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

Dissertação para obtenção do Grau de Mestre em Biotecnologia

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

Dissertação para obtenção do Grau de Mestre em Biotecnologia

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.

Mechanisms underlying intracellular delivery

AuNPs are versatile systems used for different biomedical application including imaging, drug and gene delivery. These systems support the intracellular transport of active molecules, a step that is considered one of the crucial problems in drug delivery. Nevertheless, in order to design optimal multifunctional AuNPs for specific and efficient nanomedicine applications, the mechanism by which AuNPs interact with living cells must be fully understand. The main goal of this work consisted in the assessment of the cellular uptake mechanism of 14 nm spherical AuNPs by A549 cells, through fluorescent spectroscopy and microscopy, in combination with quantitative analysis by ICP-MS. TAMRA labeled AuNPs were characterized by UV-visible and fluorescent spectroscopy and the final hydrodynamic diameter of 22.5 ± 0.33 nm was obtained by DLS. Regarding the cellular uptake studies, the AuNPs presented a fast cellular uptake kinetics reaching a saturation point after 6 hours of incubation in A549 cells. Further investigation concerning the internalization mechanism of this AuNPs was evaluated using specific inhibitors for different endocytic pathways. Optimal inhibition was achieved using chlorpromazine, inhibitor of clathrin-mediated endocytosis, resulting in a 23.5 % inhibition of AuNPs after 1 hour of incubation. This preliminary result obtained by fluorescent spectroscopy suggests that these AuNPs were predominantly uptake by clathrin-mediated endocytosis, meaning that other endocytic pathways must be involved in the cellular uptake of this AuNPs. In what cell viability is concern, the prepared AuNPs and the endocytic inhibitors revealed no significant effect on the cell viability in A549 cell line.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Polimorfismos da região da integrase, do gene pol, em doentes infectados por VIH-1

Ciências biomédicas

Transformation of Medicago truncatula with the arginine decarboxylase gene to modify polyamine metabolism toward water deficit resistance

Dissertation submitted to obtain a Ph.D. (Doutoramento) degree in Biology at the Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Gammaretroviral and lentiviral vectors for gene therapy: stability and inactivation mechanisms

Dissertation presented to obtain a Ph.D degree in Engineering and Technology Sciences, Gene Therapy at the Instituto de Tecnologia Quimica e Biológica, Universidade Nova de Lisboa

Post-transcriptional regulation of HFE gene expression

Thesis presented to obtain the Ph.D. degree in Biology (Molecular Genetics), by the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia.

Linking the embryonic clock with temporal collinearity of Hox gene activation

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

Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

Plos Genetics, 5(7): ARTe1000566

Development of cyclodextrin-hydrogel polymeric systems in scCO2 for drug delivery

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

Gold nanoparticle-DNA conjugates for oligonucleotide vectorization towards gene silencing

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

Development of electrospun Ion jelly fibers® for drug delivery

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Heuristics and policies for online pickup and delivery problems

Master Thesis