Biogeography of the fire salamander (Salamandra salamandra, L.) in Portugal

Dissertação de mest., Biologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2008

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Multifactorial approach to non-viral gene therapy: development of an efficient system for the retina

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Desenvolvimento de vectores não virais para terapia génica

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Salamandra salamandra (Amphibia: Caudata: Salamandridae) in Portugal: not all black and yellow

The fire salamander complex is quite diverse in the Iberian Peninsula where nine subspecies of Salamandra salamandra are currently recognized. Here, we analysed the geographical distribution of the subspecies S. s. gallaica and S. s. crespoi using partial sequences of the mitochondrial cytochrome b gene of 168 individuals from 12 locations in Portugal. Our results support the existence of a deep lineage divergence between the two subspecies, with non-overlapping geographical distributions except in two contact zones: one in Sesimbra on the western coast, and another in Alcoutim on the southeastern border with Spain. Moreover, S. s. crespoi displays signs of gene flow among the sampled locations whereas S. s. gallaica shows evidence of some restriction to gene flow. Present-day genetic make-up of S. s. gallaica and S. s. crespoi is a result of past historical events, fine-tuned by contemporary Iberian geoclimate. Humid mountain areas were found to harbour increased genetic diversity possibly acting as past refugia during drier interglacial periods. To analyse wider geographical patterns and lineage splitting events within S. salamandra we performed a Bayesian dating analysis completing our data set with previously published sequences. The observed divergences were associated to successive biogeographic scenarios, and to other Iberian species showing similar trends.

Synthesis and evaluation of polymer nanoparticles for delivery in RPE cells

Ocular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.

Characterization of pro- and anti-angiogenic factors in models of diabetic retinopathy

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015