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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.
1

A microgripper for single cell manipulation

Hermosilla, Belén Solano January 2008 (has links)
This thesis presents the development of an electrothermally actuated microgripper for the manipulation of cells and other biological particles. The microgripper has been fabricated using a combination of surface and bulk micromachining techniques in a three mask process. All of the fabrication details have been chosen to enable a tri-layer, polymer (SU8) - metal (Au) - polymer (SU8), membrane to be released from the substrate stress free and without the need for sacrificial layers. An actuator design, which completely eliminates the parasitic resistance of the cold arm, is presented. When compared to standard U-shaped actuators, it improves the thermal efficiency threefold. This enables larger displacements at lower voltages and temperatures. The microgripper is demonstrated in three different configurations: normally open mode, normally closed mode, and normally open/closed mode. It has-been modelled using two coupled analytical models - electrothermal and thermomechanical - which have been custom developed for this application. Unlike previously reported models, the electrothermal model presented here includes the heat exchange between hot and cold arms of the actuators that are separated by a small air gap. A detailed electrothermomechanical characterisation of selected devices has permitted the validation of the models (also performed using finite element analysis) and the assessment of device performance. The device testing includes electrical, deflection, and temperature measurements using infrared (IR) thermography, its use in polymeric actuators reported here for the first time. Successful manipulation experiments have been conducted in both air and liquid environments. Manipulation of live cells (mice oocytes) in a standard biomanipulation station has validated the microgripper as a complementary and unique tool for the single cell experiments that are to be conducted by future generations of biologists in the areas of human reproduction and stem cell research.
2

New insights into heme peroxisases : internediates and mechanisms

Gumiero, Andrea January 2011 (has links)
Heme peroxidises catalyse the H2O2-dependent oxidation of substrates in a two-step process, through formation of two oxy-ferryl intermediates known as Compound I and Compound II. Despite the considerable effort worldwide, important aspects about the reactivity of these enzymes are still to be clarified. Amongst all, the determination of the nature of the Fe-O bond in the oxy-ferryl intermediates, as well as the mechanism by which protons are delivered to the oxy-ferryl species during turnover, are of highest relevance. In this thesis, high resolution crystal structures of both Compound I and Compound II intermediates in two heme peroxidases, cytochrome c peroxidise (CcP) and ascorbate peroxidise (APX), are presented. In order to rule out the photoreduction arising from X-ray exposure during data collection, which causes alteration of ferryl intermediate structures, a multicrystal method has been employed. Results indicate that Compound I, with an Fe-O distance of 1.63 Å for CcP and 1.73 Å for APX, is consistent with an unprotonated oxy-ferryl species (FeIV=O), whereas Compound II, with an Fe-O bond length of 1.83 Å and 1.84 Å for CcP and APX respectively, is consistent with a protonated oxy-ferryl species (FeIV-OH). Also presented in this thesis is the 2.40 Å structure of resting ferric CcP at room temperature obtained, for the first time, by neutron crystallography. This study allowed to establish the location of individual, exchangeable hydrogen atoms thus revealing the protonation states of several key active site residues in the distal (Arg48, Trp51, His52) and proximal (His163, Trp191, Asp235) heme regions. This information was used to revise the reaction mechanism of heme peroxidises and also to infer a possible delivery pathway of protons during turnover. All together, these data not only clarify long-standing inconsistencies on the nature of the oxy-ferryl species, but they also provide new insights into The reaction mechanism of heme peroxidises and provide important information which May apply to other categories of heme enzymes such as the cytochromes P450 and NO synthases.
3

The role of adenylate cyclase-associated protein in higher plant development

Dimmock, Simon Andrew January 2005 (has links)
The Actin Cytoskeleton is essential for Eukaryotic life and is involved in a diverse range of cellular functions. Cyclase Associated Protein (CAP) was first identified in yeast as a regulator of the CYR1 Adenylate Cyclase. Subsequently CAP family members have been identified in every Eukaryotic kingdom and have also been implicated in the regulation of Actin dynamics. It has been proposed that the CAP family promotes the recycling of Actin monomers by cooperating with members of the Profilin and Actin Depolymerising Factor families. This study represents an attempt to investigate the function and developmental role of AtCAP1, an Arabidopsis member of the CAP family. Arabidopsis thaliana is widely used as a model for higher plant development due to its small sequenced genome and the availability of a wide variety of mutants. The elimination of AtCAP1 expression results in a distinct developmental phenotype. Early characteristics include the absence of the root hair collar, reduced root hair initiation and extension. Later onset phenotypes include reduced plant height and a severe reduction in pollen viability. In vivo studies of the CAP-deficient cytoskeleton reveal a distinct loss of fine filamentous Actin and the appearance of dense Actin aggregates. Cell expansion is also significantly reduced. The interaction between AtCAP1 and F-Actin is demonstrated in vitro by a biochemical interaction study and a filament bundling activity is suggested. The multimerisation of AtCAP1 and its interaction with other components of the Actin Cytoskeleton are demonstrated via Yeast Two Hybrid interactions. It is concluded that AtCAP1 is essential for the organisation of the plant cells F-Actin network and that this in turn is required for correct growth and development. It is hypothesised that AtCAP1 function is mediated by regulating the interaction between F-Actin and other Actin-interacting proteins.
4

Control and operation of a spinning disc reactor

Ghiasy, Dena January 2013 (has links)
The aim of the present research is to assess the control and operation of a Spinning Disc Reactor (SDR), carried out via four separate investigations. Firstly, the effect of equipment size reduction on control is studied by comparing the performance of a PID controller applied to simulated intensified and conventional processes. It was found that superior control performance in terms of Integral of Absolute Error (IAE) is achieved for the simulated intensified system. However, the results showed that intensified systems are more susceptible to disturbances and the controlled variable exhibits larger overshoots. Furthermore, the frequency response analysis of the two systems showed that the simulated intensified system has reduced stability margins. The second part of the research investigates the task of pH control in a SDR using a PID controller by means of simulation and experimental studies. The effectiveness of a disturbance observer (DO) and a pH characteriser to compensate for the severe pH system nonlinearity is also explored in detail. The experimental studies showed that a PID controller provides adequate setpoint tracking and disturbance rejection performances. However, sluggish transient responses prevailed and the effluent pH limit cycled around the setpoint. There were indications of unstable behaviour at lower flowrates, which implied more advanced control schemas may be required to adapt to various operating regions dictated by the complex thin film hydrodynamics. The addition of the DO scheme improved the control performance by reducing the limit cycles. In the third segment of the investigations, the potential of exploiting the disc rotational speed as a manipulated variable is assessed for the process of barium sulphate precipitation. A PI controller is successfully used to regulate the conductivity of the effluent stream by adjusting the disc rotational speed. The results are immensely encouraging and show that the disc speed may be used as an extra degree of freedom in control system design. Finally, the flow regimes and wave characteristics of thin liquid films produced in a SDR are investigated by means of a thermal imaging camera. The film hydrodynamics strongly affect the heat and mass transfer processes within the processing films, and thus the intensification aspects of SDRs. Therefore, effective control and operation of such units is significantly dependent on the knowledge of film hydrodynamics and the underlying impact of the operating parameters and the manipulated variables on a given process. The results provided an interesting insight and unveiled promising potentials for characterisation of thin liquid film flow and temperature profiles across the disc by means of thermographic techniques. The present study reveals both challenges and opportunities regarding the control aspects of SDRs. It is recommended that equipment design and process control need to be considered simultaneously during the early stages of the future developments. Furthermore, intensified sensors and advanced controllers may be required to achieve an optimum control capability. Currently, the control performance is inhibited by the lack of sufficient considerations during the SDR design and manufacturing stages, and also by the characteristics of the commercially available instrumentation.
5

Quantitative analysis of the regulation and trafficking of glucose transporters in 3T3-L1 adipocytes

Hodgson, Lorna Rebecca January 2013 (has links)
Glucose uptake into adipose tissue and skeletal muscle is enhanced by the insulin-mediated translocation of glucose transporters from intracellular membranes to the plasma membrane. Insulin primarily stimulates the redistribution of glucose transporter isoform 4 (GLUT4) from a specialised pool of GLUT4 storage vesicles (GSVs) to the cell surface. In addition, stimulation of cells with insulin initiates a modest translocation of the glucose transporter, GLUT I, from the endocytic network to the plasma membrane. GLUT4 is engaged in a slow but continual cycle between the plasma membrane and various intracellular compartments, including the endosomal network and GSVs. By contrast, despite sharing many sequence similarities with GLUT4, GLUT! recycles at a considerably higher rate, predominantly localising to the endosomes and is excluded from GSVs. Thus, the overall aim of this thesis was to examine the trafficking of GLUT4 in 3T3-L I adipocytes and explore the mechanism ofGLUT4 and GLUTI segregation and the proteins involved in the sorting of GLUT4 into GSVs. A correlative light electron microscopy (CLEM) internalisation assay was developed in order to study the trafficking itinerary of internalising GLUT4 at the ultrastructural level and identify potential proteins involved in the sorting of the transporter. Using this technique, GLUT4 was shown to transit through a variety of different intracellular compartments, before concentrating in a cluster of small insulin-responsive vesicles, approximately 50-100nm in diameter. GLUT4 sorting, translocation and internalisation is mediated by a series of Rab proteins and Rab-binding effector molecules. Previous studies have demonstrated that Rab 14 is a key protein involved in the exocytosis of GLUT4 vesicles in skeletal muscle. Confocal and electron microscopy revealed that over expression of constitutively-active Rab 14 (RabI4Q70L) in 3T3-LI adipocytes resulted in the development of enlarged ' ring-like' early endosomal structures, positive for both endogenous and internalised GLUT4. Furthermore, the CLEM assay demonstrated that knockdown of Rab 14 disrupted GLUT4 transit out of the early endosomes and into the GSVs, thus implicating Rabl4 in the endosomal sorting of GLUT4 prior to the packaging of the transporter into GSVs. The process of GLUT I and GLUT4 segregation was examined using a GLUT4 mutant protein (HA.GLUT4.SQV) containing the C-terminal PDZ ligand of GLUT 1 (DSQV), which has previously been shown to interact with SNX27 in HeLa cells during the recycling of GLUTI back to the cell surface. The HA.GLUT4.SQV mutant trafficked in a manner similar to GLUT I, redistributing to the endosomal network and recycling at an increased rate. Taken together, the data suggests that SNX27 may be responsible for the fast recycling of GLUT 1 in 3T3-LI adipocytes and the interaction of the PDZ ligand located at the C-terminus of GLUT 1 with the PDZ domain ofSNX27 is responsible for the segregation of GLUT 1 away from GLUT4. Finally, the trafficking of the Rab 1 1 effector protein, Rip 11 , which has previously been shown to be important in the insulin-mediated fusion of GLUT4 vesicles, was examined. Rip II predominantly localised to the endocytic network and co-localised with GLUT4 on recycling endosomes. Insulin stimulated the accumulation of Rip II at the cell surface by inhibiting the internalisation of Rip II in a PI 3-kinase dependent and Akt independent manner.
6

Functional characterisation of expressed ɑ9ɑ10 nicotinic acetylcholine receptor channels

Bere Harding, Court Edmund de la January 2013 (has links)
The primary aim of this project was to investigate receptor and ion channel coupling in a heterologous expression system, the GH4C1 cell line. The primary focus was the interaction between the a9alO nicotinic acetylcholine receptor (nAChR) and the small conductance calcium (Ca2+) -sensitive potassium (K+) channel, subtype 2 (SK2). In both inner hair cells (rnCs) and outer hair' cells (OHCs), the a9alO nAChR and the SK2 channel are spatially and functionally associated, or 'coupled'. Both the a9alO nAChR and SK2 were transiently expressed in vitro and functionally evaluated. The channels were then coexpressed and the interaction between the two was examined electrophysiologically. Channel blockers, receptor antagonists and an intracellular Ca2+ chelator were used, in conjunction with electrophysiological methods. No evidence of the interaction between the a9alO nAChR and the SK2 channel was found. Prior to the investigation of the coupling interaction, the endogenous currents of the GH4C1 cells were also assessed. Other experiments were aimed at recapitulating the interaction of an L-type Ca2+ channel (LTCC) and the SK2 channel in vitro, which also occurs in neonatal IHCs. These channels were not found to associate, in the cell line. Additionally, the direct effect of ryanodine upon the SK2 channel was assessed in Human embryonic kidney (HEK) 293 cells, and found to have no effect.
7

Protein-protein interactions in GnRH receptor signalling

Davidson, L. January 2005 (has links)
Human embryonic kidney (HEK) 293 cells, stably expressing the rat GnRH receptor were used to investigate the mechanism of ERK activation by GnRH.  ERK activation was found to be dependent on cell adhesion to the extracellular matrix, and required an intact actin cytoskeleton. Through the use of specific pharmacological inhibitors and expression of dominant negative cDNA constructs, ERK activation was found to be mediated by the Rho family GTPase Rac1, and the non-receptor tyrosine kinases Src and focal adhesion kinase (FAK). FAK was found to function as a tyrosine phosphorylated scaffold upon which components of the ERK cascade assembled. Having established a role for Src in the activation of ERK, a proteomics study was undertaken to identify novel Src binding proteins that may be involved in the regulation of GnRH receptor signalling. Through a combination of immune-precipitation, two-dimensional gel electrophoresis, and matrix assisted laser desorption ionisation – time of flight mass spectrometry, Src was found to associate with the lipid kinase diacylglycerol kinase zeta (DGKζ). This interaction was found to be required for GnRH to stimulate DGKζ enzyme activity. By phosphorylating the second messenger molecule diacylglycerol to produce phosphatidic acid, DGKζ may play an important role in regulating GnRH receptor signalling. In this thesis, a potential mechanism of ERK activation is described for the GnRH receptor, with Src playing a key role in this pathway. In addition, Src was found to be involved in the activation of DGKζ, and is therefore implicated in the regulation of diacylglycerol signalling. This is the first report of an interaction between Src and DGKζ.
8

The lectin pathway of complement activation in cerebral ischaemia and reperfusion injury

Chrysanthou, Elvina January 2013 (has links)
The complement system constitutes a critical component of the innate immune response. The lectin pathway is one of the three activation pathways of the complement activation cascade that can recognise and respond to structures on oxygen deprived cells and contribute to ischaemia and reperfusion injury (IRI). Cerebral IRI mediated inflammation is known to be responsible for secondary damage in the penumbra region surrounding the initial area of infarct and the prevention of IRI-mediated secondary damage provides an attractive target for therapeutic intervention. Mannose binding lectin associated serine protease 2 (MASP-2) is the key effector enzyme of the lectin pathway, since depletion of this enzyme completely ablates lectin pathway function or activity. This study assessed the impact of MASP-2 deficiency on cerebral IRI and to what extent MASP-2 targeting can reduce the secondary inflammatory damage following an ischaemic insult. The 3 vessel occlusion (3-VO) model of stroke was found to be the most appropriate model to use in this study, as it was shown to have a lower degree of variability than the middle cerebral artery occlusion (MCAO) stroke model. TTC staining revealed that MASP-2 -/- mice were significantly protected from cerebral damage, showing statistically significant smaller infarct sizes when compared to age and sex matched wild type controls. MASP-2 deficient mice showed reduced C3 deposition and a lower degree of astrocytic activation in brain sections from mice undergoing 3-VO and showed higher mRNA abundance of anti-inflammatory mediators (such as IL-10) and lower abundance of pro-inflammatory mediators (such as MIP-2) when compared to wild type control mice. Subsequently, a recombinant inhibitory anti-MASP-2 antibody, AbD04211, a murine specific MASP-2 inhibitor, was assessed for the therapeutic utility of MASP-2 inhibition in the 3-VO cerebral IRI model of stroke. The results revealed that the use of MASP-2 inhibitors at a dose of 5mg/kg of body weight achieved a statistically significant protective effect, with infarct sizes reduced by up to 30% in the anti-MASP-2 treated animals.
9

The effect of modified nucleosides on DNA duplex and triplex stability

Vadhia, Sunil Jayantilal January 2007 (has links)
To date, the single most effective method of improving base pairing affinity and binding of PCR primers, fluorescent probes and triplex forming oligonucleotides (TFO) ',h,';I,,+ destabilising mismatch base pairs has been the incorporation of modified nucleoside into these oligonucleotide structures. As a consequence, significant improvements have been made in the areas of human identity testing, forensic science analysis, pharmacogenetics/pharmacogenomics and anti-gene therapy. In an effort to improve the stability of these DNA duplexes and DNA triplexes further, we have synthesised and incorporated a series of cytosine, 7-deaza adenine, thymine and 3Hfuro-[ 2, 3-d] pyrimidin-2-one base analogues. By using a combination of UV melting analysis and fluorescence melting experiments, we have demonstrated that each of the base analogues gives a significantly higher base pairing affinity and binding selectivity when compared to their corresponding natural base. In addition, we have also incorporated these base analogues into PCR primers (7-deaza adenine) and fluorescent probe sequences (cytosine, 7-deaza adenine, thymine and 3H-furo-[2, 3-d] pyrimidin-2-one). Results from peR experiments show that the 7-deaza adenine base analogue does not adversely the functioning of Taq polymerase during amplification and therefore at the very least behaves similarly to adenine within a PCR primer sequence. In addition, all of the tLUlJre~jCel1tly labelled base analogues (cytosine, 7-deaza adenine, thymine and 3H-furo-[2, pyrimidin-2-one) show a significantly higher level ofbase pairing affinity and binding selectivity a complementary target sequence over a mismatched sequence.
10

Further characterisation of substrate, inhibitor and ancillary protein specificity of MCT1, MCT2, MCT4 and MCT6

Ovens, Matthew James January 2010 (has links)
The MonoCarboxylate Transporter (MCT) family of transmembrane proteins contain 14 members of which 6 have been functionally characterized. Of these characterised MCTs only MCTs 1-4 have been shown to transport lactate. These MCTs also facilitate the movement of pyruvate and ketone bodies across the plasma membrane (PM) in cotransport with a proton. For trafficking to and function at the PM MCTl, MCT3 and MCT4 require association with the monotopic ancillary glycoprotein basigin whereas MCT2 prefers association with embigin. This thesis has investigated the sensitivity of MCTl, MCT2 and MCT4 to the highly potent and selective MCTI inhibitor, ARC155858, discovered by AstraZeneca. Chimeras of MCTI and MCT4 were constructed and expressed in Xenopus laevis oocytes for transport studies to determine their inhibitor sensitivity. These identified a region between transmembrane domains (TMs) 7 and 10 of MCTI with which AR-C155858 binds from the cytoplasmic side. ARC155858 was shown to inhibit MCT2 but sensitivity was found to be dependent on the ancillary protein with which it is associated. Co-expression with embigin decreased the sensitivity of MCT2, but not MCTl, to AR-CI55858. The MCT C-terminus was shown to playa role in the interaction between MCT and ancillary protein which is secondary to interactions between the TM of the ancillary protein and TMs3 and 6 of the MCT. Additional studies were performed to characterise the substrate specificity of the orphan transporter, MCT6. Initial work suggested that products of pyruvate decarboxylation or polymerisation will provide lead compounds in the continuing search for the physiological substrate of MCT6, with formate another potential substrate. During this work it was also discovered that MCTI can catalyse the transport of specific dicarboxylates at low pH.

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