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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.
31

Advances in magnetic resonance phase-contrast velocity mapping

Giese, Daniel January 2012 (has links)
Phase-contrast MRI is used to assess functional flow parameters of the cardiovascular system. Long scan times of three-dimensional flow encoded MRI in particular have, however, hampered the acceptance of the method in a clinical routine setting. Furthermore, concerns have recently been raised about the limited accuracy of the technique. The present work focuses on these two limitations. Phase unsteadiness in fast phase-contrast sequences and its effect on flow quantitation is analysed and a modification to the sequence design is proposed. In a next step, system imperfections resulting from uncompensated eddy currents are studied using dynamic magnetic field monitoring demonstrating that mechanical vibrations can have a large impact on flow quantification. Approaches for background phase offset correction based on field monitoring data are discussed. To address the long scan times, dual slice excitation and spatiotemporally constrained image reconstruction are implemented to enable highly undersampled phase-contrast measurements. The successful application of these methods to three-dimensional phase-contrast MRI of congenital heart disease demonstrates clinical feasibility and robustness with great potential for future adaptation of the method in a routine setting.
32

CD46-mediated signals in the regulation of T-cell cytokine production and intestinal wound healing

Cardone, John January 2012 (has links)
Complement is a key part of innate immunity and vital in the first line of defence against invading pathogens. The understanding of the role of complement in the host’s immune defence has undergone its first major change when it became clear that complement is also required for the optimal induction and function of adaptive immunity, particularly B cell activation and T cell effector responses. It is now also becoming increasingly clear that complement plays a key role in the contraction of T cell responses and thus, by definition, in immune homeostasis. The first part of this thesis focuses on such least understood role of complement. Firstly, evidence is reported for a novel mechanism by which the complement regulatory protein CD46 regulates the key T helper type 1 cytokine IFN-y and the canonical immunosuppressive cytokine IL-10. The production of both IFN-y and IL-10 is shown to be temporally regulated by TCR and CD46 signals in CD4+ T cells in an IL-2-dependent fashion and is shown to be defective in rheumatoid arthritis patients. The molecular pathways linked to the CD46-induced switch of TR! cells are largely undefined and were investigated. Gene array studies highlighted the asparagine endopeptidase (AEP) as a candidate molecule involved in the regulation of the switch of TH1 cells towards IL-10 production. The CD46-driven IFN-y secretion, but not the intracellular expression, was found to be substantially reduced by AEP inhibition; IL-10 production was consequently affected confirming the importance of IFN-y signalling in the CD46-induced "switch" mechanism. The cytokines produced by T cells activated via CD46 may also be important in the crosstalk with surrounding tissues. Here the functional consequences of CD46 signals were further investigated in the context of intestinal epithelial cells to allow the discrimination of the effects of CD46 signals in the two different cells types. A novel role for CD46 in the promotion of cellular proliferation and wound healing was reported.
33

Immune regulatory role of human CD141+ dendritic cells in humanised models of alloimmunity

Ali, Niwa January 2013 (has links)
The study of complex biological processes at a detailed level of understanding requires an in vivo model where pathological immunity can be experimentally introduced and manipulated. In human studies however, due to obvious ethical and technical constraints, these in vivo mechanisms cannot be studied. The development of humanised models has been an important advancement to aid in the effective translation of medical research into human disease mechanisms and pathogenesis into clinical application. This thesis aims to investigate the use of these model systems to explore the role of human T-cells and dendritic cells (DCs) in the regulation of human alloimmunity. The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; “Hu-PBMC mice”) are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2R!null), notably the NOD-scid IL-2R!null (NSG) and BALB/c-Rag2null IL-2R!null (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. Engrafted human lymphocyte populations in the peripheral blood, spleens,lymph nodes and bone marrow of these mice were investigated. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45+ compartment and higher engraftment levels of CD3+T-cells were observed in NSG mice, which may explain the faster rate of GvHD II development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of Tcell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (TEM) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting TEMcell driven GvHD. The second part of thesis investigates the immune regulatory role of human skin DC subsets both in vitro and in vivo. A better understanding of human skin immune homeostasis and its regulation by specialized subsets of tissue residing immune sentinels is required. Here, we identify an immunoregulatory tissue resident DC in dermis of human skin characterized by surface expression of CD141, CD14 and constitutive IL-10 secretion (CD141+ DDCs). CD141+ DDCs possess lymph node migratory capacity, induce T cell hypo-responsiveness, cross-present self-antigens to auto-reactive T-cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D3 promotes certain phenotypic and functional properties of tissue resident CD141+ DDCs from human blood DCs. These CD141+ DDC-like cells can be generated in vitro and once transferred in vivo have the capacity to inhibit xenograft versus host disease and tumour alloimmunity. These findings suggest that CD141+ DDCs play an essential role in maintenance of skin homeostasis and in the regulation of both systemic and tumour alloimmunity. Finally, vitamin D3-induced CD141+ DDC-like cells have potential clinical use for their capacity to induce immune tolerance.
34

Development of novel macrophage imaging agents

O'Neill, Alexander January 2013 (has links)
Macrophages are an important class of cell involved in host defence and homeostasis that are able to alter their phenotype in response to their surroundings and affect a wide range of biological processes. These properties make them critical to host function in normal and disease settings, leading to their utility as targets to inform upon functional status of an organ/tissue as well as for therapeutic intervention. Thus the development of macrophage-specific imaging agents has potential clinical applications in the diagnosis and measuring response to treatment of disease. This thesis sets forth the development of novel antibody-based nuclear imaging agents 99mTc-SER4, 64Cu-NOTA-SER4, and 99mTc-ED3 targeting the antigen sialoadhesin and 1111n-DTPA-ED2 targeting CD163, of which both antigens are macrophage-restricted. All the agents described herein exhibit tracking to endogenous sialoadhesin-expressing macrophage populations with exceptionally fast blood clearance times. In a heterotopic cardiac transplantation model of acute rejection the tracer 99mTc-SER4 demonstrated enhanced targeting to the rejected organ, whilst following recovery to localised bone marrow radioablation preliminary data showed increased 64Cu-NOTA­SER4 in the ablated bone marrow. The tracers 1111n-DTPA-ED2 and 99mTc-ED3 also demonstrated the feasibility of imaging distinct macrophage populations simultaneously. In this thesis, we present sialoadhesin as a candidate target for the imaging of macrophage populations in vivo, with the potential for translation into the clinic.
35

The role of human CD23 in lgE homeostasis & allergic disease

Cooper, Ali January 2013 (has links)
CD23, the low affinity receptor for IgE on B cells, exists in membrane and soluble forms. CD23 also binds CD21 with a distinct binding site to IgE. Soluble CD23 (sCD23) fragments are released from trimeric membrane CD23 (mCD23) by the endogenous metalloprotease, ADAM10. It has been suggested that trimeric sCD23 fragments can co-ligate membrane IgE (mIgE) and membrane CD21 (mCD21) on the surface of human B cells, in a similar way to C3d-antigen complexes and mIgM, to upregulate IgE synthesis and provoke allergic responses. To test this hypothesis, purified tonsil B cells were stimulated with IL-4 and anti-CD40 to induce class switching to IgE in vitro. mCD23 was up-regulated and sCD23 accumulated in the medium prior to IgE synthesis. IL-10 and IL-21 were shown to enhance IgE synthesis by increasing cell division and plasma cell differentiation. siRNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an ADAM10 inhibitor, GI254023X, were shown to suppress IgE synthesis. Addition of a recombinant trimeric sCD23, triCD23, enhanced IgE synthesis. This occurred even when endogenous mCD23 was protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. triCD23 was shown to bind to cells coexpressing mIgE and mCD21 and caused capping of these proteins on the B cell membrane. triCD23-mediated up-regulation of IgE secretion and capping of mCD21 was blocked in the presence of an anti-CD21 monoclonal antibody. Up-regulation of IgE secretion by sCD23 occurred after class switch recombination and the effects were isotype-specific. Together, these results suggest that mIgE and mCD21 co-operate in the sCD23-mediated positive regulation of IgE synthesis by IgEcommitted B cells. These results have improved our understanding of the regulation of IgE in human B cells and provide evidence for sCD23 as a potential therapeutic target in allergy and asthma.
36

Protein engineering and characterization of a stable trimeric form of CD23 and a fluorescent IgE biosensor

Kao, Wen-Pin January 2013 (has links)
Immunoglobulin E (IgE) plays a critical role in allergic diseases such as asthma and atopic dermatitis. Cross-linking of IgE bound to its high affinity receptor, FceRI, by allergen on mast cells and basophils results in the release of inflammatory mediators and induces allergic reactions. IgE can also form a complex with its low affinity receptor, CD23, and CD21 on the B cell surface to regulate IgE synthesis. Hence, there are two possible strategies in the therapy of IgE-mediated disease: blocking IgE binding to FceRI and inhibiting IgE production. Crystal log raphic and FRET studies with a fluorescently labelled IgE-Fc biosensor revealed conformational change in IgE when it binds to FceRI. It also implied that an IgE-Fc biosensor is not only a useful tool to study the binding of IgE and its receptor, but also can be used to screen drug candidates for IgE-mediated therapy. To improve the reliability of the IgE-Fc biosensor, we characterized the fluorescent molecules used to construct the biosensor and generated a biosensor by other methods. Results suggested that a biosensor made by biotinylated IgE-Fc and monovalent streptavidin, both labelled with fluorescent dyes, is a useful tool. Therapies to block IgE binding to FceRI are clearly efficacious but still have limitations. Hence, a strategy to block IgE synthesis remains an extremely important goal of current research. Since CD23 was discovered in 1975, it has been proposed to play a critical role in IgE synthesis. Soluble CD23 is released by proteolytic cleavage from membrane-bound CD23 and interacts with membrane-bound IgE and CD21 to increase IgE production. Membrane-bound CD23 can also be ligated by IgE-allergen complexes to reduce IgE production. To test this model, we produced trimeric CD23, triCD23, which was designed by adding an isoleucine-rich a-helical coiled-coil motif to its N-terminal region and characterised this protein by various biophysical methods. Biophysical characterization suggested that triCD23 has a well folded lectin head domain and forms a trimer. Surface Plasmon Resonance results indicated that triCD23 binds to IgE-Fc fragments with two different affinities, 2.7x10"6 and 1.5x10"8M. Biological functional measurements showed that it binds to IgE on human B cells and increases IgE production.
37

In vivo imaging of regulatory T cell mediated transplant tolerance

Sharif-Paghaleh, Ehsan January 2012 (has links)
Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens. These cells have been used successfully in animal models first and more recently in the clinic to prevent Graft vs Host disease and transplant rejections. However, their locations in vivo, their migratory abilities and their in vivo survival have not been extensively investigated. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used as a reporter gene to image various cell types in vivo. It has several advantages over other imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation over time and lastly it may offer the possibility to be translated to the clinic. The study presented in this thesis addresses whether SPECT/CT imaging can be used to visualise the migratory pattern and survival of Tregs in vivo. At first, CD4+ T cells were directly radiolabelled and were subsequently imaged in vivo to demonstrate that T cells can be imaged using NanoSPECT/CT. Then Treg lines derived from CD4+CD25+FoxP3+ cells \ were/ retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate (9 mTcC)4~) and exposure of these cells to radioactivity was shown not to affect cell viability, phenotype and Treg function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using 99mTcO4". After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. Later, Treg lines with direct or indirect alloantigen specificity were imaged in skin transplant models using the same technique. It was observed that adoptively transferred Tregs migrate to the site of transplant at earlytime points and then migrated to various lymph nodes. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models. Moreover, new insight into pattern of migration of Tregs was identified.
38

Combined adjuvant for stimulation of cellular immunity

Tye, Gee Jun January 2012 (has links)
Vaccination has important clinical potential in the immunotherapy of both infectious disease and cancer. The central aim of the studies reported in this thesis has been the development of vaccination strategies that will be effective for therapeutic applications in cancer. Using ovalbumin antigen in a mouse model, we have examined a combination of recently developed adjuvants referred to as CASAC (combined adjuvants for synergistic stimulation of cellular immunity) to optimise the efficacy of vaccination induced T cell mediated immunity. These studies have examined the effect of repeated rounds of vaccination with one, or alternating cycles of two different helper peptides. The hypothesis was that repeated stimulation of the same clonal population of CD4+ T helper cells with a single MHC class II peptide could induce anergy, exhaustion, clonal deletion and/or stimulation of regulatory T-cells, resulting in reduced and shorter lived immunity. These studies have also examined the effect of inclusion of other immune regulatory components, and in particular a cocktail of cytokines generated by phytohemagglutinin stimulation of human peripheral blood mononuclear cells (referred to as IRX-2). Another important issue for vaccination mediated immune therapy that was addressed is the age-associated loss of immunological competence (immunesenescence). A comparative analysis is reported of the magnitude and duration of responses to vaccination with a single MHC class-I presented peptide in combination with the same or alternating MHC class-II presented peptides. In addition, we have quantified the number of antigen specific CDS T cells, their effector and memory subsets and in vivo antigen specific cytolytic activity to examine the effect of CASAC vaccination alone or in combination with IRX-2. The induction of immunological responses in young and aged immune backgrounds was also examined. Vaccinations with ovalbumin peptides in the CASAC adjuvant have shown higher percentages and numbers of antigen specific CDS T cells with improved cytolytic activity when helper functions are stimulated by two different class-II peptides used in alternating cycles of vaccination, rather than repeated stimulation by the same class-II peptide. This conclusion can be confirmed with a repeated experiment. Analysis of T cells with a regulatory (Treg) phenotype (CD4+CD25+Foxp3+) showed that their expansion was reduced with the alternating T-helper peptide vaccination regimen. The addition of IRX-2 to the CASAC vaccination regimen was found to enhance the in vivo antigen specific cytolytic activity. This was particularly significant in the aged mice which were found to have increased levels of Tregs.
39

Development of a versatile antibody cloning and expression system

Dodev, Tihomir January 2012 (has links)
Over the last three decades, recombinant monoclonal antibodies (mAbs) have become the key tool for basic research, diagnosis and treatment of human diseases. This has required the selection of recombinant antibodies with high affinity for appropriate epitopes on the target antigen and other desirable characteristics, such as their isotype and effector functions. In Biopharmaceutical communities specialised in antibody production, expression levels of grams per litre have been reached. However, lack of a suitable manufacturing platform, which ensures consistent antibody production, has always been one of the major impediments to the development of recombinant antibody material in academia. To overcome this barrier, we have developed a unique cloning method for one-step assembly of antibody heavy- and light-chain DNAs in a single mammalian expression vector. The DNA fragments assembled in this system do not rely on restriction enzyme- and ligase-dependant methods, thus minimising the steps involved in the cloning procedure. This allows the reproducible generation of fully functional recombinant antibodies of any species and isotype with any desired specificity. In less than four weeks, tens of milligrams per litre can be achieved, from cloning through to harvesting of transfected cell supernatants, providing an unbiased manufacturing platform compared to the currently available antibody expression methods in academia. The system proved to be very efficient and readily adaptable for the high-throughput screening of melanoma patient-derived antibody candidates with clinical potential. It enabled the parallel comparative functional studies between IgE and IgGl isotypes in an in vivo xenograft model of melanoma. The IgE isotype showed superior efficacy in restricting tumour growth, which encouraged us to continue developing antibody discovery methods, and pursuing melanoma antigen-specific antibodies as future effective therapies of this disease.
40

Advanced fluorescence lifetime imaging and spectroscopy techniques for biological samples

Chung, Pei-Hua January 2012 (has links)
Intracellular viscosity is correlated with cause of diseases, and it also affects the metabolism and chemical signalling. In this thesis, fluorescence-based studies were conducted on a fluorescent molecular rotor, BODIPY-Ci2, which was used to measure the intracellular viscosity. BODIPY-C12 is insensitive to the surrounding polarity using solvatochromic methods. It was demonstrated that the fluorescence lifetime of BODIPY-Ci2 in methanol/glycerol mixtures is related to viscosity via the Forster-Hoffmann equation, and the rotational correlation time depends on viscosity based on the Stokes-Einstein-Debye equation. The intracellular viscosity is measured via FLIM, time-resolved and steady-state fluorescence anisotropy. The relationship between the fluorescence lifetime and rotational correlation time of BODIPY-Ci2 in methanol/glycerol mixtures agrees with the combination of the Forster-Hoffmann equation and the Stokes-Einstein-Debye equation and is different to that of a rigid fluorophore. From the fluorescence imaging and counterstaining experiments, it appears that BODIPY-Ci2 is located in lipid droplets and the endoplasmic reticulum of cells. Particle tracking provided further evidence of lipid droplet staining. It seems that there are two different lifetimes in lipid droplets and the endoplasmic reticulum via time-resolved fluorescence anisotropy measurements, which indicate of two different environments for both locations. The lifetime is not a function of rotational correlation time for BODIPY-Ci2 in cells. An appropriate medium has to use to create a calibration of lifetime versus viscosity for measuring the microviscosity of cells. It is also found that BODIPY-Ci2 fails to measure viscosity in non-polar silicone oils. In addition, spectroscopic studies of Nile red, Rhodamine 123, Pyrromethene 546 and BODIPY-C12 in methanol/glycerol mixtures were carried out to examine the relationship between the radiative rate constant and refractive index according to the Strickler-Berg equation and an advanced model by Toptygin et al. The transition dipole moment and the shape of the fluorophore can be analyzed using a model also by Toptygin et al. Time-resolved fluorescence imaging and spectroscopy offer a very powerful and versatile method for applications in the life sciences.

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