Doctor en Ciencias mención Biología Molecular, Celular y Neurociencias / Cell adhesion to the extracellular matrix (ECM) is key to the regulation of cellular processes such as proliferation, survival, and migration. Mesenchymal cell adhesion requires the formation of focal adhesions, defined as multiprotein complexes that allow cell binding to the ECM. Focal adhesions involve the congregation of more than 150 proteins, which include integrins, the main ECM receptors, and the Focal Adhesion Kinase (FAK), which has as a fundamental role in the turnover of focal adhesions. Formation and disassembly of focal adhesions are dynamically regulated during cell migration, and numerous studies show that increased expression or activity of focal adhesion proteins is associated with many human diseases. For example, increased levels and/or activities of FAK have been associated with oncogenic transformation in a variety of tissues and organs. Aside the roles commonly described for FAK in activating integrins, such as integrin β1 (ITGB1), and modulating integrin downstream signaling, this kinase has been shown to behave as an intracellular effector of multiple signaling pathways, including Neogenin-1 (NEO1). NEO1 is a transmembrane receptor involved in tissue growth during embryogenesis, although it is also broadly expressed in adulthood. More recently, overexpression of NEO1 has been observed in a wide variety of human cancers including those of the breast, pancreas, cervix, colon and medulloblastoma. Despite the latter, its role in tumorigenesis is still controversial and remains to be elucidated. In the context of neural development NEO1 promotes neuronal migration and axonal guidance through interaction with the extracellular Netrin (NTN) ligand family. Interestingly, NEO1 is strongly expressed in neuroblastoma (NB), a cancer derived from neural progenitors of the sympathoadrenal lineage. Therefore, it will be relevant xii determining if NEO1, by interacting with NTNs, could promote the migration and metastasis of NB cells, and if this phenomenon is mediated through the activation of FAK, thus promoting the intracellular activation of the ITGB1. It was shown that both the NEO1 receptor and its main NTN ligands family members, Netrin-1 (NTN1) and Netrin-4 (NTN4), are expressed in NB patient samples. We reveal that NEO1, in addition to promoting chemotactic migration in association with NTN4 in vitro, promotes metastasis in vivo, as demonstrated in the chicken embryo model. Recent data from the literature supports our results and allow us to suggest that the interaction between NTN4 and NEO1 might not be direct, but rather mediated by a signaling complex also made up by laminin g1 and ITGB1. NTN1, on the other hand, acts as a chemoattractant molecule and binds directly to NEO1. In addition, we show that FAK is an downstream effector of NEO1, and that the NTN1/ NEO1 signaling complex interacts with ITGB1, in the SK-N-SH NB cells. Thus, our results suggest that NTN1/ NEO1 promotes the activation of ITGB1 through FAK, most likely through the formation of a macromolecular complex, driving cell migration. All these results are consistent with our in vivo observations, which show that NEO1 promotes the metastasis of SK-N-SH cells in an immunodeficient mouse model. In summary, this thesis shows that NEO1 promotes the migration of NB cells and that its mechanism of action is via interaction with ITGB1, with FAK being an intracellular mediator of this signaling pathway. NEO1, by promoting cell migration, could play a key role in the process of NB metastasis. Therefore, the signaling complex xiii conformed by NTNs / NEO1 is proposed as a possible prognostic marker of the progression of NB.

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.