Polymersomes (synthetic polymeric vesicles), formed by the self-assembly of amphiphilic block copolymers in water attract great attention as drug delivery systems and as diagnostic/imaging tools. Our group has shown that 2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate (PMPC-PDPA) polymersomes are of special interest due to their ability to encapsulate a wide range of therapeutic molecules including anticancer compounds, antibiotics, antibodies, and nucleic acids, and their capacity to deliver their cargo intracellularly, both in vitro and in vivo, without promoting cellular toxicity or stress. The favourable uptake kinetics and toxicological profile of PMPC-PDPA polymersomes justify a thorough study on the cellular interactions and mechanisms underlying their uptake, which was the aim of this thesis. Exploring different polymersome production methods we studied the impact that the physical properties of PMPC-PDPA polymersomes (nanoparticle size and shape) have on their cellular uptake. Using flow cytometry and fluorescence microscopy we demonstrated that both spherical and tubular polymersomes could be used as intracellular delivery vectors. In addition, spherical and tubular polymersomes presented different uptake kinetic profiles, opening new avenues to modulate the temporal delivery of a cargo. In a parallel line of work we identified receptor-mediated endocytosis as a common pathway for the internalisation of PMPC-PDPA polymersome in mammalian cells. Studying polymersome uptake in the presence of antagonists and neutralising antibodies, we identified two families of transmembrane proteins mediating PMPCPDPA polymersome endocytosis and the specific receptors facilitating polymersome uptake. In addition, different endocytic pathways and molecules (i.e. dynamin, BAR domain proteins) were investigated in relation with polymersome internalisation by means of chemical inhibitors, dominant negative proteins and siRNA knockdown. Polymersome endocytosis seems to be dominated by a high level of promiscuity and the ability of PMPC-PDPA polymersome to induce their uptake, which could be translated in new therapeutic applications with a great clinical impact.

This thesis investigates the afferent mechanism that may be involved in bladder hypersensitivity. The Study primarily focuses on the role of TRPM8 on bladder sensory firing. One part of the study also aims to replicate the ice water test in an in vitro mouse model to assess the contribution of TRPM8. An interesting aspect that the study investigates is the interaction between TRPM8 and TRPV1, TRPA1 and purinergic signalling. Finally, the study investigates ERβ KO mice as a model for interstitial cystitis. Investigating these parameters may reveal more information of the sensory changes that may occur in relation to bladder hypersensitivity, hence revealing novel targets for therapy.

The aims of this research project were to manufacture and characterise PDPA-based pH-sensitive functionalised polymersomes using a medium-high content screening method, suitable for CNS drug delivery. Angiopep-2 functionalised polymersome formulations have been found that are able to penetrate the blood-brain barrier (BBB) effectively in both in vitro models and in vivo. Using Transmission Electron Microscopy, Dynamic Light Scattering, FACS analysis, and 2D in vitro screening gave information on the physical and biological features of polymersomes based on their different chemistries, including size distribution, architecture, topology, cellular localisation, cellular uptake kinetic and immune response. The studies showed the possibility of controlling cellular internalization and cargo destinations by manipulating polymersome surface chemistry and specific ligand(s). The subsequent in vitro and in vivo studies built on these screening results. Using extensive Confocal Laser Microscopy and image analysis, Ang-POEGMA-PDPA polymersomes showed effective receptor-mediated transcytosis (RMT) in the 3D in vitro BBB model established, while the RVG- functionalised formulation did not. Further in vivo studies showed that the Ang-functionalised polymersomes were able to penetrate the mouse BBB via effective RMT. Moreover, primary cargo delivery studies showed successful IgG transport into brain by Angiopep-2-functionalised POEGMA-PDPA polymersomes. The results in this thesis can provide a useful platform for further examination of CNS delivery of polymersomes and their cargos.

During formation of the brain in mammals, neural stem cells (NSCs) transit through sequential periods of expansion, neurogenesis and gliogenesis. Notch signaling maintains NSCs and blocks transcription of pro-neurogenic factors. Notch ligands are expressed by differentiating progenitors and activate lateral inhibition signals through Notch. It has long been proposed that Notch signaling occurs bi-directionally through ligands such as Jagged1 (Jag1), which are also type I membrane proteins. However, the molecular mechanisms controlling the transition from stem cell division, where Notch plays a maintenance role, to daughter cell differentiation are poorly understood. To study the role of Jag1, in niche maintenance I began by transducing NSCs lining the subventricular zone (SVZ) with full length Jag1 (Jag1FL). Surprisingly, Jag1 induced a fate switch to Sox10+ oligodendrocyte precursors. NSCs grown under differentiating conditions in vitro recapitulated this phenotype to some degree. RNA-Sequencing analysis was performed to study the transcriptome changes of Jag1FL-IRES-GFP transduced NSCs. This screen revealed an upregulation (induction) of key genes involved in oligodendroctye maturation and myelination, which were further confirmed by qRT-PCR. Ongoing NSC grafting experiments are being performed to analyze the effects of Jag1FL-IRES-GFP+ NSCs on mice exhibiting focal myelin lesions. These proof of concept experiments hold promise for elucidating an, as of yet unknown role of Jag1 in remyelination. Through biochemical analyses, we have demonstrated that the Jag1 intracellular domain (JICD) can act as a transcription factor signaling in a cell autonomous fashion upon activation. Through chromatin immunoprecipitation, I have identified potential genomic regions and Wnt signaling target genes, directly bound by nuclear JICD. Taken together with the in vivo data, this proposes a new role for Jag1 in NSCs maintenance, and displays its potential for regulating fate switch to an oligodendrocytic lineage.

Epilepsy is a common neurological disorder characterised by recurrent epileptic seizures. It affects approximately 0.7% of the worldwide population. Even though many patients respond to the available treatments, around a third of people with epilepsy do not respond to existing anti-epileptic drugs (AEDs). Therefore, there is a need to better understand epilepsy in order to develop new therapeutic strategies for the treatment of this disorder. In this study, a model of pharmacologically-induced epileptic seizures using young zebrafish larvae was developed and characterised. It was found that the brains of young zebrafish larvae exhibited altered PTZ-sensitivity in response to repeated seizure onset or exposure to stress hormone. In both cases, the severity of the PTZ-evoked locomotor convulsive response was enhanced, and expression of selected PTZ-induced genes was reduced. In order to identify more genes involved in the response to PTZ seizure-induction, and which might be involved in the adaptation of the CNS to seizure induction, a two-colour microarray analysis was carried out and many novel PTZ-responsive genes were identified. The function of a new epilepsy risk factor, sestrin 3, was also investigated using the zebrafish PTZ model of epileptic seizures, which revealed that sesn3 promoted locomotor convulsions and regulated expression of a subset of PTZ-induced genes. In addition to the studies of seizure mechanisms in the zebrafish, the new transgenic line NBT:GCaMP3 was created, in which expression of the fluorescent genetically encoded calcium indicator was targeted to the CNS, to visualize in vivo and in real time, seizure initiation, propagation and suppression by an antiepileptic compound. In the future, combining NBT:GCaMP3 with the new technologies to create zebrafish mutations in orthologues of genes mutated in human epilepsy, will enable novel experimental studies to investigate the pathogenetic mechanisms underlying epilepsy, and facilitate novel approaches to the discovery of anti-epileptic drugs.

Arterial occlusion accounts for high rates of mortality in the western world. Strategies to bypass an occlusion by activating collateral vessels could reduce the consequences of arterial diseases. This thesis investigates the genes involved in collateral vessel remodelling in the chick embryo to gain insight into the process. Ligation of the right proximal vitelline artery of HH st 17 chick embryos occluded blood flow to the right hand side of the extra-embryonic tissue and vitelline vessel network. Collateral vessels were seen to develop from the pre-existing, left (unligated) vitelline artery and extended across the midline to carry arterial blood to the un-perfused side of the extra-embryonic tissue. The remodelling process was active over 48 hours and developed many small collateral vessels into a few, main conducting arteries. The number of collateral vessels peaked at 12 hours post tied-ligation and then decreased, whilst collateral vessel diameter continued to increase over the time period. Analysis of the global transcriptional profile of collateral vessels in the chick embryo was assessed following ligation, during early stages of collateral vessel development (4 hours), at the point of pruning and remodelling of the collateral network (12 hours). Collateral vessel formation in the chick embryo was found to be associated with a unique and specific gene expression profile. Phosphodiesterase 10A (PDE10A), an cAMP hydrolysing enzyme, was upregulated in tied-ligated embryos at 4 hours post tied-ligation and hypothesised to play a role in the remodelling process. To study PDE10A papaverine hydrochloride was used to inhibit the enzyme. Papaverine had no effect on normal vessel development but significantly impaired collateral vessel diameter from 6 hours post-ligation. This effect was rescued by co-treatment with Protein Kinase A inhibitor, Rp-8-Br-cAMPS. To begin an assessment of the role of PDE10A in collateral vessel remodelling, proliferation was investigated, in vivo. However, a mechanism of action for PDE10A has yet to be elucidated.

The human peripheral nervous system has a limited ability for self-repair however trauma often results in life-long debilitation. Due to the prevalence of dislocation it is preferred that the two severed nerve ends are approximated via direct suturing or for larger injury gaps an autograft or alternatively an NGC can be implanted. NGC’s are nerve entubulation devices designed to not only act as a guide and modulator for the regenerating nerve however improved device design and manufacturing methods are required to match the efficacy of autograft and achieve clinical acceptance. Additive manufacturing is a rapidly emerging research and commercial production method for producing highly resolved polymeric structures such as NGC’s whilst allowing the incorporation of intralumenary features, rapid production rates, economic material usage and patient device specificity. The aim of the present study was to design and fabricate NGC devices utilising a bespoke laser-sourced microstereolithography system and microwave synthesised, degradable photopolymerisable liquid pre-polymers. The suitability of the structuring system and materials in this function where assessed by the fabrication of well-defined structures and using in vitro cell culture and an in vivo PNI model to confirm biocompatibility. Commercially derived materials were used in order to establish the system, with more biologically relevant degradable materials also being developed. The use of in-vitro neuronal and explant DRG cell culture with cell viability assaying and confocal microscopy confirmed a positive cellular interaction in both commercial and custom synthesised materials. Results from the PNI animal implantation model produced promising preliminary regeneration and re-innervation results in comparison to graft repair. The adaptability of the technique and materials developed in this work allowed their use for producing other biomedical devices for bone grafts and for fabrication of a highly resolved regeneration device for the central nervous system. This work establishes the applicability of this technique and the materials used for the fabrication of simple and advanced intralumenary feature containing NGC’s but also for a wide range of other biomedical and non-biomedical applications that require highly defined structures that are geometrically complex structures and devices.

Polymersomes made from the amphiphilic diblock copolymers, PMPC-PDPA, are proposed to serve as a siRNA carrier with pH-responsive property that provides endosomal escape. The main purpose of this work is to investigate the ability of polymersomes to provide effective intracellular delivery of siRNA into HeLa cells. Encapsulation of siRNA into polymersomes was performed by pH-switch and electroporation method, both techniques enable siRNA encapsulation. No alteration of polymersomes size and morphology was observed in DLS and TEM. Purification of polymersome was conducted to ensure that no free siRNA or polymer remained. Intracellular delivery was examined by using fluorescence-labelled siRNA to track the internalisation. Flow cytometry and fluorescence microscope were used to study the cellular uptake of polymersomes and siRNA. siRNA is successfully delivered with the distribution of siRNA signal throughout the cell, with stronger signal compared with Lipofectamine. Kinetic uptake of siRNA suggests that siRNA can be effectively delivered to most cells within 20 hours. In addition, evidence of endosomal escape of siRNA delivered by polymersomes was observed. Silencing activity of siRNA was determined by qPCR and Western blot, mRNA and protein expression of Lamin A/C as a target gene were not significantly decreased. Cytotoxicity and other cellular response, including pro-inflammatory response and interferon response, were investigated. Polymersomes provide very low cytotoxicity and no pro-inflammatory response, unlike Lipofectamine. Moreover, the gene expression profile of interferon response indicates the possible apoptosis occurrence in Lipofectamine treated cells, but not in polymersomes treated cells. The information suggests two possible factors that influence the silencing activity of siRNA delivered by polymersomes; the incomplete characterisation of siRNA process and the cellular response from carriers.

Drosophila melanogaster has become a versatile model organism, with high genetic tractability and ease of manipulability, mixed with low cost and low space constraints. Genetic tools with which to modify flies in myriad ways are constantly developed and updated, whilst physical tools have also become more apt for access to various biological systems. In this thesis I have used several such tools, such as the Drosophila flight simulator, High Pressure Freezing and Transmission Electron Microscopy to test visual behaviour and synaptic function, respectively. In Drosophila’s early visual system, R1-R6 and R7/R8 information channels carry visual information to the visual brain. These channels have been thought separated on the basis of their structure and function, however it is our hypothesis that these channels can functionally inform each other and that this occurs at an early stage of the visual pathway. Here I have used the flight simulator to show that the absence of ‘chromatic’ photoreceptors adversely affects visually-driven optomotor behaviour. In conjunction with other electrophysiological data, I have helped to support the idea that this influence may result from functional connection between R1-R6 and R7/R8 photoreceptors. Similarly, I have used the flight simulator to show that Ca2+-activated K+ channel mutations in post-synaptic Large Monopolar Cells can affect visual behaviour, but that these effects are often managed by homeostatic mechanisms that serve to maintain biologically-relevant function. Additionally, I have shown that the absence of dietary Polyunsaturated Fatty Acids can influence visual behaviour. Pre- and post-synaptic information at Drosophila photoreceptor synapses has been shown to adapt in accordance with changing visual conditions. I used programmes of light- and dark-adaptation, along with High Pressure Freezing and Transmission Electron Microscopy to test how these adaptations are translated at the synapse. All of these conclusions are discussed alongside electrophysiological findings acquired from the early visual system.

The increased awareness of the ability of cells in detecting mechanical cues from the external environment [1] led to consider the possibility of triggering a cellular response by applying external mechanical forces [2]. In order to drive the commitment of differentiated cells and obtain in vitro engineered implants as replacement for bone fracture sites, a scaffold closely mimicking the 3D distribution of forces acting on bone cells in vivo is required and is still ongoing research. On this purpose, a composite scaffold embedded with collagen (cPCL) is proposed in this study as structure to transmit externally applied mechanical forces to embryonic human mesenchymal stem cells (hES-MPs) through a gelatinous matrix of collagen. A collagen concentration of 2 mg/ml and plasma treatment of scaffolds were selected as optimal conditions for survival and uniform seeding distribution of cells. Then, the second part of the study allowed to fully characterize, by mechanical testing and x-ray imaging, a novel hybrid scaffold able to provide an optimal environment for controlledbone progenitor cells growth. The objective of the last part of the study focused on the evaluation of how short bursts of compressive strain, applied as series of cycles at early stages (L1) and late stages (L2) of culture, affects cellular proliferation, bone tissue formation and the osteogenic response of hES-MPs. Short bursts of compression were found to strongly affect hES-MPs proliferation, suggesting cyclic compressive loading to delay the proliferation of samples compressed once. On the other side, L2 prevented proliferation to occur over 28 days, although greatly enhancing the production of mineral which, instead, was null for samples undergoing L1. This study underlined the existence of a strong link between proliferation and mineralization potential of cells and confirms the possibility to vary their response by short bursts of compression applied on hES-MPs seeded in 3D hybrid scaffolds.