No description available.

No description available.

No description available.

Infection with human cytomegalovirus (HCMV) is thought to promote the expansion of low affinity, terminally differentiated HCMV-specific memory T cells with increasing age, thereby resulting in reduced vaccine responsiveness and poor responses to novel pathogens. This project sought to characterise and explore the relationship between the size of the HCMV-specific immune-response and general health in older age. A multi-parametric flow cytometry assay was used to characterise ex-vivo activation induced T cell responses against 19 immunogenic HCMV proteins in three groups of healthy volunteers aged between 18 to 85 years old. This approach is different to that utilised by most studies investigating the role of HCMV-specific immunity and ageing whereby focus is often limited to T cell responses against a few ‘dominant’ peptides, or proteins. The response size against the 19 frequently recognized HCMV proteins were assessed by means of intracellular cytokine staining (ICS) assay based on these functional markers; CD40L, CD107a, TNFα, IFNγ, and IL-2. The memory phenotype with respect to each protein was also explored using these markers; CD45RA and CD27. A significant increase in pp65-specific CD4+ T cells in the Oldest group was observed compared to the Young group. Also, a significant increase was observed in the size of the summated (sum of all protein-specific responses) CD8+ HCMV-specific T cell responses in the Older group compared to Young group, but no further increase was seen in the Oldest group indicating no further expansion with age. Differences in levels of HCMV-specific T cell polyfunctionality was observed between target proteins, however level of polyfunctionality was neither reduced with increasing age nor in those with very large responses. In this study no significant differences were observed in the number of infections and health related problems reported by HCMV seropositive and seronegative aged individuals keeping a health diary; however, a significant trend was observed between the size of the summated HCMV-specific CD8+ T cells and the proportion of months unwell.

No description available.

No description available.

Antibodies are a class of proteins vital in mediating immune responses in vertebrates. Their binding site is highly malleable, allowing them to bind virtually any antigen. The versatility of antibody binding sites has received much attention from the pharmaceutical industry, marking them out as the most important category of biopharmaceuticals. The development of antibodies which bind to a specific antigen has thus far been achieved by costly and time-consuming experimental screening campaigns. However, in recent years computational approaches to antibody design have started to emerge, which offer an alternative. Computational antibody design techniques focus on determination of the binding site on the antibody, antibody-modelling, antibody-antigen docking and prediction of the binding site on the antigen. Here, we explore aspects of computational antibody design with the aim of gaining a better understanding of antibody-antigen interactions and improving existing artificial antibody design tools. We start by demonstrating our structural antibody database which has become a primary resource for antibody structural information. This is followed by a detailed analysis of the antibodyantigen interactions. The information gathered from this analysis allowed us to create an antibody contact site prediction tool, Antibody i-Patch. This tool was then employed to develop a local antibody-antigen docking pipeline, which used knowledge of the binding site of the antigen. We then tackled the global antibody-antigen docking problem by developing EpiPred, antigen binding site predictor which was employed in our global antibody-antigen docking pipeline.

An effective immune response in mammalian cells relies on a network of molecular interactions to detect and respond to pathogens. The T-cell receptor (TCR) is one of the most important components of this system responsible for the outcome of the immunological response. Paramount to its role is its ability to efficiently signal a productive interaction with a peptide embedded in an MHC molecule. Important aspects of TCR structure and organization are unknown, limiting the current understanding of this process and rendering it highly controversial. The work described in this thesis seeks to define the valency, structural organization and signalling properties of the TCR in order to provide a better framework for thinking about the receptor-triggering problem. The results suggest that a largely monovalent complex diffuses at the surface of T cells, which is able to trigger intracellular signalling in the absence of large structural rearrangements of the extracellular subunits of the TCR. Moreover, a recently proposed mechanism involving conformational rearrangements of the cytoplasmic domains of the complex is shown to fail to explain the regulation of TCR phosphorylation. Steps are also taken toward investigating the role of more subtle conformational rearrangements at atomic resolution. Finally, an investigation of what controls tyrosine phosphorylation of the receptor in resting T lymphocytes led to the development of new approaches to address the role of specific phosphatases. The outcome of this analysis suggested how a finely-tuned balance between kinase and phosphatase activity, at both global and local levels, regulates TCR phosphorylation and T-cell activation.

This thesis reports a study of the effects of ultrasound on crystal nuclcation from solution and particle breakage. Firstly, the effect of ultrasound on reactive crystallization when nuclcation is consistent with a predominantly homogeneous model is studied by measuring the induction time. Barium sulphate is used as the working substance precipitated by mixing aqueous BaCl2 and Na:S04 solutions. The relationship between amplitude of ultrasonic processor and power input was determined. The experiments were carried with various ultrasonic power inputs. It is observed that increasing ultrasound has a significant effect on reducing the induction time. At a given supersaturation level, the induction time decreases with increasing ultrasonic energy. The relationship between power input (E) and the activation energy of nucleation (E ) is discussed. And the equation between total power input (E) and the nucleation coefficient (A. ) is obtained. A cluster coagulation model, which brings together the current nucleation models and the theories describing the behavior of colloidal suspensions, was applied to estimate the induction time under various power inputs. A comparison between the predictions of the model and the results of experiments shows that the number of monomers in dominating clusters ( ) in the solution remains constant with increasing power input. The mechanism of the ultrasonic effect on homogeneous nucleation is analyzed. It is found that when temperature is kept constant, the main effect of ultrasound is to increase the diffusion coefficient (D,m)- Other parameters change only slightly or remain constant within the ultrasonic field. This suggests that diffusion acceleration is the main reason for the reduction of the induction time. The relationship of DAn and power input is also discussed. A mathematical model of power input and diffusion coefficient is presented, from which it is predicted that diffusivity enhancement (DAB- DAbo) has an exponential relationship with power input. The above relationship is consistent with the results of the experiments. Induction times have also been measured with and without ultrasound to investigate its effect on induction time of spectinomycin hydrochloride, roxithromycin and sucrose during anti-solvent crystallization. The relationship between ultrasonic power input and diffusion coefficient is also investigated. It is found that at the same power input, the ultrasonic effect on DAH decreases with increased solution viscosity. The effect of ultrasound on BaSO.* nucleation is also studied by measuring the induction time when nucleation is consistent with a predominantly heterogeneous model. In these experiments, barium sulphate is used as the working substance precipitated by mixing aqueous BaCb and Na2SC > 4 solutions with relatively low concentrations compared to the experiments of homogeneous nucleation. It is observed that at a given supersaturation level, the induction time significantly decreases with increasing ultrasonic energy during heterogeneous nucleation. The mechanism of the ultrasonic effect on heterogeneous nucleation is analyzed. The effect of ultrasound is to increase the diffusion coefficient (DAb) while reducing the contact angle {0) and geometric factor if). This gives rise to an increase in the heterogeneous nucleation rate and shorter induction time. The ultrasonic effect on critical supersaturation ratio (Sfnf) is also investigated to determine the mechanism of ultrasonic effect on / and 0. It is proposed that both the force from the cavitational bubble implosion and the nucleation process occurring at bubble/solution/foreign solid interface are the predominant reasons why contact angle ( 0) decreases with the increase of power input. In addition, the effects of ultrasound on nucleation order (n) and the nucleation coefficient (ku) are also investigated, and compared with the corresponding effects during homogeneous nucleation. A cluster coagulation model is used to calculate the induction time at different levels of supersaturation when nucleation is consistent with a predominantly heterogeneous model. It is found that the dominant cluster size increases with increasing supersaturation and increasing ultrasonic energy. The effect of ultrasound on nucleation of BaS04 is studied by a Kodak Ektapro Hs motion analyzer. Firstly, the dispersion processes of ink with ultrasound and stirring are used to compare difference of the mixing processes and hydraulic flows. The vessel is divided into several sections to investigate the local mixing process by recording the movement of small silicon carbide crystals. Velocity variance is used to indicate the difference of turbulence when ultrasound is applied and when stirring is applied respectively. The cavitation distribution in different sections is also obtained. BaSC>4 is precipitated by mixing aqueous solutions of BaCb and Na2S04. The nucleation processes are observed by Kodak motion analyzer in different sections. The mechanism of nucleation by ultrasound is analyzed. Then, the induction times at different temperatures are measured with different power inputs at a given supersaturation level. The relationship between the cavitation distribution and temperature is established.

The signalling mechanisms that control differentiation of naive T cells to effector and memory cells are unclear. A key event in T cell activation by antigen is the phosphorylation of tyrosine based activation motifs in the TCR CD3 complex by the Src-family kinases, Lck and Fyn. Elucidating the exact signalling mechanisms involved in the generation of memory T cells and effector function is fundamental and has important implications particularly in therapeutics, such as in developing treatments for diseases and infection. Lck knockout mice have no T cells, and so we were unable to study the role of Lck in the peripheral T cell compartment. Using mice that express an inducible Lck transgene in T cells, which were bred to the class I MHC restricted F5 TCR transgenic line, we have investigated the role of Lck-mediated TCR signaling in antigen-specific CD8 T cell responses. Stimulation of lymphocytes in vitro showed that the response of F5 T cells to peptide is 10-100 fold less sensitive in the absence of Lck, suggesting that the threshold of triggering is raised, however once cells were activated they underwent a similar program of division. Using an in vivo model where F5 T cells are transferred with flu virus into Rag1"A recipients, we demonstrated that Lck is required for the activation and subsequent generation of functional effector F5 CD8 T cells. The ability to generate functional Cytotoxic T lymphocytes (CTL) was impaired, shown by the reduced killing of target cells in vitro and in vivo. We have also shown defects in the production of IL-2, TNFct and Granzyme B, which appear to be Lck dependent. However there is a less stringent requirement for Lck in the production of IFNy, showing varying levels of Lck requirement for eliciting effector function. In summary we have shown that Lck contributes to multiple stages of memory cell formation. It is required for the priming, expansion and differentiation of F5 CD8 memory T cells, but is not required for their survival.