Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Assembly and function of a protein cross-linking enzyme during bacterial spore morphogenesis

Sporulation in Bacillus subtilis culminates with the formation of a dormant endospore. The endospore (or spore) is one of the most resilient cell types known and can remain viable in the environment for extended periods of time. Contributing to the spore’s resistance and its ability to interact with and monitor its immediate environment is the coat, the outermost layer of B. subtilis spores. The coat is composed by over 70 different proteins, which are produced at different stages in sporulation and orderly assembled around the developing spore.(...)

Options and Requirements for a potential market entry into Portugal with renewable energy technologies that are applicable for heating and cooling of buildings

NSBE-UNL

PROTEIN FOLDING AND DISEASE THE MITOCHONDRIAL PROTEIN FRATAXIN

This dissertation focuses on the study of frataxin, a small mitochondrial protein whose deficiency is associated with the neurodegenerative disease Friedreich's ataxia (FRDA). Aiming at a better understanding of frataxin conformational and functional properties, two lines of research were followed: first, the effect of FRDA-related mutations in human frataxin (FXN) were studied and the role of oxidative stress related modification addressed; second, yeast frataxin (Yfh1) orthologue was used to explore the conformational and functional properties of the protein.(...)

A CMMI-compliant requirements management and development process

Requirements Engineering has been acknowledged an essential discipline for Software Quality. Poorly-defined processes for eliciting, analyzing, specifying and validating requirements can lead to unclear issues or misunderstandings on business needs and project’s scope. These typically result in customers’ non-satisfaction with either the products’ quality or the increase of the project’s budget and duration. Maturity models allow an organization to measure the quality of its processes and improve them according to an evolutionary path based on levels. The Capability Maturity Model Integration (CMMI) addresses the aforementioned Requirements Engineering issues. CMMI defines a set of best practices for process improvement that are divided into several process areas. Requirements Management and Requirements Development are the process areas concerned with Requirements Engineering maturity. Altran Portugal is a consulting company concerned with the quality of its software. In 2012, the Solution Center department has developed and applied successfully a set of processes aligned with CMMI-DEV v1.3, what granted them a Level 2 maturity certification. For 2015, they defined an organizational goal of addressing CMMI-DEV maturity level 3. This MSc dissertation is part of this organization effort. In particular, it is concerned with the required process areas that address the activities of Requirements Engineering. Our main goal is to contribute for the development of Altran’s internal engineering processes to conform to the guidelines of the Requirements Development process area. Throughout this dissertation, we started with an evaluation method based on CMMI and conducted a compliance assessment of Altran’s current processes. This allowed demonstrating their alignment with the CMMI Requirements Management process area and to highlight the improvements needed to conform to the Requirements Development process area. Based on the study of alternative solutions for the gaps found, we proposed a new Requirements Management and Development process that was later validated using three different approaches. The main contribution of this dissertation is the new process developed for Altran Portugal. However, given that studies on these topics are not abundant in the literature, we also expect to contribute with useful evidences to the existing body of knowledge with a survey on CMMI and requirements engineering trends. Most importantly, we hope that the implementation of the proposed processes’ improvements will minimize the risks of mishandled requirements, increasing Altran’s performance and taking them one step further to the desired maturity level.

Manipulation of host cell functions by Salmonella: the role of the bacterial effector protein SteA

Salmonella enterica serovars are Gram-negative facultative intracellular bacterial pathogens that infect a wide variety of animals. Salmonella infections are common in humans, causing usually typhoid fever and gastrointestinal diseases. Salmonella enterica serovar Typhimurium (S. Typhimurium), which is a leading cause of human gastroenteritis, has been extensively used to study the molecular pathogenesis of Salmonella, because of the availability of sophisticated genetic tools, and of suitable animal and tissue culture models mimicking different aspects of Salmonella infections.(...)

Inhibition of SNF1-related protein kinase1 by trehalose 6-phosphate and other metabolites and the interrelation with plant gorwth

All life forms need to monitor carbon and energy availability to survive and this is especially true for plants which must integrate unavoidable environmental conditions with metabolism for cellular homeostasis maintenance. Sugars, in the heart of metabolism, are now recognized as crucial signaling molecules that translate those conditions. One such signal is trehalose 6- phosphate (T6P), a phosphorylated dimer of glucose molecules which levels correlate well with those of sucrose (Suc). Central integrators of stress and energy regulation include the conserved plant Snf1-related kinase1 (SnRK1) which respond to low cellular energy levels by up-regulating energy conserving and catabolic metabolism and down-regulating energy consuming processes. In 2009 T6P was shown to inhibit SnRK1. The in vitro inhibition of SnRK1 by T6P was confirmed in vivo through the observation that genes normally induced by SnRK1 were repressed by T6P and vice-versa, promoting growth processes. These observations provided a model for the regulation of growth by sugar.(...)

Managing marginal capital requirements in solvency II: Analysis of a real insurance portfolio

This thesis provides a complete analysis of the Standard Capital Requirements given by Solvency II for a real insurance portfolio. We analyze the investment portfolio of BPI Vida e Pensões, an insurance company affiliated with a Portuguese bank BPI, both at security, sub-portfolio and asset class levels. By using the Standard Formula from EIOPA, Total SCR amounts to 239M€. This value is mostly explained by Market and Default Risk whereas the former is driven by Spread and Concentration Risks. Following the methodology of Leblanc (2011), we examine the Marginal Contribution of an asset to the SCR which allows for the evaluation of the risks of each security given its characteristics and interactions in the portfolio. The top contributors to the SCR are Corporate Bonds and Term Deposits. By exploring further the composition of the portfolio, our results show that slight changes in allocation of Term and Cash Deposits have severe impacts on the total Concentration and Default Risks, respectively. Also, diversification effects are very relevant by representing savings of 122M€. Finally, Solvency II represents an opportunity for the portfolio optimization. By constructing efficient frontiers, we find that as the target expected return increases, a shift from Term Deposits/ Commercial Papers to Eurozone/Peripheral and finally Equities occurs.

Evolutionary dynamics of Chlamydia trachomatis genome and identification of molecular patterns of hypothetical protein coding genes

The obligate intracellular bacterium Chlamydia trachomatis is a human pathogen of major public health significance. Strains can be classified into 15 main serovars (A to L3) that preferentially cause ocular infections (A-C), genital infections (D-K) or lymphogranuloma venereum (LGV) (L1-L3), but the molecular basis behind their distinct tropism, ecological success and pathogenicity is not welldefined. Most chlamydial research demands culture in eukaryotic cell lines, but it is not known if stains become laboratory adapted. By essentially using genomics and transcriptomics, we aimed to investigate the evolutionary patterns underlying the adaptation of C. trachomatis to the different human tissues, given emphasis to the identification of molecular patterns of genes encoding hypothetical proteins, and to understand the adaptive process behind the C. trachomatis in vivo to in vitro transition. Our results highlight a positive selection-driven evolution of C. trachomatis towards nichespecific adaptation, essentially targeting host-interacting proteins, namely effectors and inclusion membrane proteins, where some of them also displayed niche-specific expression patterns. We also identified potential "ocular-specific" pseudogenes, and pointed out the major gene targets of adaptive mutations associated with LGV infections. We further observed that the in vivo-derived genetic makeup of C. trachomatis is not significantly compromised by its long-term laboratory propagation. In opposition, its introduction in vitro has the potential to affect the phenotype, likely yielding virulence attenuation. In fact, we observed a "genital-specific" rampant inactivation of the virulence gene CT135, which may impact the interpretation of data derived from studies requiring culture. Globally, the findings presented in this Ph.D. thesis contribute for the understanding of C.trachomatis adaptive evolution and provides new insights into the biological role of C. trachomatishypothetical proteins. They also launch research questions for future functional studies aiming toclarify the determinants of tissue tropism, virulence or pathogenic dissimilarities among C. trachomatisstrains.

Basel III new capital requirements, impacts and bank behavior

For some years, researchers could not find a clear effect of capital adequacy on the risk profile of banks, as shareholders could increase the riskiness of the assets (qualitative effect), crowding-out the effect of reduced leverage (volume effect). Some shareholders might have the will to increase the riskiness of the assets, but they may lack the power to do so. Considering only ”powerful” shareholders, definitive conclusions were drawn but with constant ownership profile. In this paper I investigate whether there is a significant change in the type of shareholders in response to regulatory capital shocks and, if so, will the banking system be in the hands of more “desired” shareholders. I find that ownership profile responds to a regulatory shock, changing the risk appetite of the ruling power at the bank. I find more banks and the government in the ownership of undercapitalised banks and much less institutional shareholders and free float. I claim that these new shareholders may not the desired ones, given the objective of the regulatory change, as they are associated with a preference for more leverage. One possible explanation for this crowding-out effect is that regulators are trying to contain idiosyncratic risk (more linked to the riskiness of the assets) with a rule that contains systematic risk (capital adequacy). This has a distorting effect on ownership. Another insight can be drawn from the tests: supervisors should be aware of significant ownership movements that cause the crowding-out.

Capital requirements and loan market conditions insight into Portuguese Banking system (2004-2011)

Capital Requirements have been gaining importance in the current macroeconomic and financial environment and Portugal is no exception. Nonetheless, despite the several media articles on this subject, the associations with Loan Market Conditions, namely availability and pricing are still unstudied. Thus, this project adds to the existing literature a characterization of Portuguese four biggest banks on capital reporting and requirements fulfillment. It is concluded that banks under analysis need to increase capital and that there is an association between the variables under study: Share Capital is negatively correlated with Credit Volume, and it is positively correlated with Net Commercial Income.

Staying under the radar - the multifunctional LANA Protein

Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposi´s Sarcoma associated herpesvirus (KSHV), first discovered in Kaposi´s Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-κB and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses.

Study of the role of wall teichoic acids in the localization Staphylococcus aureus cell wall synthesis protein PBP4

The cell wall of Staphylococcus aureus is a highly complex network mainly composed of highly cross-linked peptidoglycan (PG) and teichoic acids (TAs), both important for the maintenance of the integrity and viability of bacteria. The penicillin binding proteins (PBPs), which catalyse the final stage of PG biosynthesis, are targets of β-lactam antibiotics and have been a key focus of antibacterial research. S. aureus has four native PBPs, PBP1-4 carried by both methicillin-sensitive (MSSA) and –resistant (MRSA) strains. PBP4 is required for the synthesis of the highly cross-linked PG and, as shown in recent studies, is essential for the expression of β-lactam resistance in community-acquired strains (CA-MRSA). This protein has a septal localization that seems to be spatially and temporally regulated by an unknown intermediate of the wall teichoic acids (WTA) biosynthesis pathway. Therefore, if WTA synthesis is compromised, PBP4 becomes dispersed throughout the entire cell membrane. The aim of this project was to identify the WTA precursor responsible for the septal recruitment of PBP4. In order to do so, inducible mutants of tarB and tarL genes in the background of NCTCPBP4-YFP were constructed allowing for the study of PBP4 localization in the presence and absence of these specific tar genes.With this work we were able to show that the absence of TarB or TarL leads to the delocalization of PBP4, indicating that TarL or a protein/WTA precursor whose localization/synthesis is dependent on TarL is responsible for the recruitment of PBP4.