181 |
Pain, sensation and biological responses following human skin puncture with microneedlesHaq, Mohammed Inaam-ul January 2010 (has links)
Future work should optimise the design of microneedle devices for clinical delivery of active molecules.
|
182 |
Microfluidic encapsulation of cells for transplantation in neurodegenerative diseaseWorkman, Victoria January 2009 (has links)
Polymer encapsulation is now an accepted route into cellular therapies via implantation of therapeutically active allogeneic and xenogeneic cells. Although many methods are currently used for encapsulation of cells, no single method is capable of producing large volumes of mono-disperse beads, containing cells completely covered by the polymer of choice. The overall aim of this project was to develop a microfluidic method to encapsulate dopamine releasing cells in an alginate matrix, determine their viability in vitro and investigate implantation into a rodent model of Parkinson's disease. During the course of this study a novel microfluidic method was developed. Two cell types were encapsulated a human test line and a therapeutic cell line derived from rat brain tumour cells (PC 12). Cell viability, measured using an adapted trypan blue exclusion method, was observed to be minimally affected by the encapsulation process. Confocal images of cells encapsulated within alginate beads were collected in addition to long term viability data, up to 90 days post-encapsulation. Dopamine was still detected after PC 12 cell encapsulation through use of an ELISA. Modifications to the developed microfluidic method allowed beads of an appropriate size (<250microm in diameter) to be implantated into a rodent brain via a cannula. Upon implantation of alginate beads into rats' brains there was no evidence of beads after 7 days. Attempts were made to stabilise alginate beads further by addition of barium as a cross-linking agent and polycation secondary coating. Beads were observed to be more stable and remained visible within brain tissue for 14 days. Imaging of fluorescent alginate beads revealed that beads produced using the developed microfluidic method were homogeneous in nature. The work presented here represents the first microfluidic method to be developed which is capable of encapsulating viable cells. Moreover, the viability measurements carried out were the first such experiments to be performed on cells encapsulated using microfluidic methods. Although the structure of alginate beads produced using more commonplace methods has been shown, this has not previously been reported for beads produced using a microfluidic technique.
|
183 |
Probing amorphous and crystalline pharmaceuticals systems using NMRFoumier, Romain January 2006 (has links)
The Ή and '3C solution-state NMR spectra of the three studied drug molecules (indomethacin, nifedipine and carbamazepine) were fully assigned. This led to the characterisation and assignment of the '3C CPMAS SSNMR spectra of the stable polymorphic forms of these molecules. The signal for the с-СІ carbon of indomethacin has been studied as a function of applied magnetic field, and the observed bandshapes have been simulated. A T(_1)(^H)and T(_1p)(^H) study was undertaken on the crystalline materials, as well as on the quench-cooled amorphous and PVP/drug co-melts. This was done in order to fulfill the aim of this research, i.e. to understand the difference in stability of the amorphous compounds and also to investigate the effect of the presence of PVP on the stability and mobility of the drug. It was shown that, under the conditions of the experiments, amorphous indomethacin did not recrystallise until 110 c, whilst in the case of nifedipine and carbamazepine the recrystallisation occurred at 70 c and 75 c respectively. It was also shown that in the case of the co-melts a transition occurs consistently between 65 c and 85 c for the three materials and this seems to be due to the Tg of the co-melts. The comparison of the T(_1)(^H)and T(_1p)(^H) data for the different states showed that amorphisation increased the mobility of the sample, this being more pronounced for carbamazepine and nifedipine than for indomethacin, and also that the co-melts were more stable and slightly more mobile than the amorphous compounds. The comparison of the relaxation data between the pure compounds showed that amorphous indomethacin was more stable than the other two amorphous drugs as slope changes occurred for the latter at a temperature below Tg, whilst this happens at or around Tg for amorphous indomethacin.
|
184 |
Human urotensin-II receptor desensitisationBatuwangala, Madura Suharshana January 2009 (has links)
Human Urotensin-II (U-II) is a cyclic undecapeptide that binds to the U-II receptor UT. The desensitisation mechanisms of the UT receptor (G_{q/11} coupled GPCR) are not well defined and hampered by (1) lack of native (in-vitro) models; (2) paucity of ligands, especially non-peptides and (3) irreversible binding of U-II. There are some limited studies using rat aorta, where a U-II induced primary contractile response was reduced upon a secondary re-challenge after 5-hours. Studies were undertaken to characterise cell lines expressing native (SJCRH30) and recombinant human hUT (HEK293 and CHO) for their suitability in binding and functional assays (PI and Ca^2+). SAR studies were carried out to characterise novel analogues modified at Tyr^9 of the U-II(4-11) template. This led to the identification of [3,5-diiodoTyr^9]U-II(4-11) a partial agonist in aorta and Ca^2+ assays at rat UT. Full agonism was demonstrated at hUT in PI and Ca^2+ assays. Efforts were made to delineate functional and genomic desensitisation of hUT. There was no functional desensitisation in SJCRH30. In HEK293hUT functional heterologous desensitisation of hUT was observed, this was not so in CHOhUT; instead P_2YR was functionally attenuated. In SJCRH30 6-hr U-II treatments led to UT mRNA reduction. Genomic desensitisation was also studied in Peripheral blood mononuclear cells (PBMCs). U-II treatments alone did not affect UT mRNA. Lipolysaccharide treatment of PBMCs led to UT mRNA upregulation which was desensitised with U-II treatments. In recombinant systems UT mRNA was upregulated at 6-hr U-II treatments. In conclusion modification of the U-II(4-11) template at Tyr^9 is useful for reducing efficacy. There is a difference in desensitisation profiles of native and recombinant hUT, where native receptors are not prone to functional desensitisation while receptor mRNA is reduced. In recombinant systems, hUT undergoes desensitisation (HEK293hUT only) while receptor mRNA is increased in both systems.
|
185 |
GLP-1 and muscarinic receptor mediated activation of ERK1/2 in pancreatic β-cellsSelway, Joanne Louise January 2010 (has links)
Glucagon like peptide-1 (GLP-1) and acetylcholine (ACh), acting thourough their GPCRs on β-cells, potentiate glucose stimulated insulin secretion and mediate ERK1/2 activation. Both agonists have also been demonstrated to stimulate proliferation, neogenesis and increased transcription potentially thourough ERK1/2 dependent pathways. ERK1/2 has been shown to play an important role in regulating pancreatic β-cell function and mass and mediating gene transcription. Therefore, this thesis aims to elucidate the mechanism by which GLP-1, via the GLP-1R, and carbachol, via Gq-coupled mAChR activation, leads to the phosphorylation of ERK1/2 in the pancreatic β-cell line MIN6. I demonstrate that both GLP-1R and mAChR stimulation mediate Ca2+-dependent ERK1/2 activation requiring the activation of the L-type VGCC. GLP-1 causes a sustained activation of ERK1/2 that requires continual activation of the L-type VGCC and the sustained elevation of local Ca2+ around the mouth of the channel. Importantly, ERK1/2 activation stimulated by L-type VGCCs mediated Ca2+ influx is required for GLP-1 stimulated insulin transcription. The mechanism by which the L-type VGCC signals to ERK1/2 was also investigated. I demonstrate that L-type VGCC-dependent ERK1/2 activation mediated by local Ca2+ is Ras-independent. However, a global rise in Ca2+ mediated by a depolarising stimulus is capable of mediating ERK1/2 activation by a Ras-dependent mechanism. I have also demonstrated that the down-regulation of DAG-sensitive proteins significantly inhibits GLP-1 stimulated ERK1/2 activation, but this is not thourough the down regulation or inactivation of classical or novel PKC isoforms. mAChR activation mediates the rapid and transient phosphorylation of ERK1/2 which is dependent upon PLC and a rise in [Ca2+]i, but independent of PKC activation. Importantly, the rise in [Ca2+]i is mediated from multiple sources including: the efflux of Ca2+ from the ER by IP3R activation, the influx of extracellular Ca2+ thourough store operated channels (SOC) and L-type VGCC activation. I provide evidence that the activation of the L-type VGCC is partially mediated by the inhibition of KATP channels via PIP2 depletion, as increasing PIP2 levels partially inhibits carbachol-stimulated increases in [Ca2+]i and ERK1/2 activation. However, carbachol stimulated ERK1/2 activation appears to have, like a depolarising stimulus, a Ras-dependent and a Rasindependent pathway mediating ERK1/2 activation, potentially due to the L-type VGCC activation initiated by carbachol. Overall this thesis demonstrates that the L-type VGCC is a key mediator in ERK1/2 activation in β-cells. Both GLP-1 and mAChR stimulation requires the activity of the Ltype VGCC to mediate Ca2+-dependent ERK1/2 activation, and I have provided evidence that a Ras-independent Ca2+-dependent pathway leading to ERK1/2 activation is initiated within the microdomain of the L-type VGCC and can stimulate transcription.
|
186 |
Molecular basis of antagonist action at the P2X1 receptorEl-Ajouz, Sam January 2011 (has links)
P2X receptors are ATP-gated cation channels. P2X1 receptors are widely expressed throughout the body and have a range of functional roles, e.g. contraction of mesenteric arteries and regulation of blood clotting. The recent crystallisation of the zebra fish P2X4 receptor has provided a major advance in understanding the molecular basis of receptor properties. However, how agonists or antagonists are co-ordinated and the extent of the proposed ligand binding site have not been addressed at a structural level. A mutagenesis based approach was used to propose a model of the ATP binding site and has highlighted some residues involved in antagonist action at P2X receptors. The aim of this thesis was to investigate the molecular basis of antagonist action at the P2X1 receptor using site-directed mutagenesis and P2X receptor chimeras. The wild-type, mutant and chimaeric P2X receptors were expressed in Xenopus laevis oocytes and the currents were characterised using two electrode voltage clamp. Initially, suramin was shown to act as a competitive antagonist and PPADS as a non-competitive antagonist at the P2X1 receptor. The contribution of residues V74 to G96 to human P2X1 receptor properties were determined using cysteine scanning mutagenesis. This region contains a residue that has been shown to be important in suramin action at the P2X4 receptor (K78) but cysteine mutation of the residues V74 to G96 had either no effect or slightly increased antagonism by suramin or PPADS. Also, a further residue was found to be important in ATP potency (F92) and the use of partial agonists and modification with cysteine reactive methanethiosulfonate (MTS) reagents identified additional residues important in channel activation. Mapping these residues onto a homology model of the P2X1 receptor showed the depth of the agonist binding site and highlighted the importance of the rear/inner cavity of the binding pocket in the gating of the channel subsequent to agonist binding. The cysteine rich head region of the P2X receptor, which is adjacent to the proposed ATP binding pocket, is absent in the antagonist insensitive Dictyostelium receptors. P2X1 and P2X2 receptors have ~1400-fold difference in sensitivity to a suramin analogue NF449. Chimeras and point mutations in the cysteine rich head region were made between the P2X1 and P2X2 receptors and they identified the region between the third and fourth conserved cysteine residues of the P2X1 receptor as being important in conferring the difference in sensitivity. In particular, the positively charged residues at the base of the cysteine rich head region of the P2X1 receptor accounted for the highly selective antagonism of NF449 at the P2X1 receptor. Additionally, these residues were shown to play a role in the molecular basis of suramin and PPADS action at the P2X1 receptor. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity. In silico docking models highlighted possible sites of action for NF449 and suramin on the P2X1 receptor showing that the base of the cysteine rich head region may be important in the binding of antagonists. In summary, this research furthered understanding of ligand action at the P2X1 receptor.
|
187 |
Exploring the structure, function & regulation of the human glucagon-like peptide-1 receptorHuang, Yan January 2011 (has links)
Glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and promotes β-cell function via its receptor (GLP-1R), which therefore is a validated target for the treatment of type 2 diabetes. Due to difficulties with peptide therapeutics, it is important to find small-molecule GLP-1R agonists. This leads to a need to understand the structure, function and regulation of the receptor, particularly, differences between agonisms mediated by GLP-1 (orthosteric agonist) and small molecules. The GLP-1R contains a putative N-terminal signal peptide sequence, which is assessed here by recombinantly expressing several epitope-tagged GLP-1R constructs in HEK293 cells. The findings demonstrate that the GLP-1R is expressed predominately at the plasma membrane and also slightly cytosolic. Only fully glycosylated, mature form of the receptor is able to traffic to the cell surface and performs the function. The signal peptide sequence of the GLP-1R is essential for synthesis. After fulfilling the function, this sequence is cleaved and thus not part of the mature protein. The cleavage of signal peptide is critical for processing and trafficking of the GLP-1R. Based on one of these constructs generated here, a cell line (HEK293: GLP-1R-EGFP) with stable expression of the visible GLP-1R is established, which allows observations and determinations for ligand-mediated receptor internalisation in real time. Compound 2 (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline) has been described as a GLP-1R allosteric modulator and agonist. Findings here that compound 2-mediated agonisms on both the wild-type (WT) GLP-1R and the mutant with removal of the N-terminal domain provide direct evidence for the allosteric agonism. Interestingly, compound 2-mediated cAMP response is enhanced by orthosteric antagonist exendin 9-39, but the latter inhibits receptor internalisation mediated by compound 2. Recently, it has been hypothesised that the binding of GLP-1 allows a sequence of NRTFD (Asn63-Asp67) in the N-terminus of the GLP-1R to interact with another part of the receptor and cause agonism. This was examined here by generating receptor mutants and synthetic peptides. Findings here that Asp67 plays a key role in stabilising the N-terminal structure of the GLP-1R and thus is critical for processing and trafficking of the receptor protein do not support such hypothesis although synthetic NRTFD mediates a weak and partial agonism on the WTGLP-1R.
|
188 |
Biodegradable nanoparticles for oral delivery of CiclosporinAnkola, Dhawal Dhirajlal January 2010 (has links)
No description available.
|
189 |
Assessing the use of twin screw wet granulation in a multi stage manufacturing process for the continuous production of pharmaceutical productsHolman, James William January 2013 (has links)
Traditionally pharmaceutical manufacture is conducted on a batch basis but significant resources are being invested into the use of intensified continuous processes. This dissertation evaluates the use of a combined twin screw and segmented fluid bed drying process to produce granules on a continuous basis. The experimental program was conducted using structured Design of Experiments in three stages. • Wet granulation only: Investigated the initial relationships between liquid/solid ratio and power required for wet granulation, as well as granule structure using SEM Imaging. • Wet granulation and fluid bed drying: Concluded that the biggest control over, the measured mean granule size (d50) produced from the combined system was still the ratio of water to dry powder in the wet granulation. • Wet granulation through to compression: The effects of changes in the granulation process were not statically relevant on the final tablet for the process set up. The study also used PEPT data to assess motion within the TSG. The studies showed: • The time spent in the kneading zone directly after the liquid addition in relation to the overall time spent in the granulation process appears independent of the process j . set up at 32% ± 2%. • As the barrel speed of the granulator increases the relative time spent in the final ' breakage zone' of the TSG increases, therefore increasing breakage. Using the findings from the literature, the results of the experimental program were used to define the mechanisms occurring within the TSG. The experimental findings were input into a model to predict the outcome of collisions between particles. The model predicts agglomeration of the smaller particles to the larger ones and by calculating changes in the viscosity of the binder the subsequent secondary agglomeration of these granules can also be shown using this model The model is limited due to assumptions in deriving it. The model excludes capillary forces that if given sufficient time to form could have the same order of magnitude strength as other forces.
|
190 |
Formulation of bacteriophage for application in bacterial lung infectionPuapermpoonsiri, Utsana January 2011 (has links)
No description available.
|
Page generated in 0.8458 seconds