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The underlying mechanisms regulating the ability of skeletal muscle to regenerate after acute “damaging” eccentric or “non-damaging” concentric exercise in young human skeletal muscle is poorly defined. Age-related impairments in the regenerative mechanisms may contribute to the age-related loss of muscle mass and function, which has negative consequences for overall health and disease. Thus, the first aim of this thesis was to initially investigate multiple targeted mechanisms previously implicated in the regeneration process, over a comprehensive time-course following eccentric versus concentric exercise in young adults. Within this study it was found that post-exercise, in general, increased anabolic and repressed catabolic signalling preceded functional decline, whereas inflammation and ubiquitin proteasome system-related breakdown increased once functional recovery was initiated/achieved. Eccentric exercise led to greater anabolic signalling and inflammatory signalling response. As such, this study has provided a benchmark of muscle regeneration in young skeletal muscle, which implicates early anabolic and catabolic regulation in the rapid adaptation of muscle, whereas inflammation and ubiquitin proteasome system-related breakdown likely mediate longer term remodelling/adaptations, which may be greater following eccentric exercise. Using this benchmark, the aim of the second study was to identify age-related changes in targeted regenerative mechanisms. Concentric exercise did not cause a molecular regenerative response, whilst eccentric exercise induced anabolic signalling and satellite cell activation, prior to and at the nadir of force, respectively. Compared to the younger adults, ageing per se was associated with increased inflammation, whilst anabolic and catabolic signalling post-eccentric and concentric exercise was blunted. Interestingly, satellite cell activity was induced in the old only following eccentric exercise. These data suggest that eccentric exercise is potentially more advantageous for promoting muscle growth versus concentric exercise in older adults. Whilst, compared to the young, the old displayed blunted molecular responses which might underlie blunted muscle growth during ageing. Furthermore, the activation of satellite cells in the old might be the result of the impaired molecular mechanisms being suboptimal for repair thus, requiring additional regenerative means. In order to further characterise ageing muscle and the mechanisms of muscle regeneration, RNA sequencing was performed at the time of peak anabolic signalling to highlight more global and novel molecular networks. Ageing per se revealed genes involved in blood vessel development, plasma membrane and cell-cell junction expression were down-regulated, thus implicating these processes in age-related muscle loss. Following concentric exercise in older adults, there was an up-regulation of structural transcripts whilst there was a general down-regulation of genes related to metabolism, which might suggest impaired metabolism post-concentric exercise. Perhaps the blunted transcript responses contribute to the often observed age-related blunting of muscle mass adaptations in response to exercise training. Collectively, the data from this thesis has important implications for developing interventions for maximising hypertrophic responses and for counteracting the suboptimal regenerative responses observed in older adults.

Dendritic Cells (DC) play a pivotal role in both the initiation of immunity and its regulation through tolerance induction. They represent potential targets or tools of therapy in autoimmunity, allergy, cancer, and transplant medicine. Despite recent progress in mapping tissue DC subsets in human, their ontogeny and mechanisms of homeostasis remain elusive. The conventional view is that maintenance of tissue DC and macrophages is dependent on constant replenishment from circulating monocyte precursors. This concept was derived from in vitro data and mouse models under perturbed conditions. While these observations may reflect events in inflammation, studies in murine steady-state biology have challenged this, with data indicating at least three ways in which DC may persist independently of monocytes: 1, tissue DC are replaced by non-monocyte, blood-borne precursors; 2, embryological cells seed tissues and persist in adulthood; 3, DC, or their immediate precursors, self-renew in tissues. These mechanisms may not be mutually exclusive. Although much has been gained from the study of DC kinetics in human transplant, these conditions are, at least in part, inflammatory. To understand the steady-state homeostasis of DC and epidermal Langerhans cells (LC) in human tissues, a number of approaches were taken. Firstly, a comprehensive ‘DC profile’ of normal human tissues was developed, starting with detailed characterization of DC, monocyte and macrophage populations in human peripheral blood (PB) and skin. This lead to the identification of new DC subsets in peripheral tissue. Cells with DC precursor potential were then sought, through analysis of their growth factor receptor expression and ability to enter into cell cycle. Correlative data was gathered to suggest relationships between potential blood borne DC precursors and skin APC. The CD34+ haematopoietic compartment was examined to identify potential DC progenitors. The granulocyte/macrophage progenitor (GMP) and a more recently described multilymphoid progenitor (MLP) were identified and shown also to be present in PB. CD34+ cells were identified in peripheral tissue. While cell and gene knockout experiments have significantly advanced understanding of murine DC, these experiments are not possible in human. The second approach was therefore to undertake a search for subjects with spontaneous DC or monocyte deficiencies. Two novel syndromes were identified; Dendritic Cell, Monocyte, B and NK Lymphoid (DCML) deficiency (4 subjects) and autosomal recessive IRF8 deficiency (1 subject). In both cases, severe depletion of peripheral blood monocyte and DC subsets was associated with absence of tissue DC but preservation of LC and some tissue macrophages. Examination of CD34+ stem cell compartments revealed distinct stem cell defects resulting in loss of MLP and depletion of GMP in DCML deficiency, but accumulation of these subsets in IRF8 deficiency. As predicted by mouse models, DC deficiency was associated with a reduction in circulating regulatory T cells, in the context of elevated Flt3-ligand. Finally, lesions of Langerhans Cell Histiocytosis (LCH), a histiocytic disorder presenting with pathological accumulation of langerin+ DC in tissues, were studied. Analysis of langerin distribution in normal skin and lung identified a langerin+ DC, independent of LC. In keeping with in vitro culture data, preliminary observations show that langerin may be up-regulated on mDC in inflammatory conditions. These data suggest that langerin expression in LCH lesions may reflect its upregulation, rather than determine the LC origin of LCH cells. A variety of techniques have been used to explore DC in normal tissue, novel syndromes of DC deficiency and Langerhans cell histiocytosis. These studies provide new insights into the ontogeny and homeostasis of human DC and LC.

The hair follicle has the unique capacity to pass through periods of growth, regression and rest before regenerating itself to restart the cycle. This dynamic cycling capacity enables animals to change their coats, and for hair length to be controlled at different body sites. While progress has been made on some of the outstanding questions in hair follicle biology, many of the cellular and molecular mechanisms involved in driving hair follicle cycling remain undiscovered. The first section of work in this thesis reports on the dermal papilla, an essential component of the hair follicle with a key role in follicle regeneration. Using hanging drop cultures spheres of human dermal papilla cells were created, and the expression of cytoskeletal and extracellular matrix components characteristic of intact papillae were analysed using immunohistochemistry and real time PCR. I found evidence that over time the gene and protein expression profiles of the papilla cell spheres became more representative of intact dermal papillae, although differences were seen between papilla cells strains derived from different individuals. I then demonstrated the inductive capability of human dermal papilla cells using this spherical model in an amputated follicle assay. This data provides some of the first evidence that cultured human dermal papilla cells can retain inductive capacity without having to be combined with other cell types. This induction phenomenon may also have relevance to anagen initiation and cycling. The second section of this thesis was concentrated on the process of club fibre shedding, now widely recognised as a phase of the follicle cycle known as exogen. The vibrissa follicle, with its predictable timing of club fibre loss, was first developed as a new model for exogen, and the structural and adhesive changes associated with the progression towards club fibre release were analysed using SEM and ТЕМ. Combined with plucking experiments these showed club loss to be gradual process. The expression of proteins involved in cell adhesion, differentiation, communication and digestion, all potential mediators of fibre loss were examined around "young “and "old" club fibres using immunohistochemistry. To further assess the mechanism of exogen, and to search for signalling molecules microarray technology was utilised, enabling the identification of 75 genes that were associated with the process. Group analysis highlighted a particular role for proteases and their inhibitors in the retention and final release of the club fibre. Moreover, immunofluorescent analysis of the results from the microarray identified a process of differentiation, specific to the cell layer surrounding the club fibre prior to club fibre release. This data provides evidence that the process of exogen is progressive, associated with maturation of the cells surrounding the club fibre, and terminating with the release of the club fibre. The processes involved in club fibre release are becoming more widely recognised as an important aspect of the hair cycle and this thesis is one of the first comprehensive pieces of work that analyses club retention and release as an active phase.

The complex process of muscle differentiation is organized predominantly by the myogenic regulatory factors, Myf5, MyoD, Myf6 and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. The regulation of myogenin at the transcriptional level is well documented and has been studied in depth to reveal a multilayer, robust regulation of its expression. The possibility that myogenin could be regulated at the post transcriptional level through microRNAs has yet to be identified. MicroRNAs are non-coding RNA molecules that post-transcriptionally regulate many cellular processes. In an effort to investigate the role microRNAs play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491 , miR-329 and miR-96) were predicted to bind the myogenin 3 'untranslated region. However, luciferase assays showed only miR-186 inhibited translation and 3 'untranslated region mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during muscle cell differentiation. Functional studies demonstrated that cellular levels of miR-186 were inversely proportional to those of myogenin throughout differentiation and that its overexpression inhibited the differentiation of C2C12 and primary muscle cells. Alternatively, inhibition of miR-186 during muscle differentiation increased the capacity of muscle cells to differentiate raising the possibility that a miR- 186 inhibitor could be utilized in muscle damage therapeutics. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation, providing an additional layer of regulation to the robust regulation of myogenin expression.