos e vertigem. Afecta 5-20/100.000pessoas/ano (sobretudo adultos em fase activa na década de 40), com grande impacto na qualidade de vida. Possíveis causas incluem doenças infecciosas, circulatórias, traumáticas, imunológicas, neoplásicas, neurológicas, tóxicas e cocleares. No entanto, a causa da SS permanece desconhecida na maioria dos casos (80%), o que origina tratamentos controversos e frequentemente ineficientes. Os tratamentos disponíveis variam desde corticosteróides a antivirais, vasodilatadores, anti-agregantes, anticoagulantes, vitaminas e oxigénio hiperbárico (OHB). Atendendo a falta de informação relativa à etiologia e fisiopatologia da SS, pretendemos avaliar a evolução clínica dos doentes com SS tratados com OHB no Centro de Medicina Subaquática e Hiperbárica (CMSH) de Lisboa entre 2000 e 2005, durante um período mínimo de 5 anos, na tentativa de identificar eventuais factores de risco ou noxas clínicas com a SS. O estudo retrospectivo proposto baseia-se na revisão de processos clínicos do CMSH e na aplicação telefónica de questionários médicos de “follow-up” confidenciais – tanto a doentes (grupo de estudo), como aos respectivos esposos/companheiros/membros próximos da família (grupo de controlo) –, com particular ênfase nos antecedentes médicos e história clínica actual. Um estudo preliminar de 20 pessoas (10 doentes e 10 controlos) foi efectuado para antecipar dificuldades e estimar as necessidades logísticas. As dificuldades identificadas foram: 1) selecção dos doentes com números de telefone válidos e processos clínicos completos (com audiograma inicial e final); 2) contacto telefónico com os participantes de ambos os grupos (de estudo e controlo); 3) recursos humanos requeridos. Dado que a SS não é uma doença em si, mas um sintoma de uma doença subjacente, acreditamos que este estudo epidemiológico seja importante e útil, capaz de gerar novas luzes sobre a fisiopatologia e mecanismos desta entidade clínica.-------------ABSTRACT:Sudden sensorineural hearing loss (SSHL) is characterized by abrupt, mostly unilateral loss of hearing, frequently associated to aural fullness, tinnitus and vertigo. It affects 5-20/100.000 people/year (particularly working adults in the 40ths), with huge impact on quality of life. Possible causes include infectious, circulatory, traumatic, immunologic, metabolic, neoplastic, neurologic, toxic and unidentified cochlear diseases. Nevertheless, SSHL’s etiology remains unknown in most cases (80%), giving rise to controversial (and frequently ineffective) treatments. Available therapies range from corticosteroids to antivirals, vasodilators, antiaggregants, anticoagulants, vitamins and hyperbaric oxygen (HBO). Given the lack of data concerning SSHL’s etiology and physiopathology, we intend to evaluate clinical evolution of such patients treated with HBO in the Underwater and Hyperbaric Medical Center (UHMC) at Lisbon from 2000 to 2005 during a minimum period of 5 years, in an attempt to identify eventual risk factors or clinical associations to SSHL. The intended retrospective study is based on the review of patients’ medical charts from UHMC and confidential follow-up questionnaires applied telephonically both to patients (study group) and patients’ spouse/partner/close family member (control group), focusing past and present medical history. A preliminary study of 20 subjects (10 of each group) was performed to anticipate difficulties and to estimate the required logistics. The identified difficulties were: 1) selection of subjects with valid phone numbers and complete medical charts (with initial and final audiograms); 2) telephonic contact with subjects from the study and control group; 3) human logistics required. As it is believed that SSHL is not a disease by itself but rather a symptom of an underlying disease, we believe that this epidemiologic study is important and will hopefully generate sound scientific knowledge concerning physiopathology and mechanism of disease of SSHL.

Capillaria hepatica-induced hepatic fibrosis in rats: paradoxical effect of repeated infections

Multiple exposures to parasitic agents are considered an important factor in the genesis of the most severe forms of the diseases they cause. Capillaria hepatica-induced septal fibrosis of the liver in rats usually runs without signs of portal hypertension or hepatic failure. After determining the hepatic profile of 15 animals during the course of a single infection, we submitted 20 rats to multiple Capillaria hepatica infections to determine whether repeated exposures would augment fibrosis production, transforming septal hepatic fibrosis into a true cirrhosis. Ten single-infection rats served as controls. A total of 5 exposures, with 45-day intervals, were made. Histological changes were followed by means of surgical liver biopsies, collected prior to infection and to each re-infection. Functional changes were minimal and transient. Although a slight recrudescence of fibrosis was observed after the first two re-infections and when the single-infected control group was re-infected at the end of the experiment, subsequent re-infections failed to increase the amount of fibrosis. On the contrary, there occurred quantitative and qualitative evidence of collagen degradation and suppression of parasite development. These paradoxical results are in keeping with the hypothesis that a complex immunological modulation participates in the mechanism of hepatic fibrosis induced by Capillaria hepatica infection in rats.

Immunohistochemical expression of oestrogen and progesterone receptors during experimental acute and chronic murine Schistosomiasis mansoni

INTRODUCTION: The responsibility of Schistosoma mansoni in female infertility is still controversial. This study was conducted to evaluate the effect of acute and chronic schistosomiasis mansoni infection on the endometrium using immunohistochemical analysis of uterine hormone receptor expression. METHODS: Twenty-four nonpregnant swiss albino mice were divided into three groups: control, noninfected; acute; and chronic Schistosoma mansoni infection. Histological sections of uterine specimens were examined by light microscope with an image analyzing system to detect structural histological, estrogen receptor (ER) and progesterone receptor (PR) expression in the endometrium. RESULTS: No secretory phase was detected in the endometrium in acute and chronic Schistosoma infection. Hormone receptor expression (ER and PR) showed statistically significant differences among the groups (p< 0.05), with significant low ER hormone expression in chronic infection, compared to control proliferative, control secretory and acute infection cases, and statistically significant high PR expression in both acute and chronic infection cases compared to the control secretory cases (p< 0.05). CONCLUSIONS: Schistosomiasis mansoni seems to have an important impact on the hormone expression of affected women. Further studies to explore the mechanism of such changes are recommended.

Prevalence and clinical outcomes of episodes of ventilator-associated pneumonia caused by SPM-1-producing and non-producing imipenem-resistant Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa is a leading cause of ventilator-associated pneumonia (VAP) and exhibits high rates of resistance to several antimicrobial drugs. The carbapenens are usually the drugs of choice against this microorganism. However, the carbapenem resistance has increased among these strains worldwide. The presence of metallo-β-lactamases (MBL) has been pointed out as a major mechanism of resistance among these strains. No previous study addressed outcomes of respiratory infections caused by these strains. METHODS: Our group sought to analyze the epidemiology and clinical outcomes of patients with VAP caused by imipenem-resistant P. aeruginosa. A total of 29 clinical isolates of carbapenem-resistant Pseudomonas aeruginosa were screened for metallo-β-lactamase (MBL) genes. RESULTS: Demographic and clinical variables were similar between the SPM-1-producing and non-SPM-1-producing group. Five (17.2%) isolates were positive for blaSPM-1. No other MBL gene was found. All patients were treated with polymyxin B. The infection-related mortality was 40% and 54.2% for SPM-1-producing and -non-producing isolates, respectively. CONCLUSIONS: There were no differences in epidemiological and clinical outcomes between the two groups.

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Evolution of Teeth and Quadrate in Non-avian Theropoda (Dinosauria: Saurischia), with the Description of Torvosaurus Remains from Portugal

Theropods form a highly successful and morphologically diversified group of dinosaurs that gave rise to birds. They include most, if not all, carnivorous dinosaurs, yet many theropod clades were secondarily adapted to piscivory, omnivory and herbivory, and theropods show a large array of skull and dentition morphologies. This work aims to investigate aspects of the evolution of theropod dinosaurs by analyzing in detail both the anatomy and ontogeny of teeth and quadrates in non-avian theropods, and by studying embryonic and adult material of a new species of theropod. A standardized list of terms and notations for each anatomical entity of the tooth, quadrate, and maxilla is here proposed with the goal of facilitating descriptions of these important cranial and dental elements. The distribution of thirty dental characters among 113 theropod taxa is investigated, and a list of diagnostic dental characters is proposed. As an example, four isolated theropod teeth from the Lourinhã Formation (Kimmeridgian‒Tithonian) of Portugal are described and identified based on a cladistic analysis performed on a data matrix of 141 dentition-based characters coded in 60 taxa. Two shed teeth are referred to an abelisaurid, providing the first record of Abelisauridae in the Jurassic of Laurasia and the one of the oldest records of this clade in the world, suggesting a possible radiation of Abelisauridae in Europe well before the Upper Cretaceous. The consensus tree resulting from this phylogenetic analysis, the most extensive on theropod teeth, indicates that theropod teeth provide reliable data for identification at approximately family level, and this method will help identifying theropod teeth with more confidence. A detailed description of the dentition of Megalosauridae is also provided, and a discriminant analysis performed on a dataset of numerical data collected on the teeth of 62 theropod taxa reveals that megalosaurid teeth are hardly distinguishable from other theropod clades with ziphodont dentition. This study highlights the importance of detailing anatomical descriptions and providing additional morphometric data on teeth with the purpose of helping to identify isolated theropod teeth. In order to evaluate the phylogenetic potential and investigate the evolutionary transformations of the quadrate, a phylogenetic morphometric analysis as well as a cladistic analysis using 98 discrete quadrate related characters were conducted. The quadrate morphology by its own provides a wealth of data with strong phylogenetic signal, and the phylogenetic morphometric analysis reveals two main morphotypes of the mandibular articulation of the quadrate linked to function. As an example, six isolated quadrates from the Kem Kem beds (Cenomanian) of Morocco are determined to be from juvenile and adult individuals of Spinosaurinae based on phylogenetic, morphometric, and phylogenetic morphometric analyses. Morphofunctional analysis of the spinosaurid mandibular articulation has shown that the posterior parts of the two mandibular rami displaced laterally when the jaw was depressed due to a mediolaterally oriented intercondylar sulcus of the quadrate. Such lateral movement of the mandibular ramus was possible due to a movable mandibular symphysis in spinosaurids, allowing the pharynx to be widened. A new species of theropod from the Lourinhã Formation of Portugal, Torvosaurus gurneyi, is erected based on a right maxilla and an incomplete caudal centrum. This taxon supports the mechanism of vicariance that occurred in the Iberian Meseta during the Late Jurassic when the proto-Atlantic was already well formed. A theropod clutch containing several crushed eggs and embryonic material is also assigned to this new species of Torvosaurus. Investigation on the maxilla ontogeny in basal tetanurans reveals that crown denticles, elongation of the anterior ramus, and fusion of interdental plates appear at a posthatchling stage. On the other hand, maxillary pneumaticity is already present at an embryonic stage in non-avian theropods.

Role of sympathetic innervation in obesity

Part of the results presented in this thesis were published in the following reference (DOI 10.1016/j.cell.2015.08.055): Wenwen Zeng*, Roksana M. Pirzgalska*, Mafalda M.A. Pereira, Nadiya Kubasova, Andreia Barateiro, Elsa Seixas, Yi-Hsueh Lu, Albina Kozlova, Henning Voss, Gabriel G. Martins, Jeffrey M. Friedman and Ana I. Domingos. Sympathetic Neuro-adipose Connections Mediate Leptin-Driven Lipolysis. Cell 163, 84-94 (2015). The work was also presented through poster presentations at iMED Conference 6.0 (Lisbon, 2014), Sociedade Portuguesa de Bioquímica Meeting (Coimbra, 2014) and Sociedade Portuguesa de Neurociências Meeting (Póvoa de Varzim, 2015).

Natural transovarial transmission of dengue virus 4 in Aedes aegypti from Cuiabá, State of Mato Grosso, Brazil

INTRODUCTION: Dengue is the most prevalent arboviral disease in tropical areas. In Mato Grosso, outbreaks are reported every year, but studies on dengue in this state are scarce. METHODS: Natural transovarial infection of Aedes aegypti by a flavivirus was investigated in the Jardim Industriário neighborhood of Cuiabá, Mato Grosso. Eggs were collected with ovitraps during the dry, intermediate, and rainy seasons of 2012. After the eggs hatched and the larvae developed to adulthood, mosquitoes (n = 758) were identified and allocated to pools of 1-10 specimens according to the collection location, sex, and climatic period. After RNA extraction, multiplex semi-nested RT-PCR was performed to detect the four dengue virus (DENV) serotypes, yellow fever virus, West Nile virus and Saint Louis encephalitis virus. RESULTS: DENV-4 was the only flavivirus detected, and it was found in 8/50 pools (16.0%). Three of the positive pools contained females, and five contained males. Their nucleotide sequences presented 96-100% similarity with DENV-4 genotype II strains from Manaus, Amazonas. The minimum infection rate was 10.5 per 1000 specimens, and the maximum likelihood estimator of the infection rate was 11.6 (95% confidence interval: 4.8; 23.3). CONCLUSIONS: This study provides the first evidence of natural transovarial infection by DENV-4 in Ae. Aegypti in Mato Grosso, suggesting that this type of infection might serve as a mechanism of virus maintenance during interepidemic periods in Cuiabá, a city where dengue epidemics are reported every year. These results emphasize the need for efficient vector population control measures to prevent arbovirus outbreaks in the state.

Production of metallo-β-lactamase among Pseudomonas aeruginosa strains isolated in the State of Sergipe, Brazil

INTRODUCTION: Acquired production of metallo-β-lactamases is an important mechanism of resistance in Pseudomonas aeruginosa. The objective of this study was to investigate the production of metallo-β-lactamase and the genetic diversity among ceftazidime-resistant P. aeruginosa isolates from State of Sergipe, Brazil. METHODS: Metallo-β-lactamase was investigated using the disk approximation test and polymerase chain reaction (PCR). Genetic diversity was evaluated by pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 48 (51.6%) isolates were resistant to ceftazidime. Six (12.2%) of these were positive for metallo-β-lactamase production. Only two (4.1%) of the ceftazidime-resistant isolates carried the bla SPM-1 gene. CONCLUSIONS: Production of metallo-β-lactamases was not the main mechanism of resistance to ceftazidime and carbapenems among P. aeruginosa strains in Sergipe, Brazil.

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.

The role of arl13b in cilia length regulation and in zebrafish development

RESUMO: Arl13b é uma importante proteína ciliar, presente em cílios primários e cílios móveis. Ratinhos mutantes para Arl13b têm comprimento dos cílios reduzido e defeitos nos B-túbulos dos cílios. Como consequência destes fenótipos, deficiências na Arl13b originam, em modelos animais, várias doenças congénitas, incluindo problemas no estabelecimento do eixo esquerda-direita, malformações cerebrais e deformações corporais. Nos seres humanos, deficiências na Arl13b levam a uma doença crónica congénita chamada Síndrome de Joubert. Por outro lado, a sobreexpressão de Arl13b origina cílios mais longos, no entanto existe uma ausência da caracterização dos fenótipos celulares e durante o desenvolvimento embrionário. Neste trabalho, quisemos explorar o efeito da sobre-expressão de Arl13b em embriões de peixezebra. Descobrimos que, ao nível ciliar, a sobre-expressão de Arl13b nas células aumenta o comprimento ciliar em cílios primários e móveis, no entanto, a esses cílios falta adequada acetilação da alfa-tubulina no citoesqueleto feito por microtúbulos. Os nossos resultados mostraram que esse efeito é específico de Arl13b sobre-expressão e quando se manipularam as enzimas responsáveis pela acetilação (Mec17) e pela de-acetilação (HDAC6) encontrámos uma sinergia potencial com ambas. Testámos ainda, que o aumento no comprimento ciliar não estava causalmente relacionado com a falta de acetilação, ou seja, os cílios com menos acetilação não eram necessariamente os mais longos. Também mostrámos que a sobre-expressão de Arl13b é capaz de restaurar o comprimento dos cílios em mutantes com cílios curtos e como isso pode ser explorado para um futuro potencial papel terapêutico para Arl13b. Em seguida, foi avaliado o impacto do aumento da quantidade de Arl13b no desenvolvimento embrionário do peixe-zebra. Observou-se que a sobre-expressão de Arl13b apresentava fenótipos muito fracos, quando comparados com a perda de função dos mutantes de Arl13b. Focados no inesperado fenótipo leve no estabelecimento do eixo esquerda-direita abordámos a questão através do estabelecimento de uma colaboração com matemáticos, descobrimos que os cílios mais longos que potencialmente têm a capacidade de movimentar mais fluido são atenuados por amplitudes de batimento menores, e, como resultado, estes longos cílios não prejudicam o movimento do fluido e consequentemente não afetam o estabelecimento dos padrões de esquerda-direita. Sugerimos assim que a Arl13b é um regulador chave, do comprimento ciliar. Descobrimos uma nova interação com as enzimas de acetilação/de-acetilação e levantamos novas hipóteses quanto aos mecanismos moleculares da função da Arl13b. Propomos um novo modelo para o mecanismo molecular da Arl13b na regulação do comprimento dos cílios onde podemos integrar os nossos resultados com os relatados na literatura. Este trabalho adiciona mais conhecimento para o mecanismo de ação da Arl13b e, portanto, fornece uma importante contribuição para o campo da investigação em cílios.---------------------------------------------------------------------------------------------------------------------- ABSTRACT: Arl13b is an important ciliary protein, present in primary and motile cilia. arl13b-/- mouse mutants have reduced cilia length and cilia B-tubule defects. As a consequence of these phenotypes, Arl13b loss of function animal models suffer from several congenital disorders including left-right problems, brain malformations and body deformations. In humans Arl13b depletion leads to a congenital chronic disease called Joubert Syndrome. On the other hand, overexpressing Arl13b leads to longer cilia but the characterization of the cellular and developmental phenotypes was missing. In this work we explore the effect of Arl13b overexpression in zebrafish embryos. We found that, at the ciliary level, Arl13b overexpression from 1 cell stage produces longer primary and motile cilia, but these cilia lack proper alpha tubulin acetylation of their microtubule cytoskeleton. Our results showed that this effect is specific from Arl13b overexpression and when we manipulated the enzymes responsible for acetylation, Mec17, and de-acetylation, HDAC6, we found a potential synergy of both mec17 knockdown and HDAC6 activity with Arl13b overexpression. We tested that the ciliary increase in length was not causally related to the lack of acetylation, meaning the more de-acetylated cilia were not necessarily the longer ones. We also showed that Arl13b overexpression is able to restore cilia length in short cilia mutants and how that may be explored to a potential future therapeutic role for Arl13b. Next, we evaluated the impact of increasing the amount of Arl13b in zebrafish embryonic development. We observed that Arl13b overexpression presented very mild phenotypes when compared to the loss of function mutants. We focused on the unexpected left-right mild phenotype and by establishing a mathematical modeling collaboration, we found out that the longer cilia generated force was attenuated by smaller beating amplitudes, and as a result, these long cilia were not impairing the cilia generated flow and the establishment of left-right patterning. We suggest that Arl13b is one key cilia length regulator. We disclosed a novel interaction with the acetylation / de-acetylation enzymes and raised new hypothesis as to the mechanisms of Arl13b function. We propose a new model for the Arl13b molecular mechanism of cilia length regulation where we integrate our findings with those reported in the literature. This work adds more knowledge to the Arl13b mechanism of action and therefore provides an important contribution to the cilia research field.

Bond of NSM FRP strengthened concrete: round robin test initiative

Despite the extensive research that has been conducted on the debonding behaviour of FRP strengthening systems, no standard methodology has been yet established on its experimental characterization. In this context, to assess the performance and reliability of small scale testing on NSM (near surface mounted) FRP strengthening systems, an experimental program was carried out on a series of nine NSM FRP strengthening systems, in the framework of an international Round Robin Testing (RRT). Eleven laboratories and seven manufacturers and suppliers participated in this extensive international exercise, which regarded both NSM and EBR FRP strengthening systems. Test results obtained for the NSM systems by the participating laboratories are discussed and compared in this paper to investigate the feasibility of the adopted single/double pulling shear test method, to investigate the mechanism of bond between NSM FRP reinforcement and concrete, and to investigate the level of variability obtained between the participating laboratories testing the same material batches. It is concluded that the tested variants in the adopted single/double shear pulling test have a significant influence, stressing the importance of the level of detail of standardized test protocols for bond verification. On overall, given the variants included in this study, the obtained variation in bond stress-slip behaviour between the laboratories remained fairly limited.

Numerical Assessment of the out-of-plane response of a brick masonry structure without box behaviour

This paper presents the assessment of the out-of-plane response due to seismic loading of a masonry structure without rigid diaphragm. This structure corresponds to real scale brick masonry specimen with a main façade connected to two return walls. Two modelling approaches were defined for this evaluation. The first one consisted on macro modelling, whereas the second one on simplified micro modelling. As a first step of this study, static nonlinear analyses were conducted to the macro model aiming at evaluating the out-of-plane response and failure mechanism of the masonry structure. A sensibility analyses was performed in order to assess the mesh size and material model dependency. In addition, the macro models were subjected to dynamic nonlinear analyses with time integration in order to assess the collapse mechanism. Finally, these analyses were also applied to a simplified micro model of the masonry structure. Furthermore, these results were compared to experimental response from shaking table tests. It was observed that these numerical techniques simulate correctly the in-plane behaviour of masonry structures. However, the