Establishment and characterization of a human model of the blood-brain barrier

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

Morphometric study of the fibrosis and mast cell count in the circular colon musculature of chronic Chagas patients with and without megacolon

A morphometric study of the circular colon musculature was performed, in which the mast cell count was determined and the connective fibrous tissue in this layer was measured. The objective was to gain better understanding of Chagas megacolon morphology and contribute towards the knowledge of fibrosis pathogenesis in Chagas megas. An evaluation was made of 15 distal sigmoid rings from Chagas patients with megacolon (MCC), 15 without megacolon (CSMC) and 15 non-Chagas patients (NC). The rings were fixed in formol, embedded in paraffin, and 7mm thick sections were cut and stained using Azan-Heidenhain and Giemsa. The mast cell count and fibrosis were greater in the MCC group than in the CSMC and NC groups (p < 0.05; Kruskal-Wallis test) and there was no significant difference between the latter two. The fibrosis and increased mast cell count in the colon musculature of the MCC group possibly indicates that there is a relationship between mastocytosis and fibrosis, as has already been demonstrated in other pathologies.

Petri net based development of globally-asynchronous locally-synchronous distributed embedded systems

Dissertação para obtenção do Grau de Doutor em Engenharia Electrotécnica e de Computadores

Dissecting gastrointestinal dysfunction and inflammation in a “pre-motor” rodent model of Parkinson’s disease

Research on Parkinson’s disease (PD) has mainly focused on the degeneration of the dopaminergic neurons of nigro-striatal (NS) pathway; also, post-mortem studies have demonstrated that the noradrenergic and the serotonergic transmitter systems are also affected (Jellinger, 1999). Degeneration of these neuronal cell bodies is generally thought to start prior to the loss of dopaminergic neurons in the NS pathway and precedes the appearance of the motor symptoms that are the “hallmark” of PD. Gastrointestinal (GI) motility is often disturbed in PD, manifesting chiefly as impaired gastric emptying and constipation. These GI dysfunction symptoms may be the result of a loss in noradrenergic and serotonergic innervation. GI deficits were evaluated using an organ bath technique. Groups treated with different combinations of neurotoxins (6-OHDA alone, 6-OHDA + pCA or 6-OHDA + DSP-4) presented significant differences in gut contractility compared to control groups. Since a substantial body of literature suggests the presence of an inflammatory process in parkinsonian state (Whitton, 2007), changes in pro-inflammatory cytokines in the gut were assessed using a cytokine microarray. It has been found in this work that groups with a combined dopaminergic and noradrenergic lesion have a significant increase in both expressions of IL-13 and VEGF. IL-6 also shows a decrease in treatment groups; however this decrease did not reach statistical significance. The therapeutic value of Exendin-4 (EX-4) was evaluated. It has been previously demonstrated that EX-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is neuroprotective in rodent models of PD (Harkavyi et al., 2008). In this thesis it has been found that EX-4 was able to reverse a decrease in gut contractility obtained through intracerebral bilateral 6-OHDA injection. Although more studies are required, EX-4 could be used as a possible therapy for the GI symptoms prominent in the early stages of PD.

Understanding how dentritic cell glycans affect antitumor immune responses

RESUMO: As células dendríticas (DCs) têm a capacidade única de induzir respostas imunitárias contra as células tumorais, fagocitando antigénios tumorais e apresentando-os às células T, provocando respostas imunitárias específicas que conduzem à eliminação de células de tumorais. Por induzirem memória imunológica de longa duração, as DCs são uma estratégia atrativa para o tratamento e/ou prevenção do cancro. No entanto, os resultados terapêuticos obtidos em ensaios clínicos com DCs são escassos e pouco eficientes. O nosso grupo demonstrou que ácidos siálicos que contêm glicanos desempenham um papel funcional importante em DCs geradas ex vivo. Com o objetivo de estabelecer um modelo in vitro para avaliar a resposta anti-tumoral específica realizou-se um tratamento enzimático a DCs derivadas de monócitos (moDCs) com sialidase, enzima que cliva ácidos siálicos na superfície celular. O perfil de maturação de moDCs foi caracterizado por citometria de fluxo e expressão de citocinas. Os resultados mostram que a sialidase pode regular positivamente a expressão de moléculas co-estimuladoras na superfície de moDCs estimuladas com agonistas de Toll like receptors (TLRs). Para percebermos se o tratamento com sialidase afeta a sinalização dos TLRs foram usadas células HEK transfectadas de forma estável com TLRs 2, 4 and 7/8. Os dados mostraram que a desialilação não afeta a sinalização através estes recetores. Para investigar o impacto funcional da sialidase na capacidade de moDCs em apresentar um antigénio e ativar células T, moDCs foram tratadas, ou não, com sialidase e cultivadas com clones de células T CD8+ específicas para os péptidos derivados do antigénio tumoral gp100. Os resultados mostram que DCs HLA*02:01+ desialiladas exibem maior cross-presentation do péptido gp100280-288 às células T CD8+ específicas. Além disso o tratamento com sialidase também aumenta a capacidade de DCs de induzir a proliferação de células T CD4+. Em conjunto, os resultados indicam que moDCs com menos ácidos siálicos na superfície, têm melhor potencial imuno-estimulador, com maior capacidade de induzir respostas imunes anti-tumorais.--------------------- ABSTRACT: Dendritic cells (DCs) have a unique capacity to induce immune responses against tumor cells. They can phagocyte tumor antigens, maturate and present them to T cells, triggering antigen-specific immune responses that may lead to the elimination of tumor cells. Since they induce long-lasting immunological memory, DCs become an attractive strategy as cellular targets for vaccines in the treatment and/or prevention of cancer. However, the therapeutic results obtained in clinical trials with DCs are scarce and only few patients effectively respond to the DC vaccines. Our group has shown that sialic acid containing glycans play an important functional role in ex vivo generated DC. Here we aimed to establish an in vitro model to assess specific antitumor responses. To achieve this, an enzymatic treatment of monocyte-derived DCs (moDCs) was performed using sialidase to cleave surface sialic acids. The maturation profile of the moDCs was characterized by flow cytometry and cytokine expression. The results show that sialidase treatment can upregulate co-stimulatory molecules on surface of moDCs stimulated with Toll like receptor (TLR) agonists. To understand whether sialidase treatment affected the TLR signaling, we have used HEK cells stably transfected with TLRs 2, 4 and 7/8. The data showed that desialylation of moDCs does not affect the signaling via these receptors. To investigate the functional impact of sialidase treatment in the capacity of moDCs to present antigen and to activate antigen specific T cells, sialidase treated and untreated moDCs were co-cultured with CD8+ T cell clones specific for peptides derived from the gp100 tumor antigen. Our results show that desialylated HLA02:01+ DCs are superior in cross-presentation of the peptide to gp100280–288 specific CD8+ T cells. In addition, sialidase treatment also increased the DC capacity to induce CD4+ T cells proliferation. Together, these data indicate that moDCs with altered cell surface sialic acids, through a sialidase treatment, have a better immunostimulatory potential which could improve anti-tumor immune responses.

Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Modelling Z→ττ processes in ATLAS with τ-embedded Z→μμ data

This paper describes the concept, technical realisation and validation of a largely data-driven method to model events with Z→ττ decays. In Z→μμ events selected from proton-proton collision data recorded at s√=8 TeV with the ATLAS experiment at the LHC in 2012, the Z decay muons are replaced by τ leptons from simulated Z→ττ decays at the level of reconstructed tracks and calorimeter cells. The τ lepton kinematics are derived from the kinematics of the original muons. Thus, only the well-understood decays of the Z boson and τ leptons as well as the detector response to the τ decay products are obtained from simulation. All other aspects of the event, such as the Z boson and jet kinematics as well as effects from multiple interactions, are given by the actual data. This so-called τ-embedding method is particularly relevant for Higgs boson searches and analyses in ττ final states, where Z→ττ decays constitute a large irreducible background that cannot be obtained directly from data control samples.

Evaluation of temperature influence on embedded kinematic system

Dissertação de mestrado integrado em Engenharia Mecânica

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Zebrafish embryos as in vivo model for toxicity evaluation: screening of nanoparticle formulations DODAB:MO and DODAC:MO

Dissertação de mestrado em Molecular Genetics

Application of multivariate statistical process control to fuel cell manufacturing

Univariate statistical control charts, such as the Shewhart chart, do not satisfy the requirements for process monitoring on a high volume automated fuel cell manufacturing line. This is because of the number of variables that require monitoring. The risk of elevated false alarms, due to the nature of the process being high volume, can present problems if univariate methods are used. Multivariate statistical methods are discussed as an alternative for process monitoring and control. The research presented is conducted on a manufacturing line which evaluates the performance of a fuel cell. It has three stages of production assembly that contribute to the final end product performance. The product performance is assessed by power and energy measurements, taken at various time points throughout the discharge testing of the fuel cell. The literature review performed on these multivariate techniques are evaluated using individual and batch observations. Modern techniques using multivariate control charts on Hotellings T2 are compared to other multivariate methods, such as Principal Components Analysis (PCA). The latter, PCA, was identified as the most suitable method. Control charts such as, scores, T2 and DModX charts, are constructed from the PCA model. Diagnostic procedures, using Contribution plots, for out of control points that are detected using these control charts, are also discussed. These plots enable the investigator to perform root cause analysis. Multivariate batch techniques are compared to individual observations typically seen on continuous processes. Recommendations, for the introduction of multivariate techniques that would be appropriate for most high volume processes, are also covered.

Electrical Properties of Isolated Cardiomyocytes in a Rat Model of Thiamine Deficiency

In modern society, thiamine deficiency (TD) remains an important medical condition linked to altered cardiac function. There have been contradictory reports about the impact of TD on heart physiology, especially in the context of cardiac excitability. In order to address this particular question, we used a TD rat model and patch-clamp technique to investigate the electrical properties of isolated cardiomyocytes from epicardium and endocardium. Neither cell type showed substantial differences on the action potential waveform and transient outward potassium current. Based on our results we can conclude that TD does not induce major electrical remodeling in isolated cardiac myocytes in either endocardium or epicardium cells.

Ultrastructural characterisation of the cell wall of Arabidopsis thaliana transgenic plants

The plant cell wall is a strong fibrillar network that gives each cell its stable shape. It is constituted by a network of cellulose microfibrils embedded in a matrix of polysaccharides, such as xyloglucans. To enlarge, cells selectively loosen this network. Moreover, there is a pectin-rich intercellular material, the middle lamella, cementing together the walls of adjacent plant cells. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of enzymes involved in the reorganisation of the cellulose-xyloglucan framework by catalysing cleavage and re-ligation of the xyloglucan chains in the plant cell wall, and are considered cell wall loosening agents. In the laboratory, it has been isolated and characterised a XTH gene, ZmXTH1, from an elongation root cDNA library of maize. To address the cellular function of ZmXTH1, transgenic Arabidopsis thaliana plants over-expressing ZmXTH1 (under the control of the CaMV35S promoter) were generated. The aim of the work performed was therefore the characterisation of these transgenic plants at the ultrastructural level, by transmission electron microscopy (TEM).The detailed cellular phenotype of transgenic plants was investigated by comparing ultra-thin transverse sections of basal stem of 5-weeks old plants of wild type (Col 0) and 35S-ZmXTH1 Arabidopsis plants. Transgenic plants show modifications in the cell walls, particularly a thicker middle lamella layer with respect the wild type plants, supporting the idea that the overexpression of ZmXTH1 could imply a pronounced wall-loosening. In sum, the work carried out reinforces the idea that ZmXTH1 is involved in the cell wall loosening process in maize.  

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.