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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Alternatively activated macrophages recruited by Brugia malayi

Loke, P. January 2000 (has links)
The adult stage of the parasitic nematode <i>Brugia malayi</i> can live in the host lymphatics for many years and must have an extremely effective immuno-suppressive mechanism that prevents rejection. We have discovered that this parasite can induce alternatively activated IL-4 dependent macrophages that can block proliferation of other cells via a receptor-mediated mechanism. The proliferative block is reversible and is not a result of apoptosis. Furthermore, these suppressive cells can reduce the proliferation of a wide range of human cancer cell lines. These data demonstrates that <i>B. malayi</i> can lead to the activation of a novel mechanism of proliferative suppression, via IL-4 dependent macrophages. These macrophages may have important roles in altering host immune responses during parasitic infection and may even have the potential to reduce tumour cell growth. Another feature of <i>B. malayi </i>infection is a bias towards a type 2 immune response. To investigate the role that the <i>B. malayi </i>recruited antigen presenting cells have on naive T cells, the suppressive macrophages recruited by <i>B. malayi </i>was used to stimulate naive T cells from TCR transgenic mice. Although the naive T cells were inhibited by parasite-induced macrophages during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, naive T cells primed by suppressive macrophages differentiated into IL-4 producing Th2 cells upon secondary stimulation instead of IFN-g producing Th1 cells, as has been previously described. Th2 differentiation was associated with the inhibition of (or failure or stimulate) IFN-g production during primary stimulation. Interestingly, blocking antibodies against TGF-b (but not IL-10) restored the differentiation of IFN-g producing Th1 cells. These data indicate that T cells exposed to parasite induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies helminth infection.

Targeting the p53/MDM2 protein-protein interaction

Goffin, Sarah Anne January 2016 (has links)
The p53/MDM2 protein-protein interaction is the most widely characterised proteinprotein interaction to date. As of 2014, there are over 20 compounds that have been shown to the p53-MDM2 protein-protein interaction, however many compounds have not progressed into clinical trials due to their high hydrophobicity. Herein we describe the synthesis, molecular modelling, physical characterisation and biological testing of novel inhibitors of the p53/MDM2 protein-protein interaction based on the natural product chlorofusin. The first focus is a combinatorial library generated in the Searcey laboratory of known p53/MDM2 protein-protein interaction inhibitors with the desire to generate novel analogues and study their interactions with the protein through NMR spectroscopy and molecular modelling. These compounds were tested by in a fluorescence polarisation assay and also in cell lines overexpressing MDM2 as well as p53-null cells as a comparator. This generated two novel compounds shown to have activity selectively for the p53/MDM2 protein-protein interaction. The second chapter focuses on simplified substitutions of the azaphilone (the chromophore portion of chlorofusin, a natural product inhibitor of the p53-MDM2 proteinprotein interaction): initially with simple fused bicyclic carboxylic acids and later using click chemistry substitutions. Interestingly, in vitro studies showed that the click analogues retained activity or activity improved when the peptide portion was removed and hence further studies of the click amino acid analogues were generated. This library generated one analogue that was active in vitro as well as selectively in MDM2-overexpressing cell lines. The third chapter focusses on the azaphilone chromophore present in the natural product chlorofusin. The Sonogashira precursor used to generate azaphilone analogues was synthesised using a methodology adopted by Porco et al and subsequent analogues were generated using a novel double-Sonogashira approach followed by functionalisation published by Boger et al. Once the azaphilone was synthesised, metholodogies were trialled in order to condense the azaphilone with the chlorofusin peptide in order to create analogues containing both the peptide and small molecule portions of chlorofusin. In addition, molecular modelling was attempted to generate novel binding analogues.

Mechanisms regulating vasoactive intestinal polypeptide expression in cultured dorsal root ganglion neurons

Dobson, Stephen P. January 1996 (has links)
This study has attempted to identify regions of DNA within the VIP gene that may be responsible for spontaneous expression of VIP. Using an electrophoretic mobility shift assay, this study has shown that the rat VIP CRE is capable of binding c-Jun in a heterodimer with c-Fos. To determine the importance of these proteins in binding to the VIP CRE, an attempt was made to compete them off the endogenous rat VIP CRE. DRG neurons were transfected with constructs containing copies of this CRE ligated into the plasmid pUC18. Quantitative analysis of the effects of transfection on endogenous VIP immunoreactivity, showed that the CRE containing construct caused a selective reduction in VIP expression. Proteins binding to the CRE are therefore important for spontaneous VIP expression. To determine if the rat VIP CRE is all that is necessary for spontaneous VIP expression, it was analysed, using reporter constructs, for its ability to mediate patterns of gene expression analogous to those seen for endogenous VIP. This study has shown that the CRE is capable of increasing gene transcription from a heterologous c-fos promoter in neonatal, but not adult rat DRG neurons, in response to stimuli that raise cAMP and intracellular calcium and as such may be responsible for the synergistic increase in VIP expression that occurs in neonatal rat DRG neurons in response to these same stimuli. This study suggests that the spontaneous expression of rat VIP is dependent on protein complexes binding to the CRE, and that these complexes probably contain c-Jun. Although the CRE alone is capable of mediating the response of VIP to cAMP and calcium, and may mediate developmental differences in VIP expression, sequences are required in combination with the CRE for this spontaneous expression.

Endothelial factors and platelet activity : endothelin-1, a new modulator

Dockrell, Mark Edward Carl January 1997 (has links)
The aim of the work presented in this thesis was to investigate of effect of endothelin-1 on <I>in vitro</I> human platelet aggregation, to characterise the endothelin receptor subtype present on platelets and examine the effect of the peptide on the intracellular second messengers cyclic AMP and cyclic GMP. In addition the role of endothelin-1 in the modulation of platelet aggregation in subjects with high blood pressure has also been exmained. Analysis of the effect of endothelin-1 on <I>in vitro</I> platelet aggregation, by the use of transmittance aggregometry, has identified that 1. Endothelin-1 alone has a slight but significant aggregatory effect. 2. Endothelin-1 potentiates primary and inhibits secondary aggregation induced by adrenaline but not ADP. 3. Both endothelin-1 and sarafotoxin S6c, an ET<SUB>B</SUB> receptor selective agonist, potentiate adrenaline-induced primary aggregation in a dose dependent manner, furthermore endothelin-1 potentiation of aggregation is inhibited by BQ 788, and ET<SUB>B</SUB> receptor antagonist but not BQ 123, an ET<SUB>A</SUB> receptor antagonist. <I>In vivo</I> administration of endothelin-1 inhibited <I>ex vivo</I> secondary platelet aggregation to adrenaline but not to ADP. No significant effect was seen on primary platelet aggregation induced by either aggregating agent. In vitro aggregation using a concentration of endothelin-1 estimated to be approximately the same as that achieved by <I>in vivo</I> administration produced similar results. Endothelin-1 and sarafotoxin S6c both caused a dose dependent accumulation of cyclic GMP but not cyclic AMP in platelets. In conclusion, endothelin-1 appears to modulate human platelet aggregation in a bi-directional manner. The potentiation of primary aggregation is dependent on the platelet aggregating agent employed and appears to be mediated by the ET<SUB>B</SUB> receptor. Platelet cyclic GMP accumulation is also stimulated by ET<SUB>B</SUB> receptor agonists. In addition, platelet responsiveness to endothelin-1 is altered in subjects with high blood pressure and in patients with essential hypertension, and the potentiation of platelet aggregation by endothelin-1 may be associated with a genetic component of high blood pressure.

Protein-protein conjugation using interferon

Herrington-Symes, A. P. January 2015 (has links)
Therapeutic proteins are often potent and have rapid onsets of action. Unfortunately protein-based medicines can be immunogenic and have short half-lives. The circulation half-life of many proteins has been improved by the covalent conjugation of poly(ethylene)glycol (PEG) to the protein. For example, PEGylated interferon-2 (PEGASYS® and PEG-INTRON®) has become a first line treatment for hepatitis C. The aim of this thesis was to examine the possibility of using a homobifunctional PEG reagent to make protein dimers. Our group has developed PEGylation reagents that undergo conjugation by bis-alkylation to selectively conjugate either (i) two cysteine thiols from a native disulfide or (ii) two histidine residues in a polyhistidine tag. It was hypothesised that IFN dimers (IFN-PEG-IFN) could be prepared with higher retained activity than monoPEGylated IFN. It was also hypothesised that a heterodimer of IFN and an antibody fragment (Fab) could be made while retaining the activity of both proteins within the heterodimer. His8IFN-PEG-His8IFN and IFN-PEG-IFN homodimers were prepared by site-selective conjugation to the N-terminal 8-polyhistidine tag and to one of the disulfides of IFN respectively. These homodimer conjugates were characterised in terms of purity and in vitro activity. The in vitro cell based assays were optimised to accurately elucidate the specific activities of the IFN conjugates. The His8IFN-PEG20-His8IFN homodimer was found to retain greater activity than IFN-PEG20-IFN. The increased activity was thought to be due to conjugation to the polyhistidine tag, which is distal from the IFN binding surface. It was also found that IFN-PEG10-IFN homodimer retained greater activity than PEG10-IFN, which could be due to the presence of the second IFN molecule in the IFN-PEG10-IFN homodimer. Two different Fabs were used to prepare the IFN-PEG-Fab heterodimers. Conjugation was conducted at one disulfide of IFN and the accessible interchain disulfide of the Fab. One Fab was derived from a polyclonal antibody to albumin (Fabalb) and the rationale for this heterodimer was that a longer lasting form of IFN could be made (IFN-PEG20-Fabalb). The other Fab was derived from bevacizumab (Fabbeva) to give an IFN-PEG20-Fabbeva heterodimer that could, in principle, display antiangiogenic properties. Both heterodimers were evaluated using antiviral and antiproliferative assays to determine the activity of IFN in the conjugate. The IFN-PEG20-Fabalb conjugate displayed a 10-fold reduction in activity compared to IFN-PEG20-Fabbeva. It was thought that Fabalb underwent competitive binding with components of the media. Interestingly, the IFN-PEG20-Fabbeva heterodimer displayed greater activity than PEG20-IFN and IFN-PEG20-IFN in both the antiviral and antiproliferative assays. The binding properties of Fabbeva were determined by SPR. It was observed that the dissociation rate of IFN-PEG20-Fabbeva was similar to Fabbeva and PEG20-Fabbeva. IFN-PEG20-Fabalb was found to have a similar dissociation rate to Fabalb. However, PEG20-Fabalb was found to have a slower dissociation to both IFN-PEG20-Fabalb and Fabalb, this result requires further investigation but was thought to be due to the sample impurity. The association rates of heterodimers were found to similar to the PEG-Fab conjugates but slower than their native Fabs. This data suggests that the novel IFN-PEG20-Fabbeva and IFN-PEG20-Fabalb heterodimer conjugates have retained their binding affinities to their antigens. Overall, it was shown that a homobifunctional bis-alkylating conjugation reagent (e.g. PEG di(bis)sulfone 4) could be used successfully to prepare dimeric protein conjugates. This work highlighted the importance to ensure the homobifunctional conjugation reagent was pure, especially for the preparation of protein heterodimeric conjugates. To develop this work further, it would be important to investigate three broad areas: i) improving the purity of the starting homobifunctional reagents, ii) evaluate the in vivo efficacy of the resulting protein homo-/hetero-dimers and iii) determine the overall potential for efficient scaling of the process to make the desired protein homo-/hetero-dimers.

In vitro and in vivo radioligand binding studies for the N-methyl-D-aspartate receptor and the 5-hydroxytryptamine transporter

Shirakawa, Kiyoharu January 1996 (has links)
The thesis contains <I>in vitro</I> and <I>in vivo</I> radioligand binding studies for the N-methyl-D-aspartate (NMDA) receptor, the 5-HT1A receptor and the 5-HT transporter. A novel NMDA antagonist FR115427 ((+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride; (+)FR) was characterised using [<SUP>3</SUP>H]MK-801 binding to rat brain membranes. As with (+)MK-801, the affinity of (+)FR was increased when 10 μM L-glutamate was added in the assay buffer. (+)FR inhibited [<SUP>3</SUP>H]MK-801 binding to rat cortical synaptosomal membranes in the presence of 10 μM L-glutamate with a Ki value of 40 nM although (+)FR was 12-fold less potent than (+)MK-801. These results indicate that (+)FR is a non-competitive NMDA antagonist. [<SUP>3</SUP>H](+)FR synthesised by tritiation of (+)Cl-FR (mono-chlorinated aromatic precursor of (+)FR) was successfully purified by an open column method. <I>In vivo</I> distribution of intravenous injection of [<SUP>3</SUP>H](+)FR in rats was analysed. Intravenous injection of [<SUP>3</SUP>H](+)FR resulted in a rapid accumulation of radioactivity in brain, and the brain regional distribution of radioactivity appeared to reflect the localisation of NMDA receptors. The possibility of using derivatives of citalopram (selective 5-HT uptake inhibitor) as a photoaffinity or SPET ligand for the 5-HT transporter was evaluated using 5-azido-citalopram (5-AC) (photoaffinity ligand) and 5-iodo-citalopram (5-IC) (SPET ligand). 5-AC had high affinity (Ki = 1.65 nM) for [<SUP>3</SUP>H]citalopram binding sites being only 1.8-fold less potent than citalopram itself. In the presence of 5-AC, repeated U.V. irradiation (15W) of rat cortical membranes produced a significant 20% reduction in the Bmax value for [<SUP>3</SUP>H]citalopram binding site. Therefore radiolabelled 5-AC may provide a tool for isolation and characterisation of the 5-HT transporter.

Molecular mechanisms of bioreductive drug activation in solid tumour tissue

Spanswick, Victoria Jane January 1997 (has links)
The enzymology of mitomycin C bioactivation was studied <I>in vitro</I> in two solid mouse adenocarcinomas of the colon MAC 16 (high DTD) and MAC 26 (low DTD). Metabolism of mitomycin C by tumour subcellular fractions revealed a novel mitochondrial reductase active under hypoxia in both tumours. DTD and NADPH:cytochrome P-450 reductase activity was confirmed in MAC 16 but not MAC 26. The role of DTD was examined in a tumour homogenate system and was found to play a protective role under hypoxia in MAC 16, predominating over the other enzymes present. MAC 26 showed enhanced hypoxic metabolism due to the presence of the mitochondrial reductase. The studies were extended to a human perspective, using the human colon xenografts HT-29 (high DTD) and BE (low DTD). <I>In vivo</I> studies, again using the MAC tumours, revealed comparable mitomycin C metabolism and antitumour activity in both tumours. Such a result has profound implications on the use of DTD as a target of enzyme-directed bioreactive drug therapy with mitomycin C. The mitomycin C analogue indoloquinone EO9 is a promising new bioreactive drug, although little is known about its metabolism and mechanism of action. The chemical properties of the reactive intermediates of EO9 were studied under controlled conditions via pulse radiolysis, with the aim of proposing a mechanism for its cytotoxicity. Results indicated that whether EO9 undergoes reduction via one-or two-electron reduction, the hydroquinone intermediate, product of two electron reduction via enzymes such as DTD, will predominate and dictate the pattern of cytotoxicity. This suggests a central role of DTD. <I>In vitro</I> metabolism of EO9 by the tumours described above produced a number of metabolites which proved difficult to identify via liquid-chromatography-mass spectroscopy. Unlike mitomycin C and its principle metabolite 2,7-diaminomitosene, no metabolite was found to correlate with metabolic activation under varying oxic and hypoxic conditions in conjunction with DTD activity.

Synthesis of thienopyridines and thienoazepines from thiopen or benzo[b]thiopen

Pickering, Michael Wilfred January 1975 (has links)
No description available.

Microneedle-mediated transdermal drug delivery of biotherapeutic macromolecules

Courtenay, Aaron John January 2016 (has links)
In the last 20 years biotherapeutic macromolecules have become the fastest growing sector within pharmaceutical industry. Their development, facilitated by the introduction of advanced molecular engineering techniques, has led to improved treatment options for patients with autoimmune conditions, various cancer types, and infectious disease. The complex molecular structure of these drugs render them susceptible to degradation and, as a result, many commercially available products are suitable for parenteral drug delivery only. Subsequently, the hypodermic needle and syringe has remained the device of choice for biotherapeutic delivery, despite the many drawbacks associated with this method. Transdermal delivery has been an attractive alternative for many pharmaceutical formulators. However, few drugs possess the appropriate physicochemical properties required for crossing the human skin barrier. Microneedle (MN) technology combines micro-engineering and material sciences to fabricate micron scale projections manufactured onto a platform. That, when pressed against the skin, create aqueous apertures allowing drug delivery directly into the dermal tissue. This thesis explores the development of polymeric MN-based drug delivery systems, capable of facilitating intradermal and transdermal passage of biotherapeutic macromolecules. The model protein ovalbumin was incorporated into polymeric MN systems using commonly employed industrial manufacturing techniques and sterility was successfully demonstrated. Further these MN platforms were evaluated for intradermal delivery in vivo, highlighting the potential adjuvant effects of Gantrez® S-97. Subsequently, through industrial collaboration with market leading transdermal manufacturing company, Lohmann Therapie Systeme AG, the commercially available vaccine Pentavac® was successfully incorporated into dissolving MN arrays. This study has provided significant learnings for both academia and industrial partners, in relation to industrial manufacture of MN and biotherapeutic macromolecules. Finally, polymeric MN platforms were evaluated in vitro and in vivo for transdermal delivery of a therapeutically relevant monoclonal antibody, bevacizumab. This thesis provides significant evidence to support polymeric MN arrays, as minimally invasive intradermal and transdermal delivery platforms of biotherapeutic macromolecules. Focused input from key stakeholders, including: academia, industry, regulators, healthcare professionals and patients will be needed to ensure successful MN commercialisation

Enhancing the solubility of BCS class II drugs using mesoporous silica based formulations

Madi, Atif M. Abdelsaiad January 2016 (has links)
Over the last decade, the poor water solubility of new chemical entities has become a significant issue for the pharmaceutical industry. Adequate aqueous solubility levels are essential for drugs administered via the oral route to ensure appropriate therapeutic levels. Currently, different strategies have been developed to address the issue of poor aqueous solubility of drug compounds. Those strategies are based on either chemical or physical modifications where the later strategies are preferred to overcome the poor solubility issue. In this thesis, amorphous mesoporous silica particles were used as a novel platform that could function as a carrier, solubility enhancer and amorphous form stabiliser for the poorly water soluble drug, indomethacin (INM). The principal concept was to develop novel solid dispersions manufactured via several solvent-free methods using two grades of mesoporous silica particles. Materials were firstly examined for their thermal stability and crystallinity. PEGs polymers were investigated as potential solubiliser and carrier for INM particles in some hot melt extrusion (HME) studies. Polymer-free manufacturing techniques (supercritical fluid impregnation and ball milling) were employed to investigate the possibility of manufacturing amorphous solid dispersions with better dissolution performance. Moreover, it was possible to develop polymer-free extrudates with significantly improved dissolution rates using HME. All manufactured formulations showed a significant redaction in the crystalline contents and a significant enhancement in the dissolution performance relative to raw crystalline INM. The data obtained in this PhD thesis has shown that the incorporation of mesoporous silica carrier platforms may be successfully employed as an alternative approach to generating solid dispersions of amorphous drugs to enhance the solubility of poorly water soluble drugs.

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