no / Significance: Epigenetic regulatory mechanisms are essential for epidermal homeostasis and contribute to the pathogenesis of many skin diseases, including skin cancer and psoriasis. However, while the epigenetic regulation of epidermal homeostasis is now becoming active area of research, the epigenetic mechanisms controlling the wound healing response remain relatively untouched. Recent Advances: Substantial progress achieved within the last two decades in understanding epigenetic mechanisms controlling gene expression allowed defining several levels, including covalent DNA and histone modifications, ATP-dependent and higher-order chromatin chromatin remodeling, as well as noncoding RNA- and microRNA-dependent regulation. Research pertained over the last few years suggests that epigenetic regulatory mechanisms play a pivotal role in the regulation of skin regeneration and control an execution of reparative gene expression programs in both skin epithelium and mesenchyme. Critical Issues: Epigenetic regulators appear to be inherently involved in the processes of skin repair, and are able to dynamically regulate keratinocyte proliferation, differentiation, and migration, together with influencing dermal regeneration and neoangiogenesis. This is achieved through a series of complex regulatory mechanisms that are able to both stimulate and repress gene activation to transiently alter cellular phenotype and behavior, and interact with growth factor activity. Future Directions: Understanding the molecular basis of epigenetic regulation is a priority as it represents potential therapeutic targets for the treatment of both acute and chronic skin conditions. Future research is, therefore, imperative to help distinguish epigenetic modulating drugs that can be used to improve wound healing.

No / Adiponectin reportedly stimulates proliferation and elongation of human scalp hair follicles (HFs) ex vivo. In the current study, we investigated how adiponectin oligomers produced by perifollicular dermal white adipose tissue (dWAT), a potent source of adiponectin isoforms, influence human HF proliferation and pigmentation. To do so, we treated microdissected, organ-cultured HFs in the presence or absence of dWAT with a recombinant human adiponectin oligomer mix, or inhibited dWAT-derived adiponectin using a neutralizing antibody. Multiplex qPCR (Fluidigm) revealed that adiponectin oligomers downregulated pigmentation genes KITLG, PMEL and TYRP1 and Wnt genes AXIN2, LEF1 and WNT10B. In situ hybridization showed that adiponectin downregulated AXIN2 and LEF1, and up-regulated DKK1 within the dermal papilla (DP), a highly unusual transcriptional profile for a putative hair growth-promoting agent. Adiponectin oligomers also downregulated protein expression of the HGF receptor c-Met within the matrix and DP. However, adiponectin did not alter hair matrix keratinocyte proliferation within 48 h ex vivo, irrespective of the presence/absence of dWAT; HF pigmentation (Masson-Fontana histochemistry, tyrosinase activity) was also unchanged. In contrast, neutralizing adiponectin isoforms within HF + dWAT increased proliferation, melanin content and tyrosinase activity but resulted in fewer melanocytes and melanocytic dendrites, as assessed by gp100 immunostaining. These seemingly contradictory effects suggest that adiponectin exerts complex effects upon human HF biology, likely in parallel with the pro-pigmentation effects of dWAT- and DP-derived HGF. Our data suggest that dWAT-derived ratios of adiponectin isoforms and the cleaved, globular version of adiponectin may in fact determine how adiponectin impacts upon follicular pigmentation and growth. / Biotechnology and Biological Science Research Council (BBSRC) iCASE Studentship (awardees: R.P., J.P., recipient: C.N.), co-funded by Unilever, Colworth, UK; NIHR Manchester Biomedical Research Centre, Inflammatory Hair Diseases Programme (Lead: R.P.), University of Miami start-up funds (R.P.), and Frost Endowed Scholarship to R.P. (Dept. of Dermatology).

No description available.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Deon van der Merwe / 1,4-Dioxane is a potentially carcinogenic solvent. It is a problematic groundwater contaminant because of its unique physical-chemical properties. It is found in a wide range of consumer products as a by-product contaminant. This research aimed to investigate contaminant properties and behavior of dioxane in the environment and also in the human body. The dioxane ability to decontamination by adsorption processes was evaluated with four adsorbents. The adsorption efficiencies of activated carbon (AC), metal oxide nanomaterials (TiO[subscript]2 and MgO), and diatomaceous earth (DE) were assessed in aqueous and vapor phases using infrared spectroscopy. AC showed the highest adsorptive capacity for dioxane at equilibrium in both phases. The rate and extent of dermal absorption are important in the analysis of risk from dermal exposure to dioxane. For this purpose, a new flow through diffusion system (FTDS) was developed by modifying a Bronaugh flow through diffusion cell with flow capacity in both the donor and receptor compartments and using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) as the analytical technique. FTDS can provide ‘real time’ quantitative high-density permeation data over time and is characterized by the simplicity of its use and the low cost of test samples. The in vitro dermal absorption study of dioxane across human skin showed that the absorption parameters of dioxane were 1.16 ± 0.22 hr, 5.7 X 10[superscript]-4 ± (0.62) cm/hr, 0.286 ± 0.035 mg/cm2/hr, 4.8 X 10-5 (± 0.32) cm[superscript]2/hr, and 1.99 ± 0.086 mg for lag time, permeability, steady-state flux, diffusivity, and total amount absorbed over 8 hr, respectively. The study of the effect of the surfactant sodium lauryl sulphate and solvent systems water, ethanol, propylene glycol, and ethyl acetate on permeation profiles revealed that these solvents and surfactants increased the permeation of dioxane significantly. The FT-IR spectra of stratum corneum treated with solvents showed that there was broadening of the CH[subscript]2 asymmetric stretching vibration of the CH[subscript]2 peak near 2920 cm[superscript]-1 only in samples treated with ethanol. The lipid extract precipitates were detected and were mostly composed of the stratum corneum lipid part.

Nanoparticles are thought to present a unique hazard to human health. Furthermore, the increasing use of nanomaterials in consumer products has not been accompanied by relevant risk assessments. It is conceivable that skin flexion may assist the translocation of nanoparticles across the stratum corneum. However, current in vitro methodology to study dermal absorption involves the exclusive use of immobile skin within diffusion cells. Therefore, a novel skin-flexing diffusion cell system (“CutaFlex™”) was developed to incorporate reproducible skin flexing (2 flexes min-1; 6 mm maximum amplitude). The initial aims of this Thesis were to characterise the CutaFlex™ system to eliminate the possibility of flexion-induced (experimental) skin damage, demonstrate equivalence with historical permeability data to model compounds and assess the effect of skin flexing on barrier disruption. Subsequent work aimed to investigate the hypothesis that nanoparticles require dermal flexion to penetrate intact skin. In supporting these aims, this Thesis also performed work to assess the correlation between direct measurements of skin barrier function (using tritiated water) and transepidermal water loss (TEWL), the effect of flexing on the performance of topical skin protectants (barrier creams) and to further validate in vitro diffusion cell measurements against in vivo data acquired under identical conditions. The results demonstrated that skin flexing did not alter skin barrier function and that the CutaFlex™ system was in general agreement with historical measurements of skin permeability. Furthermore, controlled chemical or physical damage to the stratum corneum was not exacerbated by skin flexing. Skin flexion did not facilitate the dermal absorption of a range of nanoparticles (quantum dots). However, differences in the partitioning of nanoparticles into the stratum corneum were observed (independent of the degree of flexing), with greater amounts of negatively charged nanoparticles found in the superficial layers of the stratum corneum in comparison with positive or neutral nanoparticles. Flexing had a modest effect on the performance of a skin barrier cream which was limited to low dose applications; an effect tentatively ascribed to flexion-induced movement of cream to previously untreated areas. A poor correlation was found between 3H2O water permeability and TEWL flux. Most importantly, there was excellent agreement between in vitro skin permeability studies and in vivo studies (which used a surrogate measure of skin permeability). To summarise, the data in this Thesis has led to the development and characterisation of a novel diffusion cell (CutaFlex™), capable of simultaneously flexing skin whilst performing dermal absorption measurements comparable with the OECD-compliant models.

No description available.

Skin wound healing presents a challenging and expensive clinical problem with nearly 20 million wounds requiring intervention leading to an annual cost of more than $8 million. Tissue engineered skin substitutes are valuable not only as a clinical therapy for chronic wounds and severe traumas, but also as in vitro 3D model systems to investigate wound healing and skin pathogenesis. However, these substitutes are limited by a lack of topography at the dermal-epidermal junction (DEJ). In contrast, the native DEJ is characterized by a series of dermal papillae which project upward into the epidermal layer and create physical topographic microniches that support keratinocyte stem cell clustering. In this thesis, we created novel 3D skin model systems to investigate the role of microtopography in regulating keratinocyte function and cell fate using scaffolds containing precisely engineered topographic features. We hypothesized that the microtopography of the DEJ creates distinct keratinocyte microniches that promote epidermal morphogenesis and modulate keratinocyte stem cell clustering which can be harnessed to create a more robust skin substitute that expedites wound closure. Using photolithographic techniques, we created micropatterned DEJ analogs and micropatterned dermal-epidermal regeneration matrices (µDERM) which couple a dermal support matrix to a micropatterned DEJ analog. We found that the incorporation of microtopography into our in vitro skin model resulted in a thicker, more robust epidermal layer. Additionally, we identified three distinct functional keratinocyte niches: the proliferative niche in narrow channels, the synthetic niche in wide channels and the keratinocyte stem cell niche in narrow channels and corner topographies. Ultimately, incorporation of both narrow and wide channels on a single construct allowed us to recreate native keratinocyte stem cell patterning in vitro. These model systems will allow us to investigate the role of cellular microniches in regulating cellular function and epidermal disease pathogenesis as well as to identify topographic cues that enhance the rate of wound healing.

Introduction: Magnesium is required by the human body in modest amounts for the maintenance of health and optimal functioning. Objectives: This portfolio of work sets out to investigate whether magnesium supplementation has hypotensive effects and to determine if habitual dietary magnesium intake or loading strategies modulate the effects of magnesium supplementation. The habitual dietary magnesium intake of hypertensive patients was also examined to ascertain adequacy of dietary magnesium in this cohort. A meta-analysis was performed on the effect of magnesium supplementation on blood pressure. Other variables such as dosage, duration and study design were considered and findings from the meta-analysis used to influence future work. A further objective was to examine the effect of supplementation on aerobic and resistance exercise and subsequent recovery. Finally, the efficacy of an alternative means of magnesium delivery in the form of a transdermal magnesium cream was investigated. Methods: A 300 mg.day-1 elemental magnesium aspartate or magnesium citrate was used as a supplementation in studies 1,2,4 and 5. Participants were instructed to continue with their normal diet and for study 6 participants were required to eat the same foods for the 24 hours prior to both laboratory blood taking sessions. With the exception of the meta-analysis, food diaries were kept for various lengths of time, detailed in the publications. Aerobic and resistance exercise protocols were carried out in studies 1,2 and 4, with both performance and cardiovascular parameters investigated for any effect from supplementation. Where supplementation was in the form of a transdermal cream, this was applied to the torso and absorption of the cream was determined by investigating changes in serum and urinary magnesium levels. Summary of results: Blood pressure decreased with magnesium supplementation of 300 mg.day-1 for 7 days with greater reductions in systolic versus diastolic blood pressure consistently evident. Magnesium supplementation of 300 mg.day-1 for 7 and 14 days increased power during resistance exercise but no changes in aerobic exercise performance were observed. A high habitual dietary magnesium intake attenuated the hypotensive effect derived from magnesium supplementation when compared to those on a low habitual dietary intake. The meta-analysis supported these results. A habitually low dietary magnesium intake was observed in a cohort of clinically diagnosed primary hypertensives. Conclusion: These studies show that there is a link between low habitual dietary magnesium intake and elevated blood pressure and that magnesium supplementation appears to be associated with blood pressure. An improvement in resistance exercise performance with magnesium supplementation was also observed. Finally, a transdermal magnesium cream was shown to increase serum magnesium levels and may provide an alternative to oral supplementation.

Thesis (Ph.D. (Pharmacy))--North-West University, Potchefstroom Campus, 2008.

A viable <i>in vitro</i> excised human skin model was developed to accurately assess cutaneous delivery and metabolism of two ester type compounds; tetracaine (TC) and methyl salicylate (MS). This model could maintain the viability of fresh skin in diffusion cells for 24 hours. Skin viability was assessed using two methods; oxygen consumption measurement and confocal laser scanning microscopy. Two fluorescent probes, calcein AM and ethidium homodimer-1, were used as live and dead markers, respectively. General morphology and localization of nonspecific esterase activity in the skin samples from diffusion cell were checked histologically. Cutaneous delivery and metabolism of MS was evaluated with this viable skin model and compared to human skin homogenate model. A sensitive high performance liquid chromatography (HPLC) assay using reversed phase ion pair was developed/refined to simultaneously analyze TC and its metabolite (4-BABA). Several factors affecting this HPLC system were identified. The limit of detection for TC and 4-BABA was 0.3 ng and 0.5 ng, respectively. The limit of quantitation for TC and 4-BABA was 10 ng and 5 ng, respectively. Linearity was in the range of 10-120 ng for TC and 5-60 ng for 4-BABA. MS was hydrolyzed to salicylic acid (SA) during absorption through fall thickness human breast skin in diffusion cells. The extent of MS hydrolysis was significantly higher in viable skin than in non viable. The extent of absorption of SA through viable and non viable skins was similar. In human skin homogenate, MS was hydrolyzed at the rate of 72.31 nmol/h/[mu]g protein while the hydrolysis in phosphate buffered saline was very low. TC hydrolysis in human skin homogenate was not extensive due to substrate inhibition. From the kinetic study of TC hydrolysis in human skin homogenate, Km was in the 11-28 [mu]M range and Vmax was in the 2.0-2.8 [mu]mol/h/[mu]g protein range. Temperature over 60°C substantially reduced esterase activity in both models therefore caution must be taken during preparation and handling of tissue samples to preserve esterase activity. The viable <i>in vitro</i> excised skin model will provide more accurate quantitation of skin metabolism and absorption of xenobiotics.