Gold nanoparticles for plasmid delivery

Gene therapy presents an ideal strategy for the treatment of genetic as well as acquired diseases, such as cancer and typically involves the insertion of a functioning gene into cells to correct a cellular dysfunction or to provide a new cellular function. Gene delivery vectors are based in two models: viral and non-viral. Viral vectors have high transfection efficiency but their major barrier is immunogenicity. Since the non-viral vectors have no immunogenicity, these have been widely studied. Gold nanoparticles have been proposed as optimal delivery systems of genetic material, due their small size, high surface-to-volume ratio and the ability to be functionalized with multiple molecules. In the present work, an AuNP-based formulation was developed to deliver a plasmid in a colorectal cancer cell line, containing as reporter gene the gene encoding to EGFP. The delivery system resulted from the functionalization of 14 nm AuNP with a PEG layer (4300114 PEG chains/AuNP), which increases stability and biocompatibility of AuNPs; quaternary ammonium groups which provide positive charges that allow electrostatic binding of plasmid, which is considered the therapeutic agent to be transported into cells. The system developed was characterized by UV-vis spectroscopy, DLS, TEM and by electrophoretic mobility, yielding a formulation with 113.5 nm.Transfection efficiency of the formulation developed was evaluated through PCR and through EGFP expression by fluorescence microscopy and fluorescence spectroscopy. The internalization was observed 3h post transfection; however a low level of EGFP expression was achieved. After 24h of incubation, EGFP expression increases just 3 times compared to non-transfected cells. The commercial system (Lipofectamine) expressed EGFP 5 times more than the system developed AuNP@PEG@R4N+@pEGFP. This difference could be related to lower translocation to the nucleus.

Autonomous behaviors for an unmanned mini-aerial vehicle

This thesis aims at addressing the development of autonomous behaviors, for search and exploration with a mini-UAV (Unmanned Aerial Vehicle), or also called MAV (Mini Aerial Vehicle) prototype, in order to gather information in rescue scenarios. The platform used in this work is a four rotor helicopter, known as quad-rotor from the German company Ascending Technologies GmbH, which is later assembled with a on-board processing unit (i.e. a tiny light weight computer) and a on-board sensor suite (i.e. 2D-LIDAR and Ultrasonic Sonar). This work can be divided into two phases. In the first phase an Indoor Position Tracking system was settled in order to obtain the Cartesian coordinates (i.e. X, Y, Z) and orientation (i.e.heading) which provides the relative position and orientation of the platform. The second phase was the design and implementation of medium/high level controllers on each command input in order to autonomously control the aircraft position, which is the first step towards an autonomous hovering flight, and any autonomous behavior (e.g. Landing, Object avoidance, Follow the wall). The main work is carried out in the Laboratory ”Intelligent Systems for Emergencies and Civil Defense”, in collaboration with ”Dipartimento di Informatica e Sistemistica” of Sapienza Univ. of Rome and ”Istituto Superiore Antincendi” of the Italian Firemen Department.

Production of recombinant protein hLIF in static and dynamic systems of animal cell culture

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Polyglutamine diseases (PolyQ): construction and characterization of yeast models

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

Establishing a high titer transient gene expression process in conditioned media for CHO-DG44 cells

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Role of ß-lactamase operon on mecA expression in Staphylococcus aureus

RESUMO: Os Staphylococcus aureus resistentes à meticilina (MRSA, do inglês “methicillin-resistant Staphylococcus aureus”) são um dos principais agentes responsáveis por infeções hospitalares. Os MRSA são resistentes a praticamente todos os antibióticos β-lactâmicos devido a dois mecanismos principais: produção de β-lactamase (bla), codificada pelo gene blaZ, e produção de uma proteína de ligação à penicilina (PBP2a, do inglês “penicillin binding protein 2”), codificada pelo gene mecA. Estes dois genes são regulados por sistemas homólogos, constituídos por um sensor-transdutor (BlaR1 e MecR1) e um repressor (BlaI e MecI), de tal modo que ambos os sistemas são capazes de co-regular os genes mecA e blaZ, embora com eficiências de indução muito diferentes. De facto, a indução mediada pelo sistema mecI-mecR1 é tão lenta que se acredita que este sistema não está funcional na maioria das estirpes MRSA. No entanto, dados recentes do nosso laboratório, demonstram a ausência de relação entre a presença do gene mecI e o nível de resistência à meticilina em estirpes MRSA epidémicas, e também que, o fenótipo de resistência da grande maioria das estirpes não é perturbado pela sobre-expressão em trans do repressor mecI. Curiosamente, as duas estirpes em que a expressão da resistência foi afetada pela sobre-expressão do mecI são negativas para o locus da β-lactamase, o que sugere que este locus pode interferir diretamente com a repressão do gene mecA mediada pelo MecI. Nesta tese de mestrado esta hipótese foi explorada usando estratégias de biologia molecular e ensaios fenotípicos da resistência aos -lactâmicos. Os resultados obtidos demonstram que a presença do plasmídeo nativo da β-lactamase não só anula a repressão mediada pelo MecI, como também aumenta o nível de resistência das estirpes parentais. Várias hipóteses foram então formuladas para explicar estas observações. Dados preliminares, em conjunto com evidências experimentais publicadas, sugerem que o BlaI forma hetero-dímeros com o MecI que, após a indução, são inativados eficientemente pelo BlaR1. Em conclusão, estes resultados apresentam novas perspetivas para o mecanismo de regulação do mecA e para uma nova importante função do operão da β-lactamase para o fenótipo das estirpes MRSA.-------------------ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and is also emerging in the community. MRSA is cross-resistant to virtually all β-lactam antibiotics and has acquired two main resistance mechanisms: production of β-lactamase (bla), coded by blaZ, and production of penicillin binding protein 2a (PBP2a), coded by mecA. Both genes are regulated by homologous sensor-transducers (BlaR1 and MecR1) and repressors (BlaI and MecI), and coregulation of mecA and blaZ by both systems has been demonstrated, although with remarkable different efficiencies. In fact, induction of mecA by mecI-mecR1 is so slow that it is believed it is not functional in most MRSA strains. However, recent data from our laboratory has unexpectedly demonstrated that not only there is no correlation between the presence of mecI gene and the resistance level in epidemic MRSA strains, but also that for most strains there were no significant changes on the resistance phenotype upon the mecI overexpression in trans. Interestingly, the two strains in which mecI overexpression affected the resistance expression were negative for the bla locus, suggesting that this locus may interfere directly with the MecI-mediated repression of mecA and account for those puzzling observations. In this master thesis we have explored this hypothesis using molecular biology strategies and phenotypic analysis of -lactam resistance. The data obtained demonstrate that the presence of a wild-type plasmid containing the bla locus not only disrupts the MecImediated repression, but also significantly enhances the expression of resistance. Several preliminary hypotheses were formulated to explain these observations and preliminary data, together with published evidence, support the working model that BlaI forms functional hetero-dimers with MecI, which upon induction are readily inactivated by BlaR1. These results provide new insights into the regulatory mechanism(s) of mecA and open new perspectives for the role of β-lactamase operon in the MRSA phenotype.

A contribuição das bombas de efluxo QacA e Smr para a multirresistência em Staphylococcus aureus

RESUMO: O efluxo de compostos antimicrobianos é um mecanismo importante na multirresistência em bactérias. Bombas de efluxo codificadas em plasmídeos, como a QacA e a Smr, estão implicadas na susceptibilidade reduzida a biocidas, geralmente utilizados na prevenção e controlo de infecções nosocomiais, incluindo as causadas por estirpes de Staphylococcus aureus resistentes à meticilina (MRSA). Neste trabalho pretendeu-se avaliar a relevância de QacA e Smr no perfil de susceptibilidade dos isolados clínicos MRSA SM39 e SM52, que transportam os plasmídeos pSM39 e pSM52 com os determinantes qacA e smr, respectivamente. A actividade de efluxo das estirpes SM39 e SM39 curada (sem pSM39) e das estirpes SM52 e RN4220:pSM52 (estirpe susceptível RN4220 transformada com pSM52) foi caracterizada por: (1) determinação da concentração mínima inibitória (CMI) de biocidas, corantes e antibióticos, na ausência e presença dos inibidores de efluxo tioridazina, clorpromazina, verapamil e reserpina; e (2) fluorometria em tempo-real. A determinação de CMIs demonstrou que a actividade de efluxo mediada por QacA e Smr está envolvida na susceptibilidade reduzida aos biocidas e corantes testados, que incluíram o brometo de hexadeciltrimetilamónio, a cetrimida, o cloreto de benzalcónio, a berberina, o cloreto de dequalínio, a pentamidina e o brometo de etídeo. Os ensaios fluorométricos confirmaram a elevada actividade de efluxo presente nas estirpes com os genes qacA ou smr. A determinação de CMIs para antibióticos β-lactâmicos em conjunto com o teste da nitrocefina revelou a presença simultânea do gene qacA e de uma β-lactamase no plasmídeo pSM39. Este trabalho evidencia a importância das bombas de efluxo QacA e Smr na resistência a biocidas em estirpes MRSA e na sobrevivência destas estirpes em ambiente hospitalar e na comunidade, para além de destacar a questão da potencial co-resistência entre biocidas e antibióticos.--------------- ABSTRACT: Drug efflux has become an important cause of multidrug resistance (MDR) in bacteria. Plasmid-encoded MDR efflux pumps, such as QacA and Smr, are implicated in reduced susceptibility to biocides, generally used in the prevention and control of nosocomial infections, including the ones caused by methicillin-resistant Staphylococcus aureus (MRSA). In this work, we aimed to evaluate the relevance of QacA and Smr to the susceptibility profile of the clinical MRSA isolates SM39 and SM52, which harbor the plasmids pSM39 and pSM52 that carry the determinants qacA and smr, respectively. Efflux activity of strain SM39 and its plasmid-free counterpart, SM39 cured, SM52 and RN4220:pSM52 (susceptible strain RN4220 transformed with pSM52) was characterized by: (1) determination of minimum inhibitory concentration (MIC) of biocides, dyes and antibiotics, in the absence and presence of the efflux inhibitors thioridazine, chlorpromazine, verapamil and reserpine; and (2) real-time fluorometry. MIC determination showed that QacA and Smr mediated efflux was involved in the reduced susceptibility profile to the biocides and dyes tested, which included hexadecyltrymethylammonium bromide, cetrimide, benzalkonium chloride, berberine, dequalinium chloride, pentamidine and ethidium bromide. Fluorometric assays confirmed the higher efflux activity present in strains harboring qacA or smr genes. Moreover, MIC determination for β-lactam antibiotics together with the nitrocefin test confirmed the presence of a β-lactamase in the plasmid carried by SM39 strain, pSM39. This work highlights the relevance of QacA and Smr to the biocide resistance in MRSA strains, and consequently to their survival and maintenance in the hospital environment and in the community. Furthermore, the presence of a β-lactamase and qacA determinants in the the same plasmid reinforces the question of the potencial biocide/antibiotic co-resistance in MRSA strains.

Electrochemical characterization of Dps, a DNA-protecting protein

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Effects of UV radiation exposure on DNA and DNA repair enzymes

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Evaluation of a new vaccine based on pDNA and recombinant protein against Helicobacter pylori

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

The impact of banks dimension on profitability, sustainability and efficiency: An analysis for the Iberian financial system

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

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia

hCES2: studies on the role of glycosylation in activity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

eVentos 2 - Autonomous sailboat control

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

Exploring deregulated signals involved in motor neuron-microglia cross-talk in ALS

Part of the results discussed in this thesis was presented in the following meetings: Cunha MI, Cunha C, Vaz AR, Brites D. Studying microglial-motoneuron cross-talk in ALS pathology. 6th iMed.UL Postgraduate Students Meeting, Lisbon, July 2, 2014. [Abstract and Poster] Vaz AR. Motoneuron degeneration and glial reactivity in ALS: insights from cellular to animal models. Neuroscience Seminars at IMM 2012, Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal, June 9, 2014. [Oral Communication (by invitation)]