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Hereditary Equine Regional Derma Asthenia (HERDA) is a painful disfiguring autosomal recessive skin disorder of Quarter Horse lineages. Affected horses cannot be ridden and most are humanely destroyed. Five years following homozygosity mapping of a putative causal mutation responsible for HERDA, it remains unclear how this mutation causes the HERDA syndrome. HERDA horses have a missense mutation in peptidyl-prolyl cis-trans isomerase B (PPIB) which encodes cyclophilin B (CYPB) and alters folding and post-translational modifications of fibrillar collagen. Loss of function mutations in CYPB recognized in other species classically present as the debilitating bone disease, severe to lethal osteogenesis imperfect (OI). Objectives of this study were to develop a novel method for cryogenic clamping of tendons and ligaments of high tensile strength and validate its performance by ultimate tensile strength testing of normal equine deep digital flexor tendon. This validated method was then used to compare tendon and ligament of HERDA vs. control horses along with great vessels and skin. We hypothesized that all tissues of high fibrillar collagen content would have altered tensile properties due to the CYPB mutation affecting fibrous connective tissue globally within HERDA horses. Based on previous studies in our laboratory identifying reduced hydroxylysine content and altered collagen crosslink ratios in the skin of HERDA affected animals that implicate lysyl hydroxylase-1 (LH1) dysfunction, we hypothesized that the HERDA PPIB mutation modified an interaction between CYPB and LH1, interfering with hydroxylysine synthesis and its availability for collagen crosslink formation. In addition, we hypothesized that mutant CYPB may also lead to modifications of other known CYPB protein complexes, such as the CYPB, prolyl-3 hydroxylase-1 (P3H1) and cartilage associated protein (CRTAP) triplex. Goals of this study were to investigate the tensile properties of tissues with high fibrillar collagen content from HERDA homozygotes, to elucidate the mechanistic relationship of the HERDA CYPB mutation to the clinical disease, and to provide evidence to substantiate a heterozygote phenotype in HERDA which could be useful to explaining the correlation between lineages that carry the HERDA allele and performance outcomes in the discipline of western cutting competition.

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Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-­sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-­9216, on scalp follicle growth in organ culture was examined. NNC55-­9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.

The thesis presents a rational path for the optimization of the selection of optical treatment parameters in photobiomodulation of human skin fibroblasts. The project begins with an extensive analysis of 90 bibliographic reports in photobiomodulation published between 1985 and 2015, and revealed major inconsistencies in optical parameters selected for clinical applications. Seeking greater clarity for optimal parameter choice, a systematic approach to disentangle the multiple factors underpinning the response of human dermal fibroblasts in vitro to visible and near-infra red (NIR) light was employed. Light-based devices were constructed to specifically and systematically screen the optical parameter window (i.e. wavelength, irradiance and dose) observed in literature. Additionally, critical culture and treatment conditions that have dramatic impact on the outcome of specific light treatment of these human skin dermal cells were identified. In particular, environmental oxygen concentration, cell confluency and serum concentration were all found to have a great effect on the response of dermal fibroblasts to light. In parallel, the induction of reactive oxygen species (ROS) by short visible wavelengths on two dermal fibroblast sub-populations or lineage, reticular and papillary, was monitored by live-cell imaging. The ROS species were found to be created in or close to mitochondria. Lastly, gene expression studies revealed a strong impact of short visible wavelengths, as compared to long and NIR wavelengths on both subpopulations of human dermal fibroblasts. In particular, blue light (450 nm) specifically down-regulated proliferation, metabolism and protein synthesis molecular pathways. At the protein level, 450-nm light inhibited the production of procollagen I in human reticular and papillary fibroblasts in a dose-dependent manner. Gene expression results were in agreement i.e., the same light parameter down-regulated collagen fiber genes, integrins and up-regulated collagenase MMP1. This thesis concludes with a chapter presenting a characterization of the accuracy of a potential translation tool for the prediction of optical photon density inside human skin. / Marie Skłodowska-Curie Actions.

Traditional methods for dermal wound closure such as sutures and staples are invasive and can result in soft tissue trauma, increasing the likelihood of localized inflammation and infections. Alternately, while tissue adhesive alternatives can effectively seal and adhere to the wounds, they can also present safety concerns relating to immunogenic responses and tissue toxicity. Herein, we fabricate injectable, adhesive, and cytocompatible poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-dopamine (DA) hydrogels co-crosslinked via hydrazone and self-polymerized dopamine crosslinks that exhibit high water retention, improved tissue adhesiveness, and effective tissue regeneration properties. POEGMA-DA hydrogels exhibit independently tunable gelation properties based on their dual crosslinking mechanism, allowing for gelation as fast as 24 s (allowing for injection and rapid filling of irregularly-shaped wounds) while achieving relevant compressive moduli of up to 37 kPa and in vitro skin adhesion strengths of up to 1.2 kPa. The POEGMA-DA hydrogels induced no significant inflammation while demonstrating high interfacial adhesiveness in a stented skin excisional mouse model, enabling efficient dermal tissue regeneration by supporting collagen remodelling and enabling the regeneration of hair follicles, sebaceous glands, and blood vessels at the excision site over the 14-day study timeline. As such, injectable POEGMA-DA hydrogels represent a relevant non-toxic and adhesive alternative wound closure system for treating deep dermal wounds. / Thesis / Master of Applied Science (MASc) / Effective wound healing and subsequent tissue regeneration after a physical injury requires a moist sterile environment, the presence of oxygen, nutrients and enzymes, an efficient blood supply to the wound site, and a controlled inflammatory response to initiate the healing process. External methods of closing the wound to prevent infection aid in faster healing like sutures, staples, and liquid sealants which can result in infections and/or the stimulation of an inflammatory response that can hinder tissue restoration. Hydrogels, water-swellable polymer networks, represent an alternative solution that can both suppress infection while simultaneously promoting wound healing. Hydrogels have a similar structure to soft tissues like skin and can thus provide a supportive environment for cells to promote tissue regeneration and restore tissue structure and function. The swelling of hydrogels in water is highly beneficial for providing moisture at the wound site; however, this high degree of water retention also means they have a hard time sticking to tissues. To address this challenge, hydrogels can be modified with a component naturally derived from marine mussels that allows them to stick to their wet habitats, helping hydrogels to stick to the wound site while healing. In this thesis, mussel-inspired hydrogels are designed and can spontaneously gel and stick to a wound site to accelerate the restoration of the structure and function of skin. These biodegradable and injectable hydrogels are effective in accelerating wound closure with minimal evidence of scarring while suppressing negative inflammatory reactions and restoring the structure of skin by promoting the regeneration of hair follicles, sebaceous glands and blood vessels.

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Yes / The prevalence of Type 2 diabetes mellitus (T2DM) is escalating globally. Patients suffer from multiple complications including the development of chronic wounds that can lead to amputation. These wounds are characterised by an inflammatory environment including elevated tumour necrosis factor alpha (TNF-α). Dermal fibroblasts (DF) are critical for effective wound healing, so we sought to establish whether there were any differences in DF cultured from T2DM donors or those without diabetes (ND-DF). ND- and T2DM-DF when cultured similarly in vitro secreted comparable concentrations of TNF-α. Functionally, pre-treatment with TNF-α reduced the proliferation of ND-DF and transiently altered ND-DF morphology; however, T2DM-DF were resistant to these TNF-α induced changes. In contrast, TNF-α inhibited ND- and T2DM-DF migration and matrix metalloprotease expression to the same degree, although T2DM-DF expressed significantly higher levels of tissue inhibitor of metalloproteases (TIMP)-2. Finally, TNF-α significantly increased the secretion of pro-inflammatory cytokines (including CCL2, CXCL1 and SERPINE1) in ND-DF, whilst this effect in T2DM-DF was blunted, presumably due to the tendency to higher baseline pro-inflammatory cytokine expression observed in this cell type. Collectively, these data demonstrate that T2DM-DF exhibit a selective loss of responsiveness to TNF-α, particularly regarding proliferative and secretory functions. This highlights important phenotypic changes in T2DM-DF that may explain the susceptibility to chronic wounds in these patients. / This study was funded by an Iraqi government studentship to AHAA-R.