Investigation of micro-devices for neurobiological applications

The aim of this project is to integrate neuronal cell culture with commercial or in-house built micro-electrode arrays and MEMS devices. The resulting device is intended to support neuronal cell culture on its surface, expose specific portions of a neuronal population to different environments using microfluidic gradients and stimulate/record neuronal electrical activity using micro-electrode arrays. Additionally, through integration of chemical surface patterning, such device can be used to build neuronal cell networks of specific size, conformation and composition. The design of this device takes inspiration from the nervous system because its development and regeneration are heavily influenced by surface chemistry and fluidic gradients. Hence, this device is intended to be a step forward in neuroscience research because it utilizes similar concepts to those found in nature. The large part of this research revolved around solving technical issues associated with integration of biology, surface chemistry, electrophysiology and microfluidics. Commercially available microelectrode arrays (MEAs) are mechanically and chemically brittle making them unsuitable for certain surface modification and micro-fluidic integration techniques described in the literature. In order to successfully integrate all the aspects into one device, some techniques were heavily modified to ensure that their effects on MEA were minimal. In terms of experimental work, this thesis consists of 3 parts. The first part dealt with characterization and optimization of surface patterning and micro-fluidic perfusion. Through extensive image analysis, the optimal conditions required for micro-contact printing and micro-fluidic perfusion were determined. The second part used a number of optimized techniques and successfully applied these to culturing patterned neural cells on a range of substrates including: Pyrex, cyclo-olefin and SiN coated Pyrex. The second part also described culturing neurons on MEAs and recording electrophysiological activity. The third part of the thesis described integration of MEAs with patterned neuronal culture and microfluidic devices. Although integration of all methodologies proved difficult, a large amount of data relating to biocompatibility, neuronal patterning, electrophysiology and integration was collected. Original solutions were successfully applied to solve a number of issues relating to consistency of micro printing and microfluidic integration leading to successful integration of techniques and device components.

Molecular and cellular aspects of the SREBP pathway

The SREBP (sterol response element binding proteins) transcription factors are central to regulating de novo biosynthesis of cholesterol and fatty acids. The SREBPs are regulated by retention or escape from the ER to the Golgi where they are proteolytically cleaved into active forms. The SREBP cleavage activating protein (SCAP) and the INSIG proteins are essential in this regulatory process. The aim of this thesis is to further characterise the molecular and cellular aspects surrounding regulation of SREBP processing. SREBP and SCAP are known to interact via their carboxy-terminal regulatory domains (CTDs) but this interaction is poorly characterised. Significant steps were achieved in this thesis towards specific mapping of the interaction site. These included cloning and over expression and partial purification of tagged SREBP1 and SREBP2 CTDs and probing of a SCAP peptide array with the CTDs. Results from the SREBP2 probing were difficult to interpret due to insolubility issues with the protein, however, probing with SREBP1 revealed five potential binding sites which were detected reproducibly. Further research is necessary to overcome SREBP2 insolubility issues and to confirm the identified SREBP1 interaction site(s) on SCAP. INSIG1 has a central role in regulating SREBP processing and in regulating stability of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a rate limiting enzyme in cholesterol biosynthesis. There are two protein isoforms of human INSIG1 produced through the use of two in-frame alternative start sites. Bioinformatic analysis indicated that the presence of two in-frame start sites within the 5-prime region of INSIG1 mRNA is highly conserved and that production of two isoforms of INSIG1is likely a conserved event. Functional differences between these two isoforms were explored. No difference in either the regulation of SREBP processing or HMGCR degradation between the INSIG1 isoforms was observed and the functional significance of the two isoforms is as yet unclear. The final part of this thesis focused on enhancing the cytotoxicity of statins by targeted inhibition of SREBP processing by oxysterols. Statins have significant potential as anti-cancer agents as they inhibit the activity of HMGCR leading to a deficiency in mevalonate which is essential for cell survival. The levels of HMGCR fluctuate widely due to cholesterol feedback of SREBP processing. The relationship between sterol feedback and statin mediated cell death was investigated in depth in HeLa cells. Down regulation of SREBP processing by sterols significantly enhanced the efficacy of statin mediated cell death. Investigation of sterol feedback in additional cancer cell lines showed that sterol feedback was absent in cell lines A- 498, DU-145, MCF-7 and MeWo but was present in cell lines HT-29, HepG2 and KYSE-70. In the latter inhibition of SREBP processing using oxysterols significantly enhanced statin cytotoxicity. The results indicate that this approach is valid to enhance statin cytotoxicity in cancer cells, but may be limited by deregulation of SREBP processing and off target effects of statins, which were observed for some of the cancer cell lines screened.

Immunotherapy for the treatment of poor prognosis cancers

Cancer is amongst the leading causes of death worldwide and the number one cause in the developed world. Every year there are close to 10 million cancer related deaths and this corresponds to hundreds of millions of euro in health care costs and lost productivity, placing a substantial drain on the economy. The efficacy of traditional treatment modalities for cancer therapy, such as surgery, radiotherapy and chemotherapy has plateaued, and while they are undoubtedly effective at prolonging patient lifespan, there is a high rate of adverse side effects and fatal reoccurrence. Currently, there is a huge amount of interest in the areas of cancer immunosurveillance and cancer immuno-editing, which explain some of the complex interactions between the host immune system and cancer. If left unchecked, cancerous malignancies have the ability to generate an immunosuppressive microenvironment, effectively shielding themselves from elimination and promoting tumour growth and progression. To overcome this, the potential of the immune system must be harnessed and the work undertaken in this thesis sought to contribute to this goal. Focus was placed on using novel therapies, combining tumour ablation with immune-modulating antibodies to maximise tumour elimination in an immune dependent manner, to overcome immunosuppression and promote immune activation. Chapter 2 focuses on the use of ECT as a method of tumour ablation and its effects on the immune system. ECT proved to be effective at inhibiting the tumour growth both in vitro and in vivo, and conferred significant survival advantages in both small and large animal models. More importantly, ECT proved to cause tumour death in an immune dependent manner, displaying the hallmarks of Immunogenic Cell Death, increases in immune cell infiltration and generating tumour-specific immune responses. Chapter 3 focuses on combining ECT with immune checkpoint blockade inhibitors; anti- CTLA-4 and anti-PD-1. Both combinations proved to be effective at inhibiting both primary and distal tumour growth, indicating the generation of tumour specific immune responses and prolonged animal survival. In addition, the treatments caused increases in the levels of certain intra-tumoural immune cell subsets and modulated the cytokine profile of treated animals in a way that was favourable overall. Chapter 4 focuses on the combining ECT with an anti-iCOS agonist antibody, capable of causing immune co-stimulation. This novel combinational therapy proved to be the most effective by far, with a high cure rate achieved across a number of different in vivo tumour models. Total regression was seen in both primary and distal tumours, as well as spontaneous metastases, with the tumour specific immune response generated conferring total protection to animals on tumour rechallenge. Overall the data presented here adds further insight into the area of cancer immunotherapy with some of the novel combinational therapies demonstrating substantial clinic potential.