▼ The synthesis and processing of melanosomes, the pigmented organelles of the follicular and interfollicular epidermis, is of major interest in the field of cutaneous biology. Additionally, clarification of the biological and cellular processes of cutaneous pigmentation has several therapeutic and cosmetic based applications, including the alleviation of skin hyperpigmentation. Modification of skin complexion coloration has traditionally been accomplished by inhibition of the rate limiting enzyme of melanogenesis, tyrosinase, or attenuation of melanosome transfer from melanocytes to keratinocytes. The post transfer modification of pigmented melanosomes, the main focus of this dissertation project, provides an attractive and distinct avenue of modulating skin pigmentation. There is currently limited information on how epidermal keratinocyte process recipient melanosomes during terminal differentiation. Furthermore, the variability of melanosomal degradation seen between light and dark skin remains to be clearly established. Therefore, we have developed a novel model system to investigate the degradation of isolated melanosomes by cultured human keratinocytes. Fluorescently labeled and isolated melanosomes, cultured in the presence of light and dark skin derived keratinocyte cultures, were assessed for degradation. The extent of degradation has been qualitatively assessed, using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Within 48 hours of melanosome incorporation, indirect immunofluorescence and confocal microscopy images suggest that light derived keratinocytes may have accelerated melanosome degradation compared to dark keratinocytes. This time dependent decrease in fluorescence was then quantitatively analyzed using flow cytometry analysis. Consistent with the results of the confocal analysis, over a 48 hour time frame, light keratinocytes appear to degrade melanosomes more efficiently than the dark skin derived keratinocytes, P=0.039. This methodology offers a novel mechanism to address the differential ability of light and dark keratinocytes to degrade melanosomes. To further delineate the process of melanosome degradation between light and dark skin, we focused on hydrolytic enzymes that have been implicated in epidermal differentiation and potentially melanosome degradation. To investigate this, we performed preliminary microarray analysis on suprabasal epidermis derived from light and dark skin, by laser capture microscopy (LCM). Data analysis of the microarray experimentation showed over-expression of various hydrolytic enzyme genes in the suprabasal epidermal layers, when comparing light to dark skin. Western blot analysis performed to confirm the expression pattern of hydrolytic enzymes from either light or dark skin derived epidermal lysates, demonstrated that cathepsin L2 was reproducibly upregulated in light skin, P=0.048. In addition, immunofluorescence analysis of cathepsin L2 in light and dark foreskin cryosections confirmed this differential expression and demonstrated that this enzyme was expressed throughout the epidermal layers. Biochemical analysis of cathepsin L2 activity in the two complexion types confirms an elevated enzyme activity in light compared to dark skin complexion samples, 1.75 fold higher activity in light skin compared to dark skin, P=0.03. Taken together these results confirm the differential expression of the acid hydrolase cathepsin L2 in light and dark skin at the gene and protein level. These results may have identified a specific acid hydrolase that may play a role in melanosome degradation and pigment processing. Advisors/Committee Members: Wickett, R. Randall.

▼ Human scalp hair is exposed to water for a significant portion of its lifetime. Acting as an ion-exchange resin, hair can extract cations of water hardness metals, calcium and magnesium, from the tap water used during hygiene practices and chemical treatments. The characteristics and consequences of the interaction between water hardness metals and inert substrates such as pipes and bathroom fixtures are well understood and recognized. However, less work has been conducted to elucidate the nature of the interaction between water hardness metals and human hair, a chemically reactive substrate. Thus, we endeavored to investigate this interaction, and our research efforts and findings are summarized in this dissertation. First, the variables that influence the interaction between water hardness metals and human hair were examined. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to quantify the metal content of hair, which was studied as a function of the following variables: hair condition (oxidative damage), level of water hardness, and water pH. It was found that the condition of the hair, a key representation of the binding capacity (available anionic sites), was most influential. Water hardness level had a smaller effect than one would expect; hair became saturated with notable amounts of water hardness metals even after repeated exposure to soft water depending on the level of damage. Water pH was positively correlated with metal uptake. A pilot study was conducted to determine the strength of the laboratory data vs. human subject data. The study consisted of thirty-five women who were grouped based on their residential water hardness (soft, hard, or very hard) and hair type (virgin, colored/bleached). The data from this study agreed with the laboratory experiments, and also provided information on consumer perception of water hardness effects on hair. Next, the hair structural implications of water hardness metal uptake were explored. Hair mechanics and styling were examined by technical measures of single fiber torsional and tensile properties, combability, and style retention as a function of the calcium and magnesium content of virgin and bleached hair. Fiber stiffening was induced by the presence of water hardness metals in the fibers of both virgin and bleached hair, and this was accompanied by a reduction in combing forces. The long-term style retention of virgin hair was significantly improved by water hardness metals, while that of bleached hair was directionally reduced. Finally, the key technical approaches to the management of water hardness metal uptake, chelation and acidic treatment, were evaluated. This work was completed through the testing of three commercial products that utilized one or both of these approaches and acidic surfactant solutions with and without a chelant. It was concluded that the most effective way to control the water hardness metal uptake was to first reduce the levels in hair with an extended contact, rinse-off product, and then use a shampoo that can either maintain the reduced levels or slow down uptake during subsequent wash cycles. Both products should be acidic (pH < 6) and contain chelants. Advisors/Committee Members: Wickett, R. Randall.

▼ Treatment of dyslipidemia in HIV-infected persons is commonly required with the use of protease inhibitors (PIs) which may be restricted by drug-drug interactions between antiretroviral agents and statins. We hypothesized that darunavir/ritonavir (DRV/rtv) modulate the pharmacokinetics of rosuvastatin (RSV) when co-administered by altering its hepatic disposition. The plausible genetic influence on the plasma exposure of RSV was also explored. To investigate this hypothesis, we first conducted a clinical drug interaction study and subsequently performed in vitro correlative/mechanistic analysis. In the open label, crossover study, 12 healthy volunteers were randomized to receive either RSV 10 mg/day or DRV/rtv 600/100 mg (bid) and in combination for 7 days followed by a crossover to the other regimen for 7 more days. The fasting lipids were obtained at baseline and on days 7, 21 and 35 along with intensive PK sampling for 24 hours post-dose. The PK parameters such as Cmax, t1/2 and AUC0-24 were determined by non-compartmental analysis using WinNonLin 5.2. Statistical analyses were performed using Paired t test. Drug levels were compared with OATP1B1 c.521T > C, 388A>G, BCRP c.421 C > A, MRP2 c. -24C>T, 1249G>A, 3972C>T single nucleotide polymorphisms (SNPs) to investigate potential outliers in the drug interaction study. Next we assessed the influence of DRV on the activity of multidrug transporters OATP1B1, BCRP and Pgp in the transporter over-expressing CHO and MDCK cells and primary human hepatocyte cultures. The AUC0-24 and Cmax of RSV before and after administration of DRV/rtv showed a significant increase by 1.48 fold ( P=0.003) and 2.44 fold (P<0.001) without a change in the t1/2 (P=0.176). However the AUC, Cmax, and t1/2 of DRV and RTV were unaltered. The total cholesterol increased by a median 4% (P=0.002) when RSV was given in combination than when given alone. Furthermore, we observed a trend toward lower plasma exposure of RSV in subjects with OATP1B1 *1a/*15 than OATP1B1*1a/*1a genotypes. And no adverse events were attributable to the drug interaction. Our in vitro findings suggest that DRV/rtv led to an inhibition of OATP1B1 (IC50 = 10 µM) and BCRP (IC50 = 77 µM) and an induction in Pgp activity (p= 0.05) Co-administration of DRV/rtv significantly increased the plasma exposure of RSV and consequently led to a decline in the lipid-lowering effects of RSV. This study warrants large-scale interaction in HIV-infected population. Advisors/Committee Members: Desai, Pankaj.