Diabetes is a lifelong condition caused by an inability of the body to break down glucose due to a defect in either insulin synthesis or the target cells becoming resistant to secreted insulin. There are two main types of diabetes, type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus. T2DM mainly occurs due to insulin resistant, inability of cells to respond to normal levels of insulin, the treatment of T2DM usually involves exercise, diet control, drugs and in some cases insulin is needed. In T1DM, the immune system starts to attack the β-cells, the cells responsible for insulin hormone synthesis and secretion from the endocrine pancreas. The aim of T1DM management is to restore carbohydrate metabolism as close to the normal condition as possible. To achieve this goal insulin hormone must be provided daily. Insulin is given in different ways such as injection (which is the most common method), pump and inhalation. Insulin sources can be either recombinant or animal-based. Treatment of T1DM is continuous and even with the best treatment options people with diabetes can develop serious complications such as acute diabetic coma or other long-term complications. These include diabetic cardiovascular disease, diabetic retinopathy, and diabetic nephropathy. Injection of insulin and daily monitoring of glucose levels presents a substantial burden to both the diabetic patient and also to associated dependents or carers i.e paediatric diabetes. So there is a strong need to develop an alternative treatment to reduce the burden, enhance control, and even ultimately cure diabetes mellitus. A recent medical invention is the use of stem cells in the treatment of many disease conditions. Stem cells have a remarkable ability to develop into many different cell types. Stem cells therapy offers a new method for treating disease such as diabetes. The results of many studies demonstrate the capability of mesenchymal stem cells (MSCs) in the treatment of bone disease, cardiac disorder, and multiple sclerosis. However, much work remains to be done in the clinic and laboratory to optimise the use of these cells. The main aim of this study was to explore the therapeutic effectiveness of MSCs conditioned media to restore the viability and function of β-cells. In order to establish an in vitro model of cytokine driven β-cells apoptosis, pancreatic β-cells were treated with rising concentrations of pro-inflammatory cytokines TNF-α, IFN-γ and IL-1β and endotoxin LPS for 24 h. The optimal concentration of each cytokine or endotoxin was assessed by MTT assay. Optimal concentrations were deemed to be those that induced an approximate reduction in cell viability of 50%. The cells were treated with the optimal concentration and cell viability was monitored over time in addition to assessment of anti-apoptotic gene induction via qPCR assay. Mesenchymal stem cells (MSCs) secretome was collected as conditioned media and the β-cells cultured in non-conditioned and conditioned media during cytokine-driven apoptotic induction. β-cell viability and anti-apoptotic gene expression was determined to evaluate the therapeutic effectiveness of mesenchymal stem cells conditioned media (MSC-CM) in protecting β-cells from pro-inflammatory cytokines. We observed a significant increase in the viability of pancreatic β-cell lines cultured in conditioned media when compared to those cultured in non-conditioned media. After that, we sought to identify a possible candidate that is present in the MSC-CM and help the cells to overcome the effect of pro-inflammatory cytokines. We found a high concentration of IL-10 in our conditioned media, in addition to the presence of IL-4, PIGF and VEGF in variable amount. Based on our present findings, cytokine-induced apoptosis is mediated through the TRAIL-dependent pathway. However, the addition of MSC-CM blocked cytokine-induced apoptosis and downregulated the genetic expression of A20 and TRAIL. Also, IL-10 was able to block IFN-γ and TNF-α-induced apoptosis.

Pulmonary arterial hypertension (PAH) is characterised by elevated pulmonary arterial pressures and obstructive lesions in the distal vasculature. As a result, the right ventricle is placed under excessive strain resulting in adaptive hypertrophy, progressing to maladaptive hypertrophy and failure. Women develop PAH more frequently than men. It is postulated that 17β-estradiol (E2) plays a role in disease pathogenesis and/or the E2 metabolic axis may be dysregulated in PAH. Growing evidence also implicates a role for ROS and oxidative stress in PAH, yet mechanisms linking these systems are elusive. We hypothesised that either E2 or the E2 metabolite, 16α-hydroxyestrone (16αOHE1), stimulates Nox-induced ROS generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMC) and that, in PAH, aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of E2-Nox-dependent processes was studied in female Nox1-/- and Nox4-/- mice exposed to chronic hypoxia. HPASMCs from female non-PAH individuals (control hPASMC) and female PAH patients (PAH-hPASMC) were exposed to E2 and 16αOHE1 in the presence/absence of inhibitors of Nox1, Nox2 and Nox4, cytochrome P450 1B1 (CYP1B1) and estrogen receptors (ER), ERα, ERβ and G-protein coupled estrogen receptor (GPER). E2, through ERβ, increased Nox1 and Nox4-derived O2- and redox-sensitive growth in control hPASMCs. 16αOHE1, through ERα activation, stimulated O2- production in control hPASMCs and PAH-hPASMCs. E2- -stimulated O2- production was inhibited by CYP1B1 blockade. Basal expression of Nox1 and Nox4 was potentiated in PAH-hPASMCs. In control hPASMCs, 16αOHE1 increased p47phox and poldip2 and Nox1 expression. In PAH-hPASMCs, 16αOHE1 decreased nuclear factor erythroid-2-related factor-2 (Nrf-2) activity and expression of Nrf-2-regulated antioxidant genes in PAH-hPASMCs. Female Nox1-/-, but not Nox4-/- mice were protected against chronic hypoxia-induced pulmonary hypertension and vascular remodeling. Expression of CYP1B1 was increased in pulmonary arteries of wild-type and Nox4-/- mice exposed to hypoxia, yet this induction in CYP1B1 expression was absent in those arteries from hypoxic Nox1-/- mice. Findings detailed in Chapter 3 show that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth through both Nox1 and Nox4. In vivo studies exhibited protection against pulmonary hypertension specifically in Nox1-/- mice. This study provides new insights through Nox1/ROS and Nrf-2 whereby 16αOHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH.

No description available.

No description available.

This thesis examines the use of hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose (NaCMC) or ethylcellulose, alone or in combination with other adjuncts, to control the release of propranolol hydrochloride from matrices. Gels were characterized by U tube viscometry and their cloud points of gels. The properties of polymers and their mixture in matrices or in gels were investigated by differential scanning calorimetry (DSC). Compendial dissolution methodology was used to determine drug release from matrices and their release exponents. Propranolol hydrochloride increased the solubility of HPMC and altered the water distribution in their gels. The release of propranolol hydrochloride from HPMC matrices was dependent on the square root of time. Release exponents were - 0.6, indicating that diffusion and erosion contributed to drug release. The release rates of propranolol hydrochloride from NaCMC matrices decreased as the NaCMC content increased. Addition of propranolol hydrochloride to NaCMC gels produced an insoluble complex which, in matrices, controlled the drug release. The interaction between propranolol hydrochloride and NaCMC was confirmed by DSC and dialysis. The viscosity grade of NaCMC affected the drug release. NaCMC matrices showed fast erosion. The release of sodium ions from matrices containing NaCMC was enhanced propranolol hydrochloride, confirming the occurrence of the interaction in matrices. The release of propranolol hydrochloride from NaCMC matrices was not dependent on either the square root of time or time. Large increases in release rates from matrices containing NaCMC in acidic media implied the polymer was unable to gel provide or a sustained release of propranolol hydrochloride at low pH. A synergistic increase in viscosity in gels containing HPMC and NaCMC probably played a minor role in propranolol release from matrices containing both polymers. NaCMC decreased the cloud point of HPMC. Drug release from matrices containing HPMC and NaCMC was very complicated, but zero order release was achieved from matrices containing 285 mg of 1: 3 HPMC : NaCMC. Addition of HPMC to NaCMC matrices suppressed an initial burst release of propranolol. The release of propranolol hydrochloride from matrices containing HPMC and NaCMC was dependent on pH. Ethylcellulose was capable of binding = 14% w/w water. Matrices containing ethylcellulose 7 cP (<125 pm) showed lower release rates than matrices containing ethylcellulose 10 cP, or at greater particle sizes. Compaction pressure generally did not affect drug release. The release exponent from matrices containing ethylcellulose was 0.44 - 0.49 indicating diffusion predominated drug release. Admixture of ethylcellulose with HPMC did not change the release exponent (0.59 < n < 0.61) from that of HPMC alone, whereas the exponents of NaCMC:ethylcellulose matrices was altere. Addition of ethylcellulose to HPMC increased the initial uptake of water. The incorporation of a complex of propranolol and β-cyclodextrin failed to retard the release of drug or alter the release exponent.

A high speed hydraulic press has been developed into a computer controlled high speed compression Simulator, capable of reproducing displacement time profiles seen on any production tabletting machine. The system has been validated to monitor punch displacements to ?12pm and loads to ?O.05% of full scale. Confidence in the results obtained using the Simulator were enhanced by comparison with other operational Simulators. The established Simulator was then used to investigate the effects of compression on ibuprofen. Ibuprofen was found to consolidate mainly by plastic deformation with a lesser contribution from the melting of asperities. A significant amount of pressure induced melting and subsequent fusion bonding occurred at higher pressures. Ibuprofen was found to be sensitive to the magnitude and rate of application of the compression pressure. The extent of plastic flow exhibited by ibuprofen during compression was, found to decrease as the compression speed increased. Lamination and capping of ibuprofen compacts at high compression speeds was considered to be due to a combination of air entrapment and the inability of the compact to withstand the stresses of decompression. When ibuprofen was mixed with a second material and compressed, the consolidation mechanism and properties of the compacts formed were found to follow complex relationships. The relationships were dependant on the proportion of each material and the speed of compression. For ibuprofen microcrystalline cellulose mixtures positive interactions were considered to occur due to bonding between the two materials. For ibuprofen and lactose mixtures the interactions observed were considered to be a balance between the plastic deformation by the ibuprofen relieving the applied load preventing the cri tical force required for fracture of the lactose being attained, and the lactose fragments bearing the applied load reducing plastic flow by ibuprofen. The Simulator was then employed to investigate different aspects of tabletting machine design. A simple ibuprofen microcrystalline cellulose mixture and a commercial ibuprofen formulation were compressed to a constant load and then to a constant thickness and the properties of the compacts compared. Tablets prepared under a constant maximum applied load, with fill weights varied over the B.P uniformity of weight limits, had relatively constant disintegration times and radial tensile strengths. This was considered an advantage over the tablets prepared to a constant thickness which showed considerable varia?tion under the same conditions. The second aspect of tabletting machine design to be investigated was the use of relatively high precompression pressures using a commercially available paracetamol granulation. The maximum compression pressure exerted during the tableting cycle was found to be the major factor contributing to the tensile strength of the tablets. The use of a second compression either before or after the main compression was found to produce a significant increase in tablet tensile strength. The greater the magnitude of the second compression, the greater its effect on tensile strength. The contribution of a second compression towards the tablet tensile strength was attributed to the effective increase in dwell time it generated. The orientation of the greater and lesser compression pressures during tableting was found to influence the tablet tensile strength. Stronger tablets resulted if the greater pressure was exerted first. This was considered to be a function of temperature increases within the tablet and the disruptive effects of the second compression.

The resurgence of tuberculosis cases world-wide over the last two decades has led to one third of the population being infected and an ever increasing number of deaths (World Health Organisation, 2006). Little is known about the pathogenicity of the infectious agent, Tubercule bacillus, and resistance to the key chemotherapeutic drugs is widespread. Increasing research effort aiming to curtail the spread of this disease has been aided by the work of Cole et al. (1998 and 2002), which provided genomic annotations of the H37Rv strain of Mycobacterium tuberculosis. Subsequent structural genomics projects have identified hundreds of potential targets for structure-based drug design. The research presented in this thesis focuses on the expression and characterisation of targets from the Mycobacterium tuberculosis genome. Cell-free expression trials of 36 unique targets were performed. Initial screening resulted in soluble expression for 30 % of the targets and inclusion of additives, such as molecular chaperones or detergents, increased this to 67 %. Milligram quantities of protein were obtained for eleven targets. As a comparison, four targets were chosen for expression trials using an E. coli in vivo system. Similar results were obtained for three of the targets using the cell-free or in vivo expression systems. However, significant quantities of soluble Rv3545c, a cytochrome P450 125, were only produced using the in vivo method. Proteins that were expressed in sufficient quantities were progressed into crystallisation trials, one of which yielded crystals suitable for X-ray diffraction. The crystal structure of Rv3628, an inorganic pyrophosphatase (Mtb-PPase), was refined to 2.7 A resolution in space group P3221. Inorganic pyrophosphatases (PPases) are ubiquitous metalloenzymes which belong to the phosphatase superfarnily, and play an essential role in biosynthetic reactions (Teplyakov et aL, 1994). 'ne refined crystal structure of Mtb- PPase was found to exhibit a similar overall fold and oligomeric form to existing type I PPase structures. Comparison with two recent Mtb-PPase structures, both in space group P6322 (Tammenkoski et al., 2005 and Benini and Wilson, to be published), highlighted a possible pH-dependent role of His93 within the active site. The characterisation of Rv3545c, a predicted cytochrome P450 125 (Mtb-CYP125), is also described in this thesis. Cytochrome P450s are a superfamily of haern-thiolate proteins (50 to 60 kDa) which monooxygenate hydrophobic substrates as part of electron transport chains (Nebert and Gonzalez, 1987 and Chapple, 1998). P450s have recently been implicated as novel antimycobacterial targets (Munro et al., 2003). Spectroscopy was used to confirm the cytochrome P450 annotation of Rv3545c, with the ferrous enzyme exhibiting a Soret peak at 450 nm in the presence of CO. A high-to-low spin-shift was observed by UV/visible and EPR spectroscopy, upon imidazole-inhibition of ferric Mtb-CYP125. Secondary structural elements were determined by circular dichroism (CD) to be - 33 % a-helix and - 14 % P-sheet. Finally, dark brown/red crystals of Mtb-CYP125 were obtained, but it was not possible to collect a full data set. This was primarily due to the crystals forming clusters which were impossible to separate. Despite this, weak diffraction data to 3 A resolution were measured, and further optimisation of the crystallisation conditions may prove successful.

This thesis examines the use of hydroxypropylmethylcellulose (HPMC) of different viscosities and ethylcellulose aqueous dispersion (Surelease), alone or in combination with each other, to control the release of metoclopramide hydrochloride or diclofenac sodium from coated pellets or matrices. The glass transitions of the polymeric films were determined by thermomechanical analysis and used as a guideline to select the inlet air temperature in coating operation. The coating procedure was performed using Accela-Cota. Matrices were prepared either by direct compression or wet granulation. Compendial dissolution methodology was used to determine drug release from coated pellets as well as matrices. Release exponents indicating release mechanisms were calculated from the dissolution data. The release of both drugs from coated pellets decreased as the coating load of HPMC increased. However HPMC did not control drug release rate and the majority of both drugs released in less than 1 h. The release exponents for metoclopramide hydrochloride release from HPMC E5 and HPMC E15 coated pellets were -0.45 and -0.46 respectively. The corresponding value for diclofenac sodium was -0.50. These values of n indicate that diffusion is the predominant mechanism for drug release from HPMC coated pellets. The release of both drugs controlled with application of Surelease on drug-layered pellets. Increasing coating load of Surelease extensively decreased the release rates of both drugs and increased the lag times before controlled release was achieved. The release exponent for metoclopramide hydrochloride was independent of coating load and the mean value was -0.60 indicating predominantly diffusion controlled release. However the value of n for diclofenac sodium was higher at low coating loads suggestmg erosion controlled mechanism and decreased as the coating load increased, indicating more diffusion controlled mechanism. The mean value was -0.70. Inclusion of HPMC increased the release rates of both drugs. The Surelease:HPMC ratio had a major role in the release rates of drugs. Addition of HPMC into Surelease did not change the release exponent for metoclopramide hydrochloride (-0.57) from that of Surelease alone and diffusion remained the main mechanism controlling drug release. However the release exponent (-1.28) increased for diclofenac sodium release upon addition of HPMC indicating erosion controlled mechanism. Application of 2% seal-coat of HPMC E5 prior to Surelease resulted in decrease in the release rates of both drugs. However the exponent n and consequently release mechanism remained unchanged for either metoclopramide hydrochloride (-0.60) or diclofenac sodium (-0.69). Generally release of diclofenac sodium from Surelease or Surelease/HPMC coated pellets was faster than metoclopramide hydrochloride. This was attributed mainly to the interaction of the latter drug with the anionic surfactant ammonium oleate present in the Surelease coat. The interaction of metoclopramide hydrochloride with ammonium oleate was confirmed by dialysis studies. Drug release from HPMC matrices was controlled by the polymer content and viscosity. The drug release was also dependent on the solubility of the drugs. Metoclopramide hydrochloride released faster than diclofenac sodium. The release exponent for metoclopramide hydrochloride was in the range of 0.53-0.6.4. The corresponding value for diclofenac sodium was O.59-0.8O. Therefore a combination of diffusion and erosion controlled the release of either drug. The higher value of n for diclofenac sodium may indicate the greater role for erosion than was the case for metoclopramide hydrochloride. The incorporation of Surelease into HPMC K4M matrices considerably decreased the release rate of metoclopramide hydrochloride while that of diclofenac sodium was less affected. The conversion of metoclopramide hydrochloride to its base form was proposed as an explanation. The release exponents for Surelease granulated matrices was -0.56 for both drugs indicating diffusion mainly controlled the mechanism of release.

Endophytic fungi from Cassia fistula or golden shower, a well known medicinal plant in Thailand and Asia, were isolated from trees growing in three geographical separate sites. These locations were Kanchanaburi, Nakhon Ratchasima and Bangkok and were selected to allow comparisons between their endophytic assemblages and to evaluate these data in relation to differences in plant diversity and density and local environment. Kanchanaburi which was the site closest to a natural forest situation provided the highest number of isolates with Bangkok, where the trees were isolated individuals, having the least. Members of the Xylariaceae proved to be common and frequent isolates especially species of Xylaria and Daldinia but Nemania and Hypoxylon were also obtained. Phomopsis was also well represented and clearly was dominant at the Kanchanaburi site. Species of Fusarium, Colletotrichum, Penicillium, Nigrospora, Coprinus and Psathyrella were also identified but were occasional isolates. Differences in endophytic assemblages between samples obtained early in the rainy season (July, 2001) with those sampled towards the end of the rainy season (December, 2001) were found to occur in the Nakhon Ratchasima samples with over twice as many isolates obtained from the December samples. This is likely to be a reflection on the longer exposure period to the potential inoculum of these leaves. A total of 956 endophytic isolates were obtained from the three sites with samples from Kanchanaburi (December 2000) and Bangkok and Nakhon Ratchasima in July 2001 with a further samples from Nakhon Ratchasima in December, 2001. Isolations were also made from different anatomical regions of the leaf, leaf lamina, midrib and veins. There were no appreciable differences in either the number of isolates obtained or an association between leaf area and specific fungal species. Identification of many xylariaceous endophytic isolates is well known to be problematic since Xylaria species rarely produce their anamorphic form in culture and virtually no members of the Xylariaceae develop their teleomorph in culture. Therefore molecular techniques were used to compare DNA sequences of the ITS region from a selection of endophyes with sequences obtained from teleomorphic material, or cultures derived from teleomorphs of identified and authenticated Xylariaceae. Comparisons were also made with data held in GenBank. This enabled the identity of a number of taxa to be made although more sequences from Xylaria species are required for future investigations. A number of non-xylariaceous taxa were also named as a result of DNA sequence comparisons. Secondary metabolites from the xylariaceae were also investigated and their metabolite profiles used to support identifications. The metabolite profiles proved to be a useful tool to confirm doubtful endophytic isolates when their DNA sequences could not place them with certainty in a right group. Together with extracts from other endophytic species, their inhibitory effects on bacteria and fungi were tested. Cassia endophytes were found to show low antimicrobial activity. However, they may later be shown to have other activities when when tested e. g. anti-malarial, anti-cancer and anti-HIV.

Streptococcus pneumoniae is the leading bacterial cause of pneumococcal diseases, of which pneumonia is the main cause of death amongst the immunocompromised, elderly over the age of 50 and children under the age of 5. Although vaccines such as pneumococcal polysaccharide vaccine 23, pneumococcal conjugate vaccine 7, 10 and 13 are available, they are expensive to produce and distribute. Moreover, the variation in serotype distribution across geographical locations and rise in dominance of disease due to non-vaccine serotype coverage has led to significant attention towards the development of alternate vaccine candidates such as pneumococcal surface protein A (PspA). A potential dry powder vaccine formulation containing polymeric nanoparticles (NPs) adsorbed with PspA4Pro and formulated into nanocomposite microparticles (NCMPs) using L-leucine (L-leu) to be delivered via inhalation was developed. Poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, NPs with either anionic or cationic surface charge of optimum size (~200-250 nm) to be effectively taken up by the lung dendritic cells (DCs) were successfully produced. The NPs were then surface adsorbed with PspA4Pro (~20 µg of PspA4Pro per mg of NPs) and spray-dried using L-leu as a microcarrier to produce NCMPs with a product yield of 55.55±6.64% for the PspA4Pro adsorbed anionic NPs/NCMPs and 53.98±2.23% for the PspA4Pro adsorbed cationic NPs/NCMPs. The NCMPs produced had a corrugated and wrinkled surface morphology. The aerosol properties of anionic NPs/NCMPs determined using a Next Generation Impactor displayed a fine particle fraction (FPF) of 74.31±1.32% and mass median aerodynamic diameter (MMAD) of 1.70±0.03 μm indicating that the majority of the dose would be deposited in the respirable airways of the lungs. The anionic and cationic PGA-co-PDL NPs upon incubation with DCs for 1 h showed an effective uptake as visualised using confocal microscopy. Furthermore, the anionic NPs/NCMPs were well tolerated by the A549 cell line with a cell viability of 87.01±14.11% at 1.25 mg/ml concentration, whereas the cationic NPs/NCMPs showed a cell viability of 75.76±03.55% at 156.25 µg/ml concentration upon 24 h exposure. The PspA4Pro released from the optimised formulations largely maintained its structure as determined using SDS-PAGE and circular dichroism, and the relative antigenicity measured using ELISA was 0.97±0.20 and 0.85±0.05 for anionic and cationic formulations, respectively. Overall, the results obtained indicate the use of these NPs as novel carriers for pulmonary vaccine delivery against pneumococcal diseases.