Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Master of Science / Department of Kinesiology / Brett J. Wong / Mechanisms underlying the cutaneous vasodilation response to local skin heating and whole body heating in humans remain unresolved. Although nitric oxide (NO) is known to contribute to these responses, it remains unclear as to the source of NO. Adenosine receptors induce vasodilation in many human tissues and may work, in part, through NO. As these receptors are also known to be located in the cutaneous vasculature, the studies contained in this thesis were designed to investigate a potential contribution of adenosine receptor activation to the rise in skin blood flow elicited by local skin and whole body heating. The study presented in chapter IV was designed to determine a potential role for adenosine receptors in contributing to cutaneous thermal hyperemia. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments designed to elucidate the contribution of A[subscript]1/A[subscript]2 adenosine receptors during local skin heating. Each site was locally heated from a baseline temperature of 33°C to 42°C at a rate of 1°C/10 s and skin blood flow was monitored via laser-Doppler flowmetry (LDF). The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation directly contributes to cutaneous thermal hyperemia. These data further suggest a portion of the NO response may be explained by A[subscript]1/A[subscript]2 adenosine receptor activation; however, a substantial portion of the NO response is independent of the adenosine receptor contribution. The study presented in chapter V was designed to determine a potential role for A[subscript]1/A[subscript]2 adenosine receptors in contributing to cutaneous active vasodilation. Four cutaneous microdialysis sites were randomly assigned one of four drug treatments, as above, and skin blood flow was monitored via LDF. Whole body heat stress, sufficient to raise oral temperature at least 0.8°C above baseline, was induced via water-perfused suits. The data obtained from these experiments suggest A[subscript]1/A[subscript]2 adenosine receptor activation does not directly contribute to cutaneous active vasodilation; however, a role for A[subscript]1/A[subscript]2 adenosine receptor activation is unmasked when NO synthase is inhibited. The data from this study further suggest that A[subscript]1/A[subscript]2 adenosine receptor activation may be responsible for a portion of the known NO component of cutaneous active vasodilation.

In the present study non-5-HT₁(A)/non-5-HT₁(C) binding sites in the rabbit caudate nucleus (CN) were examined to determine if they might be identical to the recently discovered 5-HT₁(D) sites in the bovine CN. The characterizations were carried out measuring high-affinity [³H]5-HT binding under conditions where 5-HT₁(A) and 5-HT₁(C) sites were pharmacologically masked in both tissues. Comparison of the pharmacologic profiles of the bovine 5-HT₁(D) and rabbit non-5-HT₁(A)/non-5-HT₁(C) sites revealed similarities, but showed distinct differences. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by GTP than was [³H]5-HT binding in the rabbit CN, and this effect was differentially sensitive to calcium and other divalent cations (i.e., Mg²⁺, Mn²+)⁺in the two tissues. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by NEM than it was in the rabbit CN. Thus, it may be concluded that the non-5-HT₁(A)/non-5-HT₁(C) [³H]5-HT binding sites in rabbit CN are distinct from those in the bovine CN, and we propose that they be tentatively identified as 5-HT₁(R) to distinguish them from the 5-HT₁(D) site.

Th17 cells are implicated in a variety of inflammatory disorders including Spondyloarthritis (SpA). Ankylosing spondylitis (AS), the most common SpA, is genetically associated with HLA-B27 and IL-23R polymorphisms, although the link remains unexplained. In addition to classically folded heterotrimers, HLA-B27 can form β2m-free homodimers (B272). B272, as well as free heavy chains, are able to interact with KIR3DL2. I have shown an expansion of KIR3DL2+ CD4+ T cells in peripheral blood of AS patients and HLA-B27+ healthy controls. Although KIR3DL2+ CD4+ T cells comprise a minority of CD4+ T cells in peripheral blood, KIR3DL2+ CD4+ T cells account for the majority of peripheral blood CD4+ T cell IL-23R expression. KIR3DL2+ CD4+ T cells from synovial fluid are also enriched for IL-23R expression compared to matched peripheral blood samples. Moreover, treatment exposure to rIL-1/23 significantly increases IL-17 production by KIR3DL2+ CD4+ T cells in AS patients but not RA patients and healthy controls. KIR3DL2+ CD4+ T cells from AS patients produce more IL-17 than KIR3DL2+ CD4+ T cells from HLA-B27- healthy controls. KIR3DL2+ CD4+ T cells from AS patients and healthy controls are also enriched for dual production of IL-17 and IFNγ, consistent with the theory that AS is a Th17 or a Th17/1 driven disease. I have shown that naïve CD4+ T cells express KIR3DL2 de novo after activation. Interaction with B272-expressing cells acts to maintain and/or further increase KIR3DL2 expression. Interaction with B272-expressing cells also increases expression of Bcl-2. I propose that interaction of KIR3DL2+ CD4+ T cells with B272 preferentially promotes the survival of these pro-inflammatory cytokine-producing KIR3DL2+ CD4+ T cells in SpA. HD6, a B272-specific monoclonal antibody, decreases IL-17 production by PBMCs from AS patients, making it an attractive candidate therapeutic reagent in the treatment of AS and other HLA-B27-associated SpA.

Endocrine and metabolic disease or dysfunctions are of growing concern in modern societies across the globe, underlining the need for continued focus on the development of pharmaceuticals. Subsequent scientific research has revealed a trend in the increase of such abnormalities and expansion of chemical industries, highlighting concerns that these disorders may, in part, be caused by exposure to environmental pollutants. This has led to changes in legislation concerning chemicals safety testing involving an increasing number of vertebrate animal tests as a part of environmental risk assessment process, at significant financial and ethical costs. A solution that is appropriate and aligned with the three R’s (reduction, refinement and replacement) in relation to animal research is to exploit the use of small invertebrate organisms as possible replacements for mammals. In line with the above approach/solution, this thesis is based on the null hypothesis that common genes, proteins and processes in gastropod molluscs and humans underlie the response of male reproductive organs to androgenic chemicals. Using a freshwater pulmonate snail, Biomphalaria glabrata, physiological effects of two steroid androgens on the development of mollusc secondary sexual organs were studied. Furthermore, an exhaustive investigation on the mollusc nuclear receptor repertoire and reproductive type neuropeptides was conducted. This also included the study of the evolutionary degree of conservation of these genes in non-model molluscs. The results obtained suggest that the snails did not respond to, and were not affected by exposure to the androgens. These results were supported by the absence of the members of subfamily 3C of nuclear receptors, which includes some of the “vertebrate” steroid hormone targets, suggesting that this mollusc may be an inappropriate model for steroid hormone mediated mammalian endocrine function. The nuclear receptor (NR) repertoire of B. glabrata comprised of 39 nuclear receptors representing all the known subfamilies of the NR superfamily. 21 reproductive type neuropeptide genes were identified encoding precursors that are predicted to release over 124 bioactive cleavage products. The consequence of these findings is significant in the context of the development of alternative model organisms for chemical testing as well as elucidating the taxonomic scope of nuclear receptor mediated endocrine disruption.

Androgen receptor (AR) signalling is essential for the development and function of the male reproductive system. Conditional gene ablation using the Cre-loxP system has previously assisted in the elucidation of the role of AR in different cell types. The aim of this study was to examine the effects of the ablation of AR in previously untargeted cell types, with the hypothesis that this will have significant and novel effects on reproductive development and function that have not been previously documented by current models of androgen disruption. In these studies, three Cre recombinase lines were empirically validated for action in the male reproductive system, before being used to ablate AR and the phenotypes of the resulting lines were characterised. Endothelial-specific receptor tyrosine kinase (Tie2)-Cre was shown to target the vascular and endothelial cells of the testis, and used to ablate AR in these cells. The testes of the resulting Tie2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. Aquaporin 2 (Aqp2)-Cre was shown to target the post-meiotic germ cells of the testis, and was used to ablate AR in these cells. The testes of the resulting Aqp2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. It was concluded that the Ar gene was dispensable in the endothelial cells and post-meiotic germ cells of the testis for normal spermatogenesis. Forkhead box protein G1 (FoxG1)-Cre was shown to target the caput epididymal epithelium and pituitary, and used to ablate AR in these cells. d100 FoxG1-ARKO mice had a severe testicular phenotype, with sloughing of the seminiferous epithelium, atrophy of some seminiferous tubules and distension of the rete testis with spermatozoa. Despite the severe testis phenotype, ablation in the testis was incomplete and restricted to a small percentage of Leydig cells, with no ablation in Sertoli cells. Ablation of AR in the embryonic pituitary did not cause adult serum testosterone or LH concentrations to change, nor did it cause changes in other pituitary hormone transcripts. Mosaic ablation of AR in the caput epididymal epithelium was shown to impair epididymal development, with failure of initial segment (segment I) development and a significant decrease in epithelial cell height and lumen diameter in the remaining proximal caput epididymis (segment II). Dysfunction of the caput epididymis resulted in the failure of spermatozoa to transit the efferent ducts into the epididymis correctly: instead they were found to stall in the efferent ducts and produce a block. The testicular phenotype could be explained as the result of fluid backpressure effects resulting from the efferent duct block. Consequently, low concentration of spermatozoa in the cauda epididymis resulted in infertility in the FoxG1-ARKO, which represents a new model of obstructive azoospermia.

Aquaculture is known as a major food-producing industry and Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the major cultured species in Scotland. However, disease outbreaks in aquaculture have been reported and are commonly associated with intensive fish farming, which results in a tremendous cost in the industry. Hence, understanding what immune-related genes and cells are present, their responses, mechanisms and functions in these farmed animals is a first requirement for potent vaccine design, selection of disease-resistant breeds and disease outbreak prevention. The innate immune system is the first line of defence against microbes which use germline-encoded pattern recognition receptors (PRRs) to recognise specific, conserved and constitutive products of invading pathogens, called pathogen-associated molecular patterns (PAMPs) that are important for survival of the microorganism and are thus hard for the microorganism to change. This thesis focuses on the identification and characterisation of a family of PRR called toll-like receptors (TLRs) and a TLR accessory protein UNC93B1 using different approaches. In Chapters 2, 3 and 5, eleven TLR genes and UNC93B1 were identified from Atlantic salmon whole-genome shotgun (WGS) contigs. These genes were cloned and sequenced and their putative domain structure, gene synteny and homology to other genes were determined by bioinformatics analysis. In addition, the constitutive expression profile of these genes was examined in different tissues from healthy salmon using real-time PCR. The potential modulation of these genes was examined in different in vitro and in vivo models which provide information to help understand the role(s) of these genes during inflammation or in the immune responses against pathogens. Several of these TLRs are so-called non-mammalian TLRs (TLR19, TLR20a and TLR20d) and are therefore particularly interesting to study. The sub-cellular localization was also investigated in TLR-GFP expression plasmid transfected Salmon Head Kidney-1 (SHK-1) cells. Lastly, attempts were made to develop a Human Embryonic Kidney (HEK) 293T cell line based platform to study TLR signalling and ligand specificity (Chapter 4).

Genetic variation within the cannabinoid 1 receptor (CB1R) locus (CNR1) has been repeatedly associated with drug addiction pathologies. Genomic annotation of CNR1 indicates the vast majority of this genetic variation likely results in altered transcriptional regulation of the CNR1 gene as a mechanistic link to the disease phenotype. There is a lack of information describing the regulation of CNR1 transcription and the potential impact of disease-associated variation within the CNR1 locus on its transcriptional regulation. This study investigates the impact of an evolutionary conserved regulatory region of CNR1, termed ECR1, and the disease-associated variation contained within, on the transcriptional activity of the cognate CNR1 promoter region. Reporter assays conducted in primary hippocampal cells demonstrate that CNR1 promoter exhibits variable transcriptional activity during periods of CB1R signalling and cell depolarisation. Coupled to allelic variants of ECR1, the CNR1 promoter shows significant changes in transcriptional activity under resting conditions indicating that disease-associated variation within ECR1 may decrease CNR1 transcription. Further, alleles of ECR1 can drive allele-specific transcriptional responses from the CNR1 promoter during periods of CB1R stimulation and cell depolarisation. The results highlight the potential for disease-associated regulatory variation of the CNR1 locus to create stratified transcriptional responses to specific cell signalling scenarios and putatively to clinical strategies employing pharmacological agents. Furthermore, investigation of DNA-protein interactions at the allelic ECR1 region demonstrate that disease-associated variation within ECR1 alters DNA-protein interactions within the nucleus consistent with a decrease in transcriptional activity in the disease-associated allele variant. Collectively the current work supports the hypothesis that disease-associated variation within the ECR1 regulatory region of the CNR1 locus has the capacity to significantly impact on CNR1 promoter transcriptional activity. It is posited that allele-specific transcriptional effects may have a major impact on the susceptibility of individuals to drug addiction or on responses to clinical pharmacological treatments.

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published_or_final_version / Zoology / Doctoral / Doctor of Philosophy