Recent studies and clinical trials have shown the potential of cell-based therapies for the treatment of a number of diseases and organ/ tissue damages. However, limited availability of some therapeutically important cells (i.e. adult stem cells) still remain as main challenges in the development of tissue engineering through to the clinic. Healthy cells are required in large numbers to form a tissue-engineered construct and primary cells must therefore be expanded in vitro for both scientific and clinical applications. Various strategies have been developed to expand cells in vitro with increasing emphasis on 3D matrices because it can provide microenvironments which more closely mimic in vivo systems. In this way the inherent difficulties associated with 2D culture such as loss of phenotype could be overcome. Moreover, 3D matrices provide higher surface areas to support expansion of larger cell numbers compared to monolayer culture. Although each 3D method has certain advantages, there is no single technique that can be used to produce material assemblies that address all the fundamental problems linked to 3D cell seeding (penetration into the scaffold), passaging (use of enzymes), and harvesting (cell yield). Recently, thermally reversibly-associating particles have been studied for the growth and support of multiple cell types and for delivery of therapeutic cells. But coupling of thermoresponsive properties to magnetic microspheres would enhance the 3D culture and expansion of multiple cell types, and facilitate rapid recovery of the expanded cell population by simple magnetic separation. In this study, it was proposed that the thermoresponsive properties would allow simple cell seeding at temperatures below the LCST of polymer stabiliser when the suspension is flowing and upon heating to above the LCST cells would be encapsulated and cultured within the particle gels (every cells surrounded by a number of particles, as the size of the particles are much smaller than the cells). The magnetic responsive property would allow efficient and scaffold free cell recovery after expansion without the need for using trypsin or enzymatic treatment. The ‘switchable’ component of reversibly associating colloidal microparticles were prepared via two different strategies. In the first strategy, thermoresponsive PDEGMA was physically adsorbed onto the surface of PS microspheres, whereas, in the second strategy, PDEGMA was chemically grafted from functionalised PCMS microspheres via SI-ATRP. The most simple method i.e. physical adsorption is rapid and can be adapted to many microparticle surfaces but has the drawback of possible desorption of polymer chains during extended use. The chemical grafting method i.e. the formation of covalent bonds between the polymer corona and the microparticle core provides robust and well defined materials but is more complex and time-consuming. In both cases, particle aggregation in their suspensions occurred on increasing the temperature to above the LCST of PDEGMA, but could be reversed by cooling the suspensions back to below the LCST. This confirmed the presence of the thermoresponsive polymer on the surface of the microspheres using both methods (adsorption and grafting). Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Cell culture studies showed that these microparticle based scaffolds can support expansion of clinically relevant cell types (human MSC) and allowed efficient cell recovery after proliferation without the need for using trypsin or enzymatic treatment. Overall, those results suggest that the designed scaffolds had great potential for 3D in vitro cell expansion. The new developed materials have excellent biocompatibility, allow simple and rapid cell seeding and cell recovery after expansion, and possess mechanical strength and stability to support cell growth and proliferation. The materials developed and studied in this thesis may represent a significant contribution to the fields of biomaterials, tissue engineering, 3D cell culture and even bio-separation.

Chemotherapy has been highly successful in treating many forms of cancer; however there are increasing reports that this treatment causes cognitive declines in cancer survivors. These effects have been called “chemobrain” and while not affecting all patients, can persist for many years after the completion of treatment. The symptoms of chemobrain include a decline in concentration, memory and attention which are associated with a lower quality of life and in ability to return to work. Very little is known about the mechanisms behind these changes, or even the brain regions that are affected. Animal studies have found that systemic chemotherapy causes a decrease in the proliferation of neural stem cells (NSCs) in the subgranular zone (SGZ) neurogenic niche of the hippocampus and a decline in spatial memory. As hippocampal neurogenesis is required for a number of memory functions including the consolidation of long term memory, a decline in neurogenesis is likely to be one of the causes of the cognitive decline experienced by patients after chemotherapy. Previous animal work has shown that chronic treatment with the antidepressant fluoxetine prevents the decrease in neurogenesis and the associated cognitive decline. In the absence of fluoxetine chemotherapy spares dividing cells which are in contact with the surface of blood vessels in the SGZ of the hippocampus. This project used in vitro techniques to firstly look at whether fluoxetine has a direct effect on the sensitivity of neural cells to chemotherapy or whether treatment of astrocytes cells with fluoxetine produces an indirect effect on sensitivity. Secondly the effect of contact between NSCs and either astrocytes or endothelial cells was investigated to see if these cell types could provide protection to NSCs from chemotherapy. In the third part of the project, fluorescence activated cell sorting (FACS) and measurements of oxygen consumption after different drug treatments were used to investigate DNA damage, apoptosis and changes in the cell cycle and the metabolic response of different cell types to chemotherapy respectively. To do this we evaluated the relative sensitivity of primary NSCs, neural N2a cells, endothelial cells HBMEC, primary astrocytes, C6 astrocytes and 3T3 cells to the chemotherapy drug 5-fluorouracil (5-FU). NSCs were found to be more sensitive to 5-FU than other cell types. A concentration of 5 μM 5-FU was found to reduce NSC viability by 50% but largely to spare astrocytes and endothelial cells. FACS analysis showed that 5-FU was causing DNA damage and inducing apoptosis with some cells arresting in the G1 stage of the cell cycle. Measurements of oxygen consumption were not conclusive in explaining the differences in sensitivity between different cell types. Treatment with fluoxetine, either directly to neural cell cultures or via conditioned media from fluoxetine treated astrocytes or endothelial cells, was not found to be protective against 5-FU treatment. In contrast co-culture of NSCs with astrocytes or endothelial cells, protected NSCs from chemotherapy treatment. This effect was specific to cells in the neurogenic niche as co-culture with 3T3 fibroblasts did not provide protection. Direct cell contact between neural cells and astrocytes or endothelial cells was required for protection as neither conditioned media nor co-culture separated by a porous membrane was found to be effective. To investigate the mechanism behind astrocyte or endothelial contact dependent protection of neural cells, the gap junction protein Cx34 was stained for and found to be present on neural, astrocytic and endothelial cells. Fluorescent dye loading of marked cells showed that gap junctions were functional and dye could pass between them. Blocking gap junctions with the gap junction inhibitor carbenoxolone (CBX) abolished the protection provided by contact with astrocytes or endothelial cells. I hypothesise that the protection provided in vivo by fluoxetine may be mediated by its anti-inflammatory effect on the brain and is not a direct effect on cells in the stem cell niche. Contact with astrocytes or endothelial cells may provide Ca2+ buffering via gap junctions and in this way protect the more sensitive neural cells from the effects of chemotherapy.

Great progress has been made towards the understanding of the molecular mech- anisms driving factor induced somatic cell reprogramming to pluripotency since the discovery by Takahashi and Yamanaka. However this process remains highly stochastic and inef cient. More study is needed in order to achieve a more de- terministic cell fate conversion which could further improve the quality of stem cells generated, essential for prospective therapeutic applications. The work presented here was developed under the premise that natural embryonic devel- opment can serve as a guide to achieve more ef cient pluripotency induction. It was observed that the histone variant H2A.Z, which has a role in pluripotency in embryonic stem cells, is highly expressed in the oocyte and upon over-expression, together with Pou5f1, Sox2, Klf4 and Myc, was able to increase the ef ciency of somatic cell reprogramming to induced pluripotency. A gene co-expression network analysis of somatic cells being reprogrammed identi ed hub genes as- sociated with H2Af.z and chromatin remodelling related genes which could be tested for further improving the reprogramming ef ciency induced by H2Af.z over-expression. Moreover, the study of genetic networks from pre-implantation embryos identi ed preserved genetic circuits also present during the course of reprogramming. The most preserved network modules are associated with the nal stages of pluripotency induction. However the analysis also identi ed a genetic network associated with the zygotic genome activation in the totipotent embryo stage which is also found in a sub-population of pluripotent stem cells characterised by the expression of genes from the Zscan4 family, Tet1, Etv5 and Mga among other genes. This provocative observation led me to hypothesise that during the course of reprogramming the forced activation of hub genes from such network may help improve its ef ciency, possibly by recapitulating the natural embryonic processes which induces totipotency prior to pluripotency. The identi- cation of preserved network modules and its hub genes presented in this work may serve a platform for further reprogramming studies in a quest for improved cocktails of reprogramming factors capable of more ef ciently generating induced pluripotent stem cells.

Current culture methods to generate large quantities of cells destined for tissue engineering and regenerative medicine commonly use enzymatic digestion. However, this method is not desirable for subsequent cell transfer to the body due to the destruction of important cell-surface proteins and the risk of enzymatic contamination [1]. Therefore, research has led to the development of thermo-responsive surfaces for the continued culture of mammalian cells, with passaging achieved via a drop in the culture temperature. Recognising that the three-dimensional (3D) culture environment influences the cell phenotype, our aim was to generate a thermo-responsive 3D fibre-based scaffold, using electrospinning, to create an enzyme-free 3D culture surface for mammalian cell expansion that would be suitable for cells destined for the clinic. Thermo-responsive poly (poly (ethylene glycol) methacrylate), poly (PEGMA188), with lower critical solution temperature (LCST) of 26°C has been proposed for use within this thesis. It was used in combination with poly (lactic-co-glycolic acid) (PLGA) and poly (ethylene terephthalate) (PET) polymers in order to create 3D thermo-responsive non-woven electrospun fibrous scaffolds, on which different cell types could be cultured and passaged. Poly (PEGMA188) was prepared by free radical polymerization, and then incorporated with PLGA and PET polymers via four different methods: (i) surface adsorption, (ii) NaOH surface treatment, (iii) surface entrapment and (iv) blend-electrospinning. Blend-electrospinning was chosen over the other methods as it produced nano-PET and micro-PLGA bead-less fibres with responsive behaviour. The biocompatibility was assessed via the adhesion and proliferation of different mammalian cell types, including (i) red fluorescent protein (RFP)-expressing 3T3 fibroblasts, (ii) green fluorescent protein (GFP)-expressing primary immortalized human mesenchymal stem cells (ihMSCs), (iii) human colon adenocarcinoma cells (Caco2) and (iv) primary human corneal stromal stem cells (hCSSCs). The cell viability (Alamar Blue assay) was determined to measure the difference in cell populations adherent to the scaffolds while changing the culture temperature. These thermo-responsive scaffolds were able to support cell adhesion and proliferation at 37°C (hydrophobic surface). Furthermore, it was possible to detach the cells from the scaffolds by decreasing the temperature to 17°C (hydrophilic surface). Irrespective of the concentration of poly (PEGMA188) used, all scaffolds exhibited thermo-responsive proprieties; the cells were viable and proliferated in a similar manner to those cultured on control surfaces (PLGA or PET scaffolds). Finally, the effects of the thermo-responsive polymer and 3D culture environment on the hCSSC phenotype were assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunocytochemistry. The application of 3D environments can promote the reversion of activated corneal stromal cells’ ‘fibroblastic phenotype’ to a desirable quiescent keratocyte phenotype. Therefore, seven thermal and enzymatic passages on responsive 3D scaffolds and 2D TCPS, respectively, were performed. Cell culture on the 3D scaffolds promoted the quiescent keratocyte phenotype, with the increased expression of the keratocyte markers, CD34 and ALDH, and decreased expression of the myofibroblast marker, ACTA2, when compared with cells cultured on the 2D culture flasks. In this thesis, the preparation and application of first generation, biocompatible thermo-responsive fibrous scaffolds are described. The combination of ease of preparation, positive cell response and the expansion of a desirable cell phenotype make the thermo-responsive fibres promising as a new class of materials for application in cell culture. The materials developed and studied in this thesis are believed to represent a significant contribution to the fields of biomaterials and tissue engineering.

The use of protein microfluidic systems is of growing interest for a variety of applications, including but not limited to tissue engineering, drug delivery and biosensors. The means by which to control chemistries on substrates for biological and medical applications is therefore in high demand. Here the creation of a bio-functional gradient on silica and polymeric surfaces using a micro fluidic technique, for the guidance of cell adhesion and functionality, using AFM tools for protein imaging and force spectroscopy investigation is reported. Atomic force microscopy (AFM) is a high resolution microscopic technique highly used in biological investigations, allowing conformational elucidation of protein deposition on the substrate. In this work application of the techniques of AFM, fluorescence microscopy and cell adhesion studies were used to assess the protein deposition along the microfluidic system. From the fluorescence analysis, it was immediately observed that successful protein immobilization on both substrates was achieved. Differences in fluorescence intensity were also registered along the microfluidic channel (start and end point) suggesting a variation in protein adsorption along the channel. The AFM imaging analysis conducted on the same samples revealed a difference in surface coverage considering the injection and end point (from 70% to 14% respectively) of the protein pattern. The difference in protein density registered along the fibronectin pattern was tested using a functionalised probe AFM technique, allowing molecular resolution of ligands in a physiological environment. A difference in the percentage of observed adhesion events was registered considering the start and end point of the microfluidic pattern, from 90% to 37% respectively. This is likely due to the fact that at the higher surface concentration there is a higher probability of the functionalised tip interacting with multiple fibronectin molecules, as confirmed from the presence of multiple adhesions at start point with a higher adhesion force of 82 pN ± 7.4 pN. To complement the AFM force measurements, protein functionality was tested by investigating the cell adhesion, shape and migration on the protein pattern. The fibronectin protein gradient was shown to control cell adhesion and migration along the patterns, demonstrating that this system can be used for biological applications to monitor the cell behaviour using difference protein concentration and cell density all in the same microfluidic channel. The ability to control cell adhesion and migration on substrates could be of significant interest when researching possible applications in future tissue engineering and biological studies. The combination of AFM and fluorescence microscopy techniques for protein density investigation used in this work demonstrated that protein deposition and arrangement on substrate play an important role in cell adhesion and migration studies.

Previous studies on the mechanism of action of pyrethroids have confirmed that voltage-gated sodium channels (VGSC) in the axon membrane are the major target site of these compounds. The use of pyrethroids to control maize weevils, Sitophilus zeamais, a major pest of stored maize in Brazil, has led to the occurrence of resistance. The work described here seeks to establish whether changes in VGSC of S.zeamais can explain pyrethroid resistance. The S. zeamais homologue of the Drosophila para VGSC was identified using degenerate primers and sequenced. Resistance mutations were examined by sequencing the IIS4-IIS6 region of the gene from laboratory strains of susceptible and resistant insects, revealing one amino acid replacement (T929I). The T929I mutation has been identified in other insects but always associated with a second mutation together producing a highly resistant phenotype. The occurrence of T929I in isolation is rare. DNA-based diagnostic assays were designed to screen weevils for the T929I mutation and analyse Brazilian field populations revealing a low frequency of heterozygous individuals carrying the mutation. The effect of the T929I mutation on VGSC function was investigated using whole cell patch clamping on cultured neurons isolated from thoracic ganglia of wild-type and resistant weevils. Inward currents were recorded by depolarizing the neuron to test potentials in the range -70mV to +70mV in 10mV increments for 25ms from a holding potential of -80mV. Current amplitudes were similar in cells from resistant weevils however other changes were apparent, notably a significant depolarizing shift in the voltage-dependence of activation of sodium currents in the resistant animals (P<0.05). Mutant neurons are also less sensitive to deltamethrin than the wild types.

3, 11-difluoro-6, 8, 13-trimethyl-8H- quino [4, 3, 2-kl] acridinium methosulfate (RHPS4) is a member of a series of pentacyclic acridines developed at the University of Nottingham, which bind to, and stabilise the structure of G-quadruplex DNA and inhibit the action of telomerase at sub-micromolar concentrations in the cell free TRAP assay and limit cancer cell growth therefore leading to the conclusion that RHPS4 has potential anti-tumour activity. Previous biological studies, however, have suggested that the mechanism of action of RHPS4 may be much more complicated than previously anticipated. Exposure of human melanoma cell lines to low doses of RHPS4 reveal an irreversible cessation of growth and telomere erosion. When used at higher doses, RHPS4 elicits short-term apoptosis/senescence concurrent with an increased number of telomere fusions and this is not a result of telomere shortening suggesting that the mechanism of action of RHPS4 is more than that of a simple telomerase inhibitor. In this study, we have developed a systems biology approach to the analysis and integration of biological data from investigations of the activity of RHPS4. The aim of this approach was to link the emergent properties of the biological studies of RHPS4 to a potential molecular target. In the laboratory, we are able to quickly derive data with respect to the time- and concentration- effects of RHPS4 on cell cycle distribution, population doublings and senescence levels, however, the kinetics of the cell cycle are neglected. A mathematical model has been derived of adequate complexity to reproduce the biological results of the laboratory and yet sufficient simplicity to yield predictive information and biological insights into the action of this complex molecule has been developed allowing us to integrate the data we can derive. The mathematical model has five compartments, those being X (representative of G1/G0 phase), Y (representative of S phase), Z (representative of G2/M phase), Σ (representative of senescence) and A (representative of apoptosis) with rate and movement between the compartments denoted by the rate parameters kxy, kyz, kzx, kxΣ and kΣA, respectively. Parameterisation of the mathematical model requires robust data from a well-characterised cell lines and initially four colorectal cell lines were chosen, namely HCT116, HT-29, KM12 and HCC-2998 to determine the short- and long-term effects of RHPS4 on their cell cycle distributions, population doublings and senescence levels. The aim was to determine which of the cell lines displayed good sensitivity to RHPS4, good growth characteristics in the presence and absence of RHPS4 and tractability in a range of biological assays. Initial studies revealed the HCT116 cell line was the most relevant for the further study of RHPS4. Data regarding the effects of RHPS4 on cell cycle distribution, growth rate and senescence over 21 days was derived and fitted to the five-compartment mathematical model to reveal aspects of the action of RHPS4 that would be difficult to appreciate without it. The model suggests that RHPS4 increases the rate of cells moving into a senescent state (indicated by an increase in kxƩ relative to control) and there is inhibition of apoptosis (indicated by a reduction in kƩA relative to control). Data with respect to doxorubicin was also applied to the model and suggested that consistent with RHPS4 there was inhibition of apoptosis relative to control, however, unlike RHPS4, there were initial reductions followed by increases in the rates of cells moving between G1/G0 and S by kxy, S and G2/M by kyz and G2/M and G1/G0 by kzx relative to control in a concentration- and time-dependant manner. This study has highlighted the important role of mathematics in the understanding of biomolecular processes and this is not the end as we have simply allowed the data to speak for itself. This project is the starting point for further, more focussed studies of RHPS4, and other anti-tumour agents. This approach to the analysis of anti-tumour agents is general, however, allows us preliminary insight into the mode of action of RHPS4 and to relate changes observed to potential underlying molecular targets and further the understanding of this complex molecule.

The development of microspheres for the sustained delivery of protein and small drug delivery has been utilised in tissue engineering and drug delivery applications. However problems exist in obtaining a controlled and predictable release pattern of the encapsulated molecules from these materials. In this study, microspheres with a zero order release kinetic profile and no lag phase were developed from a novel PLGA based polymer blend. The novel PLGA based polymer blend was made from blending PLGA with varying compositions of the triblock co-polymer PLGA-PEG-PLGA. These blends were subsequently used in the fabrication of lysozyme and dexamethasone loaded microspheres. Blending of the triblock copolymer with PLGA resulted in a reduction of the glass transition temperature (36.1ºC against 59.7ºC) and an increased mechanical strength (25.25 ± 1.26MPa against 0.26 ± 0.05MPa) for PLGA and 30% triblock w/w microspheres respectively. An incremental increase in the triblock composition within the Triblock/PLGA blends resulted in a corresponding reduction in glass transition temperature of the microspheres. Varying the triblock composition within the microspheres showed no significant effect on entrapment efficiency (EE) of lysozyme (protein) and dexamethasone (drug) within fabricated microspheres (EE ~ 60% for and 75% for loading weight 5% w/w for lysozyme and dexamethasone microspheres respectively). Controlled release experiments showed incorporation of the triblock increased the burst release of the protein and drug molecules from the microspheres and improved their release kinetics, with zero-order release profile (post burst phase) observed at a triblock composition of 30% w/w. A positive correlation between the amount of triblock within the triblock / PLGA blend and the rate of protein and drug release was also observed. The induction of osteogenesis and chondrogenesis within stem cells seeded on dexamethasone and ascorbate phosphate, and TGF-β3 loaded scaffolds was successfully demonstrated. Zonal release of TGF-β3 and BMP4 proteins from a bilayered scaffold was also demonstrated. However experiments conducted to demonstrate the tissue zonation within a bone cartilage bilayered construct developed from embryonic stem cell seeded TGF-β3 and BMP4 loaded bilayered scaffolds yielded inconclusive data. These results suggests that protein and drug loaded injectable microspheres for tissue engineering applications can be formed from triblock/PLGA blends, and that by varying the triblock composition, the temperature at which the microspheres form scaffolds, the release kinetics and the mechanical strength of the resulting scaffolds can be controlled.

The establishment of stable immortal ES cell lines using embryos as a source of isolation in domesticated farm animals, in particular for pigs, which are closer to humans than other ungulates, has not been reported; hence this information could contribute to the improvement of regenerative medicine in humans, biotechnology and agriculture. Therefore, the discovery of effective protocols to derive and maintain ES cells and the induction of purified somatic cells from ES cells in pigs is of importance. The objectives of this study were to produce pES-like cells and direct differentiation of the ES-like cells obtained by improving the culture conditions. In vivo-derived porcine blastocysts at day 6-8 were classified into two groups distinguished by the exhibition of ICMs and epiblasts of the embryos. In each group, intact blastocysts and isolated ICMs or epiblasts were designed to culture in either KO4bh or DM40bh medium on mitotically inactivated MEFs under the humidified air of 5%CO2 at 39°C until the primary outgrowth of ES-like cells was observed. Two morphologically distinct pES-like cells, pESA-like and pESB-like cells were isolated from the epiblasts, whereas no cell lines were generated from ICMs. pESA-like cells were observed as individual small round cells containing one or multiple nucleoli along with a high ratio of nucleus to cytoplasm, while pESB-like cells formed dome-like colonies. The pESA-like cells were stained both negative and positive with the alkaline phosphatase enzyme, while pESB-like cells were all stained positive. With immunofluorescence staining of OCT-4 and nanog, the nuclei of pESB-like cells appeared not to be stained positive with these two antibodies, while the designed self-renewing genes such as OCT-4, nanog, SOX-2, REX-1 and DPPA-3 were detectable as similar to mES cells. Regarding the pluripotent abilities of pESB-like cells, they could be induced to form neuronal-like, neuronal supporting-like, smooth muscle-like and hepatic-like cells in a variety of desirable differentiation media under the feeder-free culture system. The cytoplasmic contents of certain induced mature cells were stained positive with nestin, α-smooth muscle actin and α-fetoprotein in association with the expression of differentiated genes specific to each germ layer such as nestin, α-smooth muscle actin, smooth muscle myosin heavy chain, α-cardiac actin, transthyretin, α-fetoprotein, albumin and HGF1β. In conclusion, pESB-like cells obtained in this study may possibly have the potential to be authentic ES cells isolated from early epiblast origin as mES cells.

Beta-Adrenoceptors (B-ARs) mediate vasodilatation by activating various mechanisms that collectively contribute to vascular smooth muscle (VSM) relaxation. It has been shown that B2-AR stimulation in cultured cells results in activation of extracellular signal-regulated kinase (ERK). As the functional relevance of this was not known, the aim of the current investigation was determine the role of ERK in beta-AR-mediated vasodilatation. Isoprenaline-induced relaxation of porcine coronary artery (PCA) segments pre-contracted with the thromboxane mimetic U46619 was significantly enhanced by inhibition of ERK activation. Relaxations to the beta2-AR agonist salbutamol, but not those to the beta1-AR agonist xamoterol or the adenylyl cyclase activator forskolin, were also enhanced. The intermediate-conductance Ca2+-activated K+ (IKCa) channel blocker TRAM-34 prevented the enhancement of beta2-AR-mediated responses. Taken together, the data indicate that ERK inhibits beta2-AR-mediated vasodilatation by interacting with a cyclic 3’, 5’-adenosine monophosphate-independent relaxation pathway involving K+ channels. This may occur through a direct regulatory action on the IKCa channel via phosphorylation. Furthermore, the finding that increased ERK activation in a rat model of Type II diabetes was associated with significantly impaired beta-AR-mediated vasodilatation raises the possibility that ERK may represent a promising therapeutic target in the treatment of disease states characterised by abnormal vascular function.