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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.

Computational studies of protein dynamics and drug resistance

Mohammedali, Hani January 2013 (has links)
Berezovsky et al.'s closed loop folding hypothesis suggests that the locks of closed loops (i.e. the ends of the interacting loop) of length ~25-35 residues interact through hydrophobic interactions and that this is a vital event in protein folding. Here we investigate a possible link between the lock residues and drug resistance. The hypothesis is that drug resistance can be limited if the drug binds to the high connectivity regions such as the lock regions. To follow this perspective, the work has moved on to develop an additional method for determining closed loops by using the X-ray crystallographic B-factors as a measure of flexibility. From previous kinetics studies, by using the correlation of folding rate with contact metrics evaluated over the locks of closed loops and their neighbours, we observed that the conservation, particularly maximum proportion is possibly the best method for determining closed loops; we also found that the closed loop hypothesis requires modification to include the neighbours of the lock residues. Berezovsky et al.'s hypothesis has been evaluated using literature results from time-resolved dynamic non-radiative excitation energy transfer measurements for bovine pancreatic ribonuclease A protein (RNase A), as studied by Haas' group. An analysis of the experimental data and molecular dynamics (MD) data in the light of Berezovsky et al.'s hypothesis shows that the MD results are consistent with the experiment results. To investigate the lock residues to look for any additional properties that may help in drug design, we investigated the lock residues of HIV reverse transcriptase and protease by analysing the positions of lock residues vis a vis positions of drug resistant mutations in both enzymes, focussing on the closed loops in accordance to the Berezovsky et al.'s hypothesis. Our results indicate that if these lock residues were to be targeted by drugs, they may be less likely to generate resistance mutations.

Functional regulation of the Discs Large Tumour Suppressor by phosphorylation

Narayan, Nisha January 2008 (has links)
The human homologue of the Drosophila Discs Large Tumour suppressor, hDlg, has been subject to various speculations concerning its role in the cell, with studies indicating roles in establishing and maintaining cellular polarity, as well as in controlling cell proliferation. The biochemical mechanism by which it might act in executing either function have, however, remained ambiguous. In this study we show that phosphorylation is a major posttranslational modification of the protein, affecting both location and function. We show that hDlg is phosphorylated both by the MAPKs and the CDKs, and both groups of kinases affect different aspects of the protein's behaviour. Post osmotic shock, the phosphorylation of hDlg by JNK leads to its accumulation in vesicular structures which we identify as endosomes, while its phosphorylation by the p38 MAPK in addition to relocating it to sites of cell-cell contact, also makes it more susceptible to degradation by the HPV18 E6 oncoprotein. Secondly, we show that hDlg is differentially regulated during the cell cycle, with each stage of the cell cycle leading to a different localisation of the protein, including its accumulation at the mitotic spindle in the M phase, as well as at the midbody during cytokinesis. We show also that the protein is phosphorylated by the Cyclin Dependent Kinases (CDK) 1 and 2, in a cell-cycle dependent manner on two sites - serine 158 and serine 442, and that these phosphorylations render the protein more stable and less susceptible to ubiquitination. Finally we show that hDig phosphorylated on serine 158 and on serine 442 is largely nuclear, and that both the HPV18 E6 and the HPV16 E6 oncoproteins, target this nuclear form for degradation. These findings help us understand the processes that regulate hDlg and how these modifications of the protein might contribute to its growth-regulatory function in the cell.

The role of the neuregulins in the nucleus

Wang, Ming January 2011 (has links)
The neuregulins (NRGs) are ligands for receptor tyrosine kinases of the ErbB family. The NRG gene family consists of four members, NRG 1- 4 encoding different isoforms due to alternative splicing. NRG signalling has been implicated in normal development and in the pathology of many diseases. Using immunohistochemical staining of tissue arrays, we detected that NRG 1 a and 1 ~ localised to the cell nuclei of a range of normal and human cancer tissues. The ~3 isoform of NRG 1 localised to two subnuclear compartments: nucleoli and spliceosomes. We tagged NRG 1 ~3 with photoactivateable GFP and demonstrated that the fusion protein re-Iocalised from nucleoli to spliceosomes over a ninety minute period. Using wild type NRG 1 ~3 and its two mutants which localised exclusively to spliceosomes or to nuc1eoH, we explored the possible functions of intranuclear NRG 1 ~3 in each of these compartments separately. We showed using an array capable of detecting 42 receptor tyrosine kinases that wild type NRG 1 and a mutant exclusively local ising to spliceosomes increased phosphorylation and/or expression of the ErbB4 and ErbB2 receptors. Using a transcriptomic analysis the same two constructs induced expression of messenger RNA of Heat Shock Protein 70B' and we confirmed its increased expression at the protein level using western blot analysis. This data supports the hypothesis that intranuclear NRGs could activate receptor signalling and alter gene expression when localised in spliceosomes.

The use of cell-penetrating peptides to deliver antibody fragments into the cell

Cheung, Jason W. C. January 2013 (has links)
Enabling the entry of therapeutic antibodies into cells for the modulation of intracellular targets has the potential to greatly expand the treatment options for diseases, such as cancer. A class of promising delivery vehicles for therapeutic antibodies is the cell-penetrating peptides (CPP), which are capable of entering cells spontaneously and promote the cellular uptake of conjugated biomolecules, such as antibodies. Demonstration of the CPP-mediated delivery of a functional protein was performed using the well-established example of p27, a cyelin-dependent kinase inhibitor, fused to the Tat delivery domain of the human immunodeficiency virus (HIV). Concomitant with established data, the Tat-p27 fusion protein entered cells, induced G1 cell cycle arrest in MCF-7 cells and induced HepG2 cells to undergo cell scattering and cytoskeletal alterations.

Ion uptake and electrical potentials in plants

Spanswick, R. M. January 1964 (has links)
No description available.

The formation and function of putative IgLON cis heterodimeric complexes

McNamee, Christine Jane January 2008 (has links)
During development individual neurons have to reach specific locations in the embryo to form a connected nervous system. Molecular complexes form between receptors expressed on the surface of the developing axon and molecular guidance cues in the extracellular environment, to guide the neuron to the correct location. Synapses then connect the axons with their target location. This thesis describes the interactions between the IgLON family of cell adhesion molecules and how these interactions affect their function during neuronal development. There are four members of the IgLON family in chick, namely LAMP, OBCAM, CEPU-I and Neurotractin. They are highly glycosylated proteins, predominantly anchored by a GPI anchor to the extracellular surface of the cell membrane. Initially, the comparative strengths of homophilic and heterophilic trans interactions of CEPU-I, OBCAM and LAMP were established. Generally trans heterophilic interactions within the family have a higher affinity than homophilic interactions, LAMP having the highest heterophilic affinity for CEPU and OBCAM. The lowest affinity of all the trans interactions tested was the LAMP homophilic interaction. Data is presented to suggest IgLONs also interact in cis to form heterodimeric complexes or Diglons. These putative Diglons affect the trans binding affinity for IgLON-FC recombinant proteins, possibly due to a conformational change altering the availability of the IgLON binding site. Biochemical and imaging studies provided additional physical evidence for the Diglon complex. Inhibition of the initiation of neurite outgrowth from chick forebrain neurons was found to be dependent on the presence of two IgLONs in the extracellular environment to suggest formation of putative Diglon heterodimeric complexes facilitates IgLONs to function as negative axon guidance cues. IgLONs have no cytoplasmic domain so are reliant on being part of a molecular complex on the surface of the cell membrane for signal transduction. Assays measuring neurite outgrowth from chick forebrain neurons suggested signal transduction for Diglons is linked to trimeric Go/i membrane proteins and/or to Rho GTPases.

Cellular Roles for Proteins Linked to Phosphatidylinositol (3,5)-bisphosphate Metabolism

Whittingham, Jane January 2008 (has links)
The phosphoinositide lipids Ptdlns(3)P and Ptdlns(3,5)P2 play important roles on the endocytic pathway. Ptdlns(3)P localises to early endosomes and multivesicular bodies (MVBs) and is proposed to recruit proteins involved in fusion of early endosomes and internalisation of ubiquitinated receptors. Ptdlns(3,5)P2 is proposed to be involved in terminal maturation of Iysosomes and endosome to Golgi transport, but the precise role of this lipid in mammalian cells remains unclear. Studies in yeast have identified various proteins associated with Ptdlns(3,5)P2 metabolism, for which mammalian homologues have been found. These are the Ptdlns(3)P5- kinase Fab1/PIKfyve, Fig4 a 5-phosphatase that dephosphorylates Ptdlns(3,5)P2, Vac14 which has been shown to act as an upstream activator of PIKfyve, and WIPI-2/Svp1 a putative downstream effector of Ptdlns(3,5)P2. In this study I have further characterised three of these proteins and examined the cellular roles of all four with respect to a variety of trafficking pathways in which Ptdlns(3,5)P2 has been implicated. siRNA was used to examine the knockdown phenotypes of each of these proteins. Furthermore, I directly compared for the first time the effects of knockdown of PIKfyve, with acute pharmacological inhibition of its enzymatic activity. Loss of PIKfyve activity caused a failure in the retrieval of a variety of different cargos to the trans-Golgi network (TGN), including mannose-6-phosphate receptors, responsible for efficient delivery of lysosomal enzymes, and the TGN resident protein TGN46, leading to their accumulation in dispersed punctae. A failure in tyrosine kinase receptor downregulation was also observed following combined knockdown of PIKfyve with either Vac14 or Fig4 or following pharmacological inhibition of PIKfyve. PIKfyve knockdown alone had no effect, suggesting a low threshold of Ptdlns(3,5)P2 is necessary and sufficient for this pathway. Svp1 is the best characterised Ptdlns(3,5)P2 effector in yeast and is also an autophagy-related gene (Atg18), thereby implicating Ptdlns(3,5)P2 in this process. A family of putative mammalian Svp1 homologues have been identified, known as the WIPI family. I investigated the role of Ptdlns(3,5)P2 and WIPI-2 in mammalian autophagy. By monitoring formation of the autophagosome marker GFP-LC3 II, PIKfyve and WIPI-2 were found to have opposite effects. Furthermore, WIPI-2 redistributes to punctate structures upon induction of autophagy, which partially colocalise with autophagosome markers, in a manner dependent not on PIKfyve but on PI(3)-kinase activity. The evidence presented suggests that Ptdlns(3,5)P2 may playa role in mediating the maturation of a subset of MVBs, leading to swelling of endosomal compartments and rendering the MVBllate endosome or autophagosomes refractory to fusion with the lysosome in cells depleted of PIKfyve.

The Biochemistry of the Latter Stages ofPeptidoglycan Biosynthesis and Modification

Clarke, Thomas Brian January 2008 (has links)
Bacterial cell integrity is maintained by peptidoglycan, a rigid polymer of alternating sugar residues (N-acetlyglucosamine and N-acetylmuramic acid) cross-linked by short peptide stems. Peptidoglycan biosynthesis is a complex three-stage process, under tight spatial and temporal control, with multiple steps exploited as antibiotic targets. The final stage occurs in the periplasm and involves the polymerisation of lipidlI (the final monomeric peptidoglycan precursor) by penicillin-binding proteins, and the attachment of cell surface proteins, containing a C-terminal LPXTG motif, by the sortase family of enzymes. The biochemical characterisation of penicillin-binding proteins and sortases has been mainly limited to investigating interactions with inhibitors and peptide analogues, due to the unavailability of the natural peptidoglycan precursors. Work presented in this thesis describes the development of protocols for the synthesis of both cytoplasmic and lipid-linked peptidoglycan intermediates. It was possible to produce approximately 8S mg of the final cytoplasmic precursor (UDP-MurNAcpentpeptide) in a single-pot incubation. UDP-MurNAc-pentapeptide was then converted to lipidII using a preparation of M jlavlls membranes. Due to the presence of cross~ linking in the peptidoglycan of many Gram-positives, a chemo-enzymatic procedure was used to attach branching amino acids. L-Ala and L-Ala-L-Ala amino acid branches, were attached to the e-amino group of L-Lys in position three of the UDP-MurNAcpentapeptide. These branched derivatives were also converted to lipidII. These substrates were then used to study the enzymology of a variety of pencillin-binding proteins. The transpeptidase activity of two high-molecular weight penicillin-binding proteins (S. aureus PBP2' and E.faecalis PBPS), with intrinsic'low affinity to ~-lactams, was investigated. However, no transpeptidase activity was detected with any monomer substrate. The requirement for the prepolymerisation of the glycan backbone prior to transpeptidation was investigated using two types of polymeric substrate (secreted uncross-linked peptidoglycan polymers and lipidII treated with a monofunctional transglycosylase to polymerise the glycan backbone of lipidII), however, no transpeptidase activity was detected using either of these polymeric substrates. The DDcarboxypeptidase and endopeptidase activity of a low-molecular weight penicillinbinding protein (E. coli PBP4) was investigated with a variety of peptidoglycan fragments. The importance of a small pocket at the base of the E. coli PBP4 transpeptidase active site for substrate recognition was demonstrated by site-direct mutagenesis. The differences in activity of these related classes of enzyme highlights the gap in the understanding of substrate recognition by the transpeptidase domain of pencillin-binding proteins. Sortase enzymes covalently attach proteins to peptidoglycan. Proteins are covalently linked to branched lipidlI, which is subsequently polymerised in to the peptidoglycan polymer. S. pnellmoniae SortaseA was crytsallised, and a fluorescence resonance energy transfer assay was used to detect transpeptidation. This work provides the basis for full structural and biochemical characterisation of Sortase mediated transpeptidation.

E-cadherin and cell adhesion : a role in architecture andfunction in the pancreatic islet

Rogers, Gareth James January 2007 (has links)
The insulin-secreting p-cells of the pancreas play a crucial role in blood glucose homeostasis. An elevation in blood glucose triggers the release of insulin into the systemic circulation, restoring euglycaemia. Diabetes mellitus arises when there is no longer an adequate supply of insulin available to regulate the concentration of blood glucose effectively. Beta-cells reside within cellular aggregates called the islets of Langerhans, the three-dimensional anatomy of which is crucial to their secretory function. It is well established that increased cell proximity augments glucose-evoked insulin secretion and several studies have highlighted the importance of homologous p-cell contacts within the islet configuration. It has been suggested that the improved secretory function is the result of communication via gap junctions and/or paracrine mediators between adjacent p-cells. However, there may be other molecules that are capable of modulating p-cell function in response to cell contact and recent work has provided evidence for a role for the Ca2+-dependent cell adhesion protein, E-cadherin. Using MIN6 pseudoislets as a model system, this study demonstrates that E-cadherin is a key regulator of p-cell function and is more than just an adhesion molecule, holding the three-dimensional islet together. It is not only important for the homotypic coupling of p-cells but also affects their secretory activity. Insulin secretion experiments demonstrate that the treatment of pre-formed pseudoislets with an immuno-neutralising E-cadherin antibody markedly reduces glucose-evoked insulin release. Furthermore, Fura-2 microfluorimtery shows that the synchronous glucoseinduced [Ca21i oscillations characteristic of pseudoislets are lost in the absence of Ecadherin- based cell contacts. Attenuation of fluorescent dye transfer between cells in the absence of cadherin ligation indicates a lack of functional gap junctions under such conditions providing evidence for an interaction between gap junctions and Ecadherin in p-cells. Overall these findings imply that E-cadherin has an important role in the regulation of intercellular communication between p-cells within the islet, an effect which appears to be gap junction dependent. The results of this study have potential implications for the in vitro generation of islets for future transplantation therapy.

Structure and properties ofa metallothionein fromwheat germ : Towards a structure/function relationship for plant metallothioneins

Leszczyszyn, Oksana Iryna January 2008 (has links)
Metallothioneins are a superfamily of small, cysteine-rich, metal-binding proteins, which have been the subject of intense scientific interest since their discovery in the late 1950s. Over the last decade, the advancement of genome sequencing, microarray and high-throughput protein identification techniques has resulted in an exponential increase in the number of plant MT sequences in protein and translated nucleotide databases. These studies show that plant metallothioneins display significant variation in sequence and are discretely expressed during various developmental stages and in different organs. Therefore, it is likely that plant metallothioneins carry out compartmentalised functions for which they possess specific properties. However, there is a relative paucity of academic literature on the structure and biochemical properties of plant MTs and this is a void that needs to be filled before we can establish structure/property and structure/function relationships for these proteins. This research has focussed on a comprehensive characterisation of the solution structure and metal binding dynamics of the wheat Eel/II metallothionein using a range of analytical and biophysical techniques. Mass spectrometry and nuclear magnetic resonance studies have identified six zinc ions in two distinct domains with hitherto unprecedented stoichiometries for MTs: Zn2Cys6 and Zn4CysllHis2. Structure calculations revealed that in addition to these unprecedented stoichiometries, the individual Ee domains possess unique structural features not previously reported in MT literature. In Domain 1, the binding of two zinc ions in a binuclear cluster was observed. When compared to Domain 2, the binuclear cluster displayed remarkable stability towards Wand EDTA attack. In Domain 2 an isolated binding site with stoichiometry ZnCys2His2 was observed, which is proposed to confer a well-defined structure for this domain. However, disruption of this site through mutation or cadmium binding abolishes ordered structure along with the defined binding of six zinc ions. These novel structural features confer distinct backbone and metal dynamic properties in each domain, and are most likely to have a functional significance. A separate avenue of investigation identified a potential role for Ee in zinc donation. A re-examination of previously published literature on the expression levels of Ee mRNA and protein demonstrated that it is conspicuously accumulated during desiccation, which suggests that it is perfectly poised to satisfy the microquota of zinc-dependent sites during rehydration processes. In conclusion, this research has made significant advances in the understanding of the structure/function relationship of the wheat Eel/II metallothionein.

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