Architecture as a Catalyst for Activity

Many of New Zealand’s smaller town centres struggle to remain viable. A common issue for these declining public realms is the hollowing out of their city centres. Numerous factors may contribute to this problem. Issues such as a lack of access, connectivity and identity within the urban fabric, or instances of privatisation, where forums that were once public have now shifted to a digital interface. One of the challenges facing cities is the diminishing number of “civic” buildings and activity located in the town centre. The Indoor Community Sports Centre (ICSC) offers a partial remedy for this problem. Even with the merging and downsizing of Council’s and their funding, Territorial Authorities continue to invest in ICSCs. This thesis investigates whether these buildings can make a positive contribution to the public domain of town centres. New Zealand ICSC’s, more often than not, are simple shed-like buildings on the periphery of cities or town centres, predominantly occupying or adjacent to large park areas, sports fields or schools. This thesis examines whether the building type can be adapted to become an “urban” building, where it will have the opportunity contribute to a revitalised town centre. A design case study based on Upper Hutt identifies three key design criteria established from initial research of Sports Centres and best-practice Urban Design. These three criteria – breaking up mass, active edges from the outside and creating a dynamic connection – allow the ICSC to become part of the civic realm. The research concludes that an ICSC can be successfully integrated into an “urban” context. In the Upper Hutt case study, success depends on two broader design strategies. First, the ICSC should be located in an area where walkability, functionality and visual and physical connectivity will benefit the public domain. Second, the ICSC should be part of a mixed-use development, which exploits the building type’s inherent flexibility. This is achieved through combining a transport hub, another essential civic amenity, as well as other commercial programmes that provide occupancy during periods of disuse. The thesis shows how a carefully adapted ICSC can turn a somewhat disconnected, hollowed out town into a functional, integrated and walkable one. The redesigned facility does so by linking existing amenities, feeding city-fringe activity back into the city centre and projecting a consciousness of place

T-cell responses to influenza virus in pigs

The tools and techniques for the study of porcine T-cells lag behind what is currently attainable in human T-cells, so this thesis was initially focused on improvements in this field. This study established long-term culture of porcine T-cells, T-cell clone procurement and relevant T-cell assays. These techniques were then used to investigate cytotoxic T-cell responses to Influenza A virus (IAV) in pigs. IAV is highly mutative and novel strains can be generated following reassortment between different viral strains. IAV is endemic in the global pig population and in some circumstances the virus can pass between humans and pigs and other animals. Pigs can therefore, potentially be a source for the generation of new and possibly pandemic influenza strains. The risk this poses to global human health, together with the negative effects of IAV infection within pig herds, highlights the need to improve our knowledge of IAV in pigs. This study identified four new MHC class I restricted IAV epitopes, derived from the viral nucleoprotein. Cytotoxic T-cells recognising these IAV epitopes were detected at high numbers ex vivo in samples from vaccinated pigs. The structures of these IAV epitopes in complex with their respective MHC class I molecules were resolved and revealed the primary anchor positions within the peptides. This enabled peptide binding motifs to be defined for two porcine MHC-I alleles. These peptide binding motifs can be utilised for efficient epitope prediction. This study also identified super-agonist ligands for two of the MHC-I restricted IAV epitopes. Overall, this work has opened up the study of porcine T-cells to a level previously unattainable and has contributed to our knowledge of IAV in pigs. It has paved the way for further experiments investigating IAV in pigs, other porcine diseases and for using pigs as an animal model for human disease

Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA

The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations

Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope

CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111–130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR–pHLA-II interactions

Characterizing and correcting immune dysfunction in non-tuberculous mycobacterial disease

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic, progressive, and growing worldwide health burden associated with mounting morbidity, mortality, and economic costs. Improvements in NTM-PD management are urgently needed, which requires a better understanding of fundamental immunopathology. Here, we examine temporal dynamics of the immune compartment during NTM-PD caused by Mycobacterium avium complex (MAC) and Mycobactereoides abscessus complex (MABS). We show that active MAC infection is characterized by elevated T cell immunoglobulin and mucin-domain containing-3 expression across multiple T cell subsets. In contrast, active MABS infection was characterized by increased expression of cytotoxic T-lymphocyte-associated protein 4. Patients who failed therapy closely mirrored the healthy individual immune phenotype, with circulating immune network appearing to ‘ignore’ infection in the lung. Interestingly, immune biosignatures were identified that could inform disease stage and infecting species with high accuracy. Additionally, programmed cell death protein 1 blockade rescued antigen-specific IFN-γ secretion in all disease stages except persistent infection, suggesting the potential to redeploy checkpoint blockade inhibitors for NTM-PD. Collectively, our results provide new insight into species-specific ‘immune chatter’ occurring during NTM-PD and provide new targets, processes and pathways for diagnostics, prognostics, and treatments needed for this emerging and difficult to treat disease

Participation demonstration projects Ethnic Minorities Recreation Project, Scunthorpe Borough Council and Humberside County Council; Phase 2 monitoring report - the second and third years

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