Novel functions of the cohesin accessory factor dPDS5 uncover a new meiotic checkpoint

Dissertation presented to obtain the Ph.D degree in Biology.

Development of novel benzimidazole- based COX Inhibitors new synthetic strategies and screening of heterocyclic structures

Dissertação para obtenção do Grau de Doutor em Química, especialidade Química Orgânica

Dynamics and kinetics of natural killer cell cytotoxicity in human malaria as evaluated by a novel stepwise cytotoxicity assay

Malaria causes important functional alterations of the immune system, but several of them are poorly defined. To evaluate thoroughly the natural killer cell cytotoxicity in patients with malaria, we developed a technique capable to assess both the dynamics and the kinetics of the process. For the kinetics assay, human peripheral blood mononuclear cells were previously incubated with K562 cells and kept in agarose medium, while for the dynamics assay both cells were maintained in suspension. NK activity from patients with vivax malaria presented a kinetics profile faster than those with falciparum malaria. NK cytotoxicity positively correlated with parasitemia in falciparum malaria. The dynamics of NK cytotoxicity of healthy individuals was elevated at the beginning of the process and then significantly decreased. In contrast, malaria patients presented successive peaks of NK activity. Our results confirmed the occurrence of alteration in NK cell function during malaria, and added new data about the NK cytotoxicity process.

Undrestanding postoperative cognitive dysfunction: novel insights

ABSTRACT: Background: Sleep is integral to biological function and sleep disruption can result in both physiological and psychological dysfunction. The acute cognitive consequences of sleep loss has been an active field of recent investigation, evidence suggests that sleep disruption in critically ill older adults can result in acute decrements in cognitive functioning. Surgery activates the innate immune system, inducing neuroinflammatory changes that interfere with cognition. The fact that patients with sleep disorders have an increased likelihood of exhibiting postoperative delirium encourages us to investigate the contribution of perioperative SF to the neuroinflammatory and cognitive responses of surgery. Methods: The effects of 24h sleep fragmentation (SF) and surgery were explored on adult C57BL/6J male mice. SF procedure started at 7 am with the home-cages being placed on a large platform orbital shaker cycled every 120 seconds (30 sec on/90 sec off). This procedure lasted for 24h. Stabilized tibia fracture was performed either before or after the 24h SF procedure. Separate cohorts of mice were tested for systemic and hippocampal inflammation and cognition. Results: Twenty-four hours of SF induced non-hippocampal memory dysfunction and increase in systemic IL-6. SF and surgery caused hippocampal-dependent memory impairment, although memory impairment was not exacerbated by combining SF with surgery. One day after either SF or surgery there was a significant increase in IL6 mRNA and TNF-alpha mRNA. These increments were more pronounced when either pre or post operative SF was combined with surgery. Conclusions: We show that while SF and surgery can independently produce significant memory impairment, perioperative SF significantly increased hippocampal inflammation without further cognitive impairment. The dissociation between neuroinflammation and cognitive decline may relate to our use of a sole memory paradigm that does not capture other aspects of cognition, especially learning.

Study of novel energy metabolism pathways in anaerobic bacteria

Energy conservation in chemotrophic anaerobic bacteria is achieved by two possible processes, substrate level phosphorylation (SLP) and electron transfer phosphorylation (ETP). This second mechanism, also known as respiration, involves chemiosmotic coupling. However, a third mechanism for energy coupling was recently proposed: the flavin-based electron bifurcation (FBEB). (...)

Chagas disease as a mechanistic model for testing a novel hypothesis

The association between depression and cardiovascular disease is well documented. Nevertheless, the process through which they are linked remains unknown, as does the direction of this relationship. Studies have suggested both that depression is a risk factor for heart disease and that heart disease is a risk factor for depression. A number of studies have established that a relationship exists between depression and inflammation, with alterations in the levels of inflammatory markers (IL-1, IL-6, TNF-alpha and others). Depressive symptoms have also been identified in many diseases characterized by inflammatory processes e.g. rheumatoid arthritis, bronchial asthma, diabetes, tuberculosis and cardiovascular diseases. In this brief viewpoint, we explain and propose how to use Chagas disease, a disorder characterized by inflammatory processes and leading to cardiovascular and autonomic problems, as a model for studying the directionality of the relationship between heart disease and depression.

Phage display as a tool for development of novel therapeutics for breast cancer

Phage display technology is a powerful platform for the generation of highly specific human monoclonal antibodies (Abs) with potential use in clinical applications. Moreover, this technique has also proven to be a reliable approach in identifying and validating new cancer-related targets. For scientific or medical applications, different types of Ab libraries can be constructed. The use of Fab Immune libraries allows the production of high quality and affinity antigen-specific Abs. In this work, two immune human phage display IgG Fab libraries were generated from the Ab repertoire of 16 breast cancer patients, in order to obtain a tool for the development of new therapeutic Abs for breast cancer, a condition that has great impact worldwide. The generated libraries are estimated to contain more than 108 independent clones and a diversity over 90%. Libraries validation was pursued by selection against BSA, a foreign and highly immunogenic protein, and HER2, a well established cancer target. Preliminary results suggested that phage pools with affinity for these antigens were selected and enriched. Individual clones were isolated, however, it was not possible to obtain enough data to further characterize them. Selection against the DLL1 protein was also performed, once it is a known ligand of the Notch pathway, whose deregulation is associated to breast cancer, making it an interesting target for the generation of function-blocking Abs. Selection resulted in the isolation of a clone with low affinity and Fab expression levels. The validation process was not completed and further effort will have to be put in this task in the future. Although immune libraries concept implies limited applicability, the library reported here has a wide range of use possibilities, since it was not restrained to a single antigen but instead thought to be used against any breast cancer associated target, thus being a valuable tool.

Human activity recognition with accelerometry: novel time and frequency features

Human Activity Recognition systems require objective and reliable methods that can be used in the daily routine and must offer consistent results according with the performed activities. These systems are under development and offer objective and personalized support for several applications such as the healthcare area. This thesis aims to create a framework for human activities recognition based on accelerometry signals. Some new features and techniques inspired in the audio recognition methodology are introduced in this work, namely Log Scale Power Bandwidth and the Markov Models application. The Forward Feature Selection was adopted as the feature selection algorithm in order to improve the clustering performances and limit the computational demands. This method selects the most suitable set of features for activities recognition in accelerometry from a 423th dimensional feature vector. Several Machine Learning algorithms were applied to the used accelerometry databases – FCHA and PAMAP databases - and these showed promising results in activities recognition. The developed algorithm set constitutes a mighty contribution for the development of reliable evaluation methods of movement disorders for diagnosis and treatment applications.

Development of novel ionic liquids based on biological molecules

Ionic Liquids (ILs) belong to a class of compounds with unusual properties: very low vapour pressure; high chemical and thermal stability and the ability to dissolve a wide range of substances. A new field in research is evaluating the possibility to use natural chiral biomolecules for the preparation of chiral ionic liquids (CILs). This important challenge in synthetic chemistry can open new avenues of research in order to avoid some problems related with the intrinsic biodegradability and toxicity associated to conventional ILs. The research work developed aimed for the synthesis of CILs, their characterization and possible applications, based on biological moieties used either as chiral cations or anions, depending on the synthetic manipulation of the derivatives. Overall, a total of 28 organic salts, including CILs were synthesized: 9 based on L-cysteine derivatives, 12 based on L-proline, 3 based on nucleosides and 4 based on nucleotides. All these new CILs were completely characterized and their chemical and physical properties were evaluated. Some CILs based on L-cysteine have been applied for discrimination processes, including resolution of racemates and as a chiral catalyst for asymmetric Aldol condensation. L-proline derived CILs were also studied as chiral catalysts for Michael reaction. In parallel, the interactions of macrocyclic oligosugars called cyclodextrins (CDs) with several ILs were studied. It was possible to improve the solubility of CDs in water and serum. Additionally, fatty acids and steroids showed an increase in water solubility when ILs-CDs systems were used. The development of efficient and selective ILs-CDs systems is indispensable to expand the range of their applications in host-guest interactions, drug delivery systems or catalytic reactions. Novel salts derived from nucleobases were used in order to enhance the fluorescence in aqueous solution. Additionally, preliminary studies regarding ethyl lactate as an alternative solvent for asymmetric organocatalysis were performed.

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Fundação para a Ciência e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cecíla Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011)

Novel Cryopreservation Strategies for Cell‐Therapies and Pre‐Clinical Research

The expanding need for complex biologics for therapeutic applications, in‐vitro pharmacology and toxicology studies and fundamental research demands the production of banks of well‐characterized and safety‐tested stocks of a large number of cell/tissue samples. This implies the development of effective cryopreservation methodologies that can cope with process scalability and automation and must reflect the biological and physical properties of the cells as these can be significantly altered by the process.

Novel ready-to-eat mango product using gellan gum as gelling agent: physico-chemical, microbial and sensory characteristics

Este trabalho foi efectuado com o apoio da Universidade de Lisboa, Instituto Superior de Agronomia com o Centro de Engenharia dos Biossistemas (CEER

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

Of Structural Denial: A Narratological Study of the Structural Disintegration of the Novel Form

If an opening to the argument of this dissertation is of imperative necessity, one might tentatively begin with Herbert Quain, born in Roscommon, Ireland, author of the novels The God of the Labyrinth (1933) and April March (1936), the short-story collection Statements (1939), and the play The Secret Mirror (undated). To a certain extent, this idiosyncratic Irish author, who hailed from the ancient province of Connacht, may be regarded as a forerunner of the type of novels which will be considered in this dissertation. Quain was, after all, the unconscious creator of one of the first structurally disintegrated novels in the history of western literature, April March. His first novel, The God of the Labyrinth, also exhibits elements which are characteristic of structurally disintegrated fiction, for it provides the reader with two possible solutions to a mysterious crime. As a matter of fact, one might suggest that Quain’s debut novel offers the reader the possibility to ignore the solution to the crime and carry on living his or her readerly life, turning a blind eye to the novel itself. It may hence be argued that Quain’s first novel is in fact a compound of three different novels. It is self-evident that the structure of Quain’s oeuvre is of an experimental nature, combining geometrical precision with authorial innovation, and one finds in it a higher consideration for formal defiance than for the text itself. In other words, the means of expression are the concern of the author and not, interestingly, the textual content. April March, for example, is a novel which regresses back into itself, its first chapter focussing on an evening which is preceded by three possible evenings which, in turn, are each preceded by three other, dissimilar, possible evenings. It is a novel of backward-movement, and it is due to this process of branching regression that April March contains within itself at least nine possible novels. Structure, therefore, paradoxically controls the text, for it allows the text to expand or contract under its formal limitations. In other words, the formal aspects of the novel, usually associated with the restrictive device of a superior design, contribute to a liberation of the novel’s discourse. It is paradoxical only in the sense that the idea of structure necessarily entails the fixation of a narrative skeleton that determines how plot and discourse interact, something which Quain flouts for the purposes of innovation. In this sense, April March’s convoluted structure allows for multiple readings and interpretations of the same text, consciously germinating narratives within itself, producing different texts from a single, unique source. Thus, text and means of expression are bonded by a structural design that, rather than limiting, liberates the text of the novel.

Late onset sepsis and intestinal bacterial colonization in very low birth weight infants receiving long-term parenteral nutrition

INTRODUCTION: The purpose of this study was to establish the late onset sepsis (LOS) rate of our service, characterize the intestinal microbiota and evaluate a possible association between gut flora and sepsis in surgical infants who were receiving parenteral nutrition (PN). METHODS: Surveillance cultures of the gut were taken at the start of PN and thereafter once a week. Specimens for blood culture were collected based on clinical criteria established by the medical staff. The central venous catheter (CVC) tip was removed under aseptic conditions. Standard laboratory methods were used to identify the microorganisms that grew on cultures of gut, blood and CVC tip. RESULTS: 74 very low birth weight infants were analyzed. All the infants were receiving PN and antibiotics when the gut culture was started. In total, 21 (28.4%) infants experienced 28 episodes of LOS with no identified source. Coagulase negative staphylococci were the most common bacteria identified, both in the intestine (74.2%) and blood (67.8%). All infections occurred in patients who received PN through a central venous catheter. Six infants experienced episodes of microbial translocation. CONCLUSIONS: In this study, LOS was the most frequent episode in neonates receiving parenteral nutrition who had been submitted to surgery; 28.6% of this infection was probably a gut-derived phenomenon and requires novel strategies for prevention.