The Melanocyte Keratinocyte Transplantation Procedure (MKTP) is an effective surgical replacement of lost melanocytes in recalcitrant vitiligo and pigmentary skin disorders. However, it is only effective in stable vitiligo lesions because active autoimmunity destroys the newly transplanted melanocytes. Despite careful selection of candidates based on the reported clinical stability, the success of the procedure is still unpredictable. MKTP candidates with non-segmental, segmental, and mixed vitiligo, as well as hypopigmented scars and Piebaldism patients were enrolled to our studies. Our aim was first, to investigate the possible immunological mechanisms responsible for the unpredictable post- transplantation outcomes, including T cell subsets and inflammatory chemokines, by correlating these biomarkers with clinical phenotypes, duration of stability, and surgical outcomes. We used suction blister biopsy, a minimally invasive technique that we developed to sample human skin. Moreover, we quantified transplanted melanocytes in the suspension using flow cytometry. Following MKTP, we corelated these biomarkers to the repigmentation score. We found that CD8+ T cells remain in some clinically stable vitiligo lesions, correlate negatively with the post-surgical score of repigmentation, and inversely impact the durability of the responses. Interestingly, the number of transplanted melanocytes in the suspension and the duration of stability do not have prognostic roles. Based on our findings and in a second group of patients, we suppressed the activity of T cells to enhance the outcomes of MKTP. We used Ruxolitinib, JAK1/2 inhibitor, in a triple blinded randomized controlled within subject study, in comparison with Tacrolimus (a calcineurin inhibitor and the standard of care treatment in vitiligo) as well as placebo control. We found lower T cell infiltrate, lower chemokines, and better skin repigmentation in lesions treated with MKTP plus Ruxolitinib or Tacrolimus than in lesions treated with MKTP plus placebo. Lastly, we compared two different types of laser in preparation of the recipient skin for MKTP - ablative versus fractional Er:YAG laser. We found that the ablative laser is combined with minimal CD8+ T cell epidermal infiltrate and superior repigmentation score in comparison to more infiltrate and lower repigmentation score with the fractional laser. Taken together, these results from our studies provide novel insight to predict the optimal surgical candidates and will improve surgical outcomes. It advances the treatment of vitiligo by uncovering the impact of autoimmunity on the success of repigmentation and discovering new approaches to optimize the surgical treatment options in patients with vitiligo and pigmentary skin disorders.

The electro–mechanical connection between under bump metallization (UBM) and solder in flip–chip bonding is achieved by the formation of brittle intermetallic compounds (IMCs) during the soldering process. These IMCs continue to grow in the solid–state during storage at room temperature and service at an elevated temperature leading to degradation of the contacts. In this thesis, the diffusion–controlled growth mechanism of the phases and the formation of the Kirkendall voids at the interface of UBM (Cu, Ni, Au, Pd, Pt) and Sn (bulk/electroplated) are studied extensively. Based on the microstructural analysis in SEM and TEM, the presence of bifurcation of the Kirkendall marker plane, a very special phenomenon discovered recently, is found in the Cu–Sn system. The estimated diffusion coefficients at these marker planes indicate one of the reasons for the growth of the Kirkendall voids, which is one of the major reliability concerns in a microelectronic component. Systematic experiments using different purity of Cu are conducted to understand the effect of impurities on the growth of the Kirkendall voids. It is conclusively shown that increase in impurity enhances the growth of voids. The growth rates of the interdiffusion zone are found to be comparable in the Cu–Sn and the Ni–Sn systems. EPMA and TEM analyses indicate the growth of a metastable phase in the Ni–Sn system in the low temperature range. Following, the role of Ni addition in Cu on the growth of IMCs in the Cu–Sn system is studied based on the quantitative diffusion analysis. The analysis of thermodynamic driving forces, microstructure and crystal structure of Cu6Sn5 shed light on the atomic mechanism of diffusion. It does not change the crystal structure of phases; however, the microstructural evolution, the diffusion rates of components and the growth of the Kirkendall voids are strongly influenced in the presence of Ni. Considering microstructure of the product phases in various Cu/Sn and Cu(Ni)/Sn diffusion couples, it has been observed that (i) phases have smaller grains and nucleate repeatedly, when they grow from Cu or Cu(Ni) alloy, and (ii) the same phases have elongated grains, when they grow from another phase. A difference in growth rate of the phases is found in bulk and electroplated diffusion couples in the Au–Sn system. The is explained in AuSn4 based on the estimated tracer diffusion coefficients, homologous temperature of the experiments, grain size distribution and crystal structure of the phase. The growth rates of the phases in the Au–Sn system are compared with the Pd–Sn and the Pt–Sn systems. Similar to the Au–Sn system, the growth rate of the interdiffusion zone is found to be parabolic in the Pd–Sn system; however, it is linear in the Pt–Sn system. Following, the effect of addition of Au, Pd and Pt in Cu is studied on growth rate of the phases. An analysis on the formation of the Kirkendall voids indicates that the addition of Pd or Pt is deleterious to the structure compared to the addition of Au. This study indicates that formation of voids is equally influenced by the presence of inorganic as well as organic impurities.

Indiana University-Purdue University Indianapolis (IUPUI) / Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.