Alopecia areata (AA) is a complex autoimmune disease in which autoreactive T cell-mediated attack of the hair follicle (HF) leads to non-scarring hair loss. Although AA is one of the most prevalent autoimmune diseases, the development of novel effective therapeutics has been limited. Standard of care remains observation for mild cases and steroids for moderate-to-severe cases, which have demonstrated only limited efficacy. The skin is a highly heterogeneous tissue at baseline, comprised of a diverse array of immune and non-immune cell types whose coordinated crosstalk is essential for homeostasis. The skin microenvironment becomes markedly altered as a result of disease-associated inflammation in AA. A pathognomonic histopathologic feature of AA is an intense lymphocytic infiltrate surrounding the lower portion of the HF in the growth phase of the hair cycle, known as anagen. We previously established that CD8+ T cells comprise the majority of this infiltrate in AA skin, and that they are necessary and sufficient to drive disease via JAK/STAT activation. While this discovery led to the pioneering use of JAK inhibitors as a novel class of therapeutics in AA, JAK inhibition is not a curative solution, since patients often experience relapse upon discontinuation of treatment. This not only underscores the continued need for translational drug discovery research in AA, but also reflects an incomplete understanding of the mechanisms that govern disease pathophysiology. Recent advances in single-cell RNA sequencing (scRNAseq) present an unprecedented opportunity to dissect the heterogeneity of complex tissues and disorders. Since its emergence, scRNAseq has proven to be a powerful tool for the discovery of rare cell types and novel therapeutic targets in a variety of contexts that range from cancer to autoimmunity. In this thesis, we leveraged scRNAseq to interrogate the cellular and molecular mechanisms underlying disease pathogenesis in AA at single-cell resolution, together with validation and functional experiments, with the goal of uncovering novel cell types and pathways that can guide the development of innovative therapeutic strategies. In Chapter 2, we performed scRNAseq of skin-infiltrating CD45+ immune cells to dissect lymphocyte heterogeneity in both murine and human AA. Our scRNAseq analyses informed a series of antibody-mediated cell depletion experiments that assessed the in vivo function of specific lymphocyte subsets in murine AA. Our results established CD8+ T cells as the predominant disease-driving cell type in AA. We identified shared mechanisms underlying CD8+ T cell heterogeneity in murine and human AA skin, in which CD8+ T cells form an “effectorness gradient” comprised of interrelated transcriptional states that culminate in increased expression of inflammatory cytokines and T cell effector function. We also demonstrated a role for CD4+ T helper cells in disease initiation, and determined that regulatory T cells possess intact immunosuppressive capacity in AA. In Chapter 3, we expanded upon the studies described in Chapter 2 and performed scRNAseq of skin-infiltrating CD45+ cells at various timepoints throughout disease course (from 3 to 24 weeks post-disease induction) in AA to analyze the temporal dynamics of lymphocyte heterogeneity in AA skin and skin-draining lymph nodes. In conjunction with scRNAseq, we also performed single-cell TCR sequencing to assess the dynamics of T cell clonality alongside changes in T cell transcriptional profiles. We observed a striking increase in CD8+ T cell clonal expansion during disease onset, which increased throughout disease progression and subsequently decreased in chronic AA, when the preclinical mouse model exhibits total body hair loss. Our single-cell analyses suggested that CD8+ T cell clonality and pathogenicity are closely linked, which we validated in vivo by demonstrating that a single expanded clonotypic population of CD8+ T cells is sufficient to induce disease in mice. In Chapter 4, we analyzed single-cell transcriptomic profiles obtained from full-thickness skin in mice with chronic AA to investigate the contributions of the HF and other non-T cell populations in disease. In this study, we also used a network biology-based approach to infer single-cell protein activity, which together with single-cell mRNA gene expression profiles uncovered a multitude of novel findings in AA. Our results revealed a role for necroptosis as a potential HF-intrinsic mechanism of pro-inflammatory signaling in AA, and also identified an MHC Class II signature specific to basal keratinocytes in AA skin. Furthermore, we uncovered a novel, rare population of disease-associated Arg1+ macrophages, which prompted us to revisit our immune-specific scRNAseq datasets described in Chapters 2 and 3 and perform an integrative analysis of this novel cell type in AA. Our preliminary in vivo studies suggested that targeting Arg1+ macrophages and/or arginine metabolism may ameliorate disease in AA. Taken together, this thesis presents a comprehensive, systematic interrogation of AA pathogenesis at single-cell resolution. Importantly, the validation and functional studies that were informed by our scRNAseq data demonstrate proof-of-concept of the use of single-cell technology to accelerate the discovery and translation of novel therapeutic targets in complex diseases. While we undertook a hypothesis-driven approach to design our studies, the data presented in this thesis was also profoundly hypothesis-generating, and has informed a number of ongoing projects in the laboratory with the shared goal of advancing our understanding of disease pathology in AA.

The nonapoptotic form of regulated cell death known as ferroptosis is an attractive target for combating numerous diseases. Ferroptosis is an iron-dependent death of cells by lipid peroxidation. Pharmacological inhibition of anti-ferroptotic pathways is a promising therapeutic avenue for treatment of cancer, and death by ferroptosis has been implicated in numerous neurodegenerative and ischemia-reperfusion-driven diseases. Therefore, demystifying the dynamics of lipid peroxidation in this cell death process opens a window to understanding disease processes and how to treat them. This dissertation makes use of ferroptosis-modulating compounds as chemical probes to elucidate the roles of different subcellular membranes in ferroptotic lipid peroxidation. Chapters two and three explore the structure-activity-distribution relationship of fatty acids and the ferroptosis inducer FINO2, respectively, and together demonstrate the endoplasmic reticulum as a driver of lipid peroxidation in ferroptosis. Chapter two makes use of stimulated Raman scattering imaging, while chapter three uses confocal fluorescence imaging. Chapter four shifts gears to focus on development of FINO2 as a drug lead, performing structure activity relationship analysis to increase the potency and pharmacological properties of the analogs. Altogether, this work answers questions about how cells die by ferroptosis, and provides footwork for how we can better modulate ferroptosis against cancer and other illnesses.

IUPUI / Fatty acid synthase (FASN) over-expression has been associated with poor prognosis and recurrence in cancer patients. In addition, it has also been found that overexpression of FASN causes resistance to DNA-damaging treatments by up-regulating the non-homologous end joining (NHEJ) repair of DNA double-strand break. Proton pump inhibitors (PPIs), were originally designed to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase. PPIs have recently been reported to reduce drug resistance in cancer cells when used in combination with other chemotherapeutics, although the mechanism of resistance reduction is uncertain. In our lab, previous investigation showed that PPIs decreased FASN thioesterase (TE) domain activity and cancer cell proliferation in a dose-dependent manner. In this study, I tested the hypothesis that PPIs sensitize breast cancer cells to doxorubicin and ionizing radiation (IR) treatments by inhibiting FASN. When administered to breast cancer cells as single-agent, lansoprazole exhibited the highest potency in inhibiting both FASN activity and breast cancer cell proliferation, among four PPIs tested. In addition, treatment of breast cancer cells with lansoprazole decreased the mRNA and protein levels of poly (ADP-ribose) polymerase-1 (PARP-1) and NHEJ activity, accompanied by elevated γ-H2AX expression. Following a 3-day treatment with lansoprazole, a dose-dependent disruption in cell cycle disruption and increased apoptosis were also detected. Combination of lansoprazole with either doxorubicin or IR caused profoundly higher levels of DNA damage accumulation than doxorubicin or IR treatment alone, suggesting synergistic effects. Taken together, our observations suggest that PPIs synergistically suppress breast cancer cells in combination with DNA damaging treatments by inhibiting FASN. These findings may provide a potential route to overcome resistance to DNA-damaging chemo/radiation treatments in refractory breast cancers.

Iron is an essential trace element required for many processes within a cell, including DNA synthesis and cell cycle progression. Moreover, it is critical for cellular respiration in mitochondria. Due to their proliferative nature, cancer cells are dependent on iron, and depleting this element via iron chelators results in the inhibition of ribonucleotide reductase, leading to cell cycle arrest and apoptosis of cancer cells. Recently, an alternative mechanism for the effect of iron chelators have been proposed, including induction of N-myc downstream regulated gene 1 (NDRG1) expression and its inhibitory effect on c-MET, EGFR, and NF-κB pathways, which can act as oncogenes in a certain context. NDRG1 is a tumour suppressor gene, which is downregulated in many cancers and its downregulation correlates with cancer progression, poor differentiation, and higher metastatic potential. It has been shown that NDRG1 expression can be regulated by intracellular iron - a decrease in intracellular iron leads to upregulation of NDRG1 at mRNA and protein level via the HIF-1-dependent mechanism by inhibiting prolyl hydroxylases. Recently, we have conceived the concept of mitochondrially targeted chelators as an effective anti-cancer agent and in this work, we focused on the evaluation of mitochondrially targeted...

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Ferroptosis is a regulated junction between cell death, metabolism, and disease, and it hasbeen implicated in many pathologies. The assorted ferroptosis pharmacology modulators offer valuable means to modulate ferroptosis in multiple diseases, to explore disease etiology, and to develop potential therapeutics. In the first part, the work focuses on inhibiting ferroptosis in a Huntington’s disease model. Ferrostatin-1 (Fer-1) is a potent small-molecule ferroptosis inhibitor that has been adopted to investigate the role of ferroptosis in many disease models. However, its further application is limited by its low potency, poor stability, possible toxicity, and lack of brain penetration. We developed the fourth and fifth generations of ferrostatins and investigated the in vitro and in vivo pharmacokinetics of lead compounds. We identified PHB4082 preferentially accumulating in the kidney as a potential candidate for kidney disease-relevant contexts. Moreover, TH-4-55-2 displayed an excellent brain penetration, preferentially accumulating in the brain at concentrations of magnitude higher than the in vitro IC50 values. In the in vivo toxicity study, it was well-tolerated over 30 days in wild-type and R6/2 mice and exhibited a protective effect against weight loss in a Huntington’s disease model, suggesting it is a strong candidate for application in HD and more neurodegenerative disease models. The second part describes the efforts to explore the therapeutic potential of inducing ferroptosis in a tumor model. Imidazole ketone erastin (IKE) induced ferroptosis by specifically inhibiting system xc– in a subcutaneous xenograft model of Diffuse Large B Cell Lymphoma (DLBCL), suggesting the potential of IKE as a therapeutic strategy for cancer. A biodegradable polyethylene glycol-poly (lactic-co-glycolic acid) nanoparticle formulation was used to aid in delivering IKE to cancer cells in vivo, exhibiting improved tumor accumulation and therapeutic index relative to free IKE, indicating its potential for treating DLBCL. In summary, this work explored the possibility to modulate ferroptosis using small molecule modulators in multiple disease models and identified some potential drug candidates and useful chemical probes.

Pancreatic cancer is the 10th most common cancer diagnosis and 4th most common cause of cancer mortality in the United States, highlighting a disparity between disease prevalence and outcome. Ineffective drug delivery to these tumors contributes to the poor prognosis for this disease, as intravenous drug delivery is hampered by poor vascularity within these tumors. Bacterial therapy, or the use of bacterial components to treat disease, is thought to be able to overcome such drug delivery challenges; through a combination of tumor homing and long-term colonization, bacteria can be utilized to produce anti-cancer molecules directly within the cores of tumors. As such, here, we interrogate the feasibility of bacterial cancer therapy for pancreatic ductal adenocarcinoma (PDAC). Before delving too deeply into bacterial therapy design, it was important to first address one major limitation in therapeutic screening models. As a therapeutic should be effective against the entirety of the tumor, without a specific emphasis on the malignant epithelia, we developed and characterized a novel protocol for culturing ex vivo (explant) murine PDAC tissue with a corresponding protocol for human PDAC tissue. We demonstrated that these tumor slice explants retain the complex cellular architecture and population complexity throughout culture, making them a useful resource for not only therapeutic screens, but also paracrine interactions, which are infeasible to explore with in vitro and in vivo models. Use of these murine and human PDAC explant models assisted in the selection of a potent, bacterial-derived cytotoxin, theta toxin, as a potential therapeutic candidate for PDAC, in both bacteria lysate and live bacteria contexts. Ultimately, we employed a strain of a probiotic bacteria, E. coli Nissle 1917, as a ‘living drug’ to selectively produce theta toxin within the confines of a PDAC tumor in a mouse model of pancreatic cancer. In in vivo studies, we demonstrated that live bacteria preferentially colonize tumor tissue following a single, direct, intratumoral injection into the primary PDAC tumor. We found that not only did the bacteria colonize the injected tumor, but also translocated to distant regions of metastasis and secondary tumors such as anogenital papillomas. However, the long-term efficacy of this strategy is in question, as bacterial colonization and therapeutic capability waned after several weeks. Despite the limited time scale of the bacterial colonization, treatment with a single dose of live, theta toxin-producing bacteria provided a nearly 3-fold improvement in overall survival compared to vehicle and standard of care chemotherapy (gemcitabine) treatment arms. Preliminary evidence suggests that this improvement is due to a combination of the direct cytotoxic effect of the theta toxin and an inherently immunostimulatory capacity of these bacteria, resulting in an influx of anti-tumor immune cells and an overall reduction in immunosuppression phenotype markers. These findings suggest that bacterial therapy could be a useful tool for the treatment of pancreatic cancer, not solely due to the direct cytotoxic effect on the tumor, but with the potential for a combination treatment with immunotherapies.

Background: Breast cancer is the most common type of cancer among women. A range of treatments are offered, all of which have side effects. Previous research has shown that women suffer from various side effects associated with breast cancer treatment. Knowledge of the women's experiences is needed, to provide support and good care. Aim: The aim of this study was to describe women’s experiences of side effects caused by breast cancer treatment. Method: This literature-based study was based on qualitative research. Friberg's five-step model was used in the analysis of the study. Eleven articles were analysed. Results: Four themes and eleven subthemes were identified. Themes that emerged were A different body, Different everyday life, Adherence to treatment and The need for knowledge and to be seen. A different body describes bodily-, sexual-, and cognitive changes. Different everyday lifedescribes how the women´s daily lives are limited by the different side effects. Adherence to treatment highlights emotions and how the emotions affected the women´s adherence to breast cancer treatment and The need for knowledge and to be seen describes experiences of lack of information and support. Conclusion: Women experience several challenges during breast cancer treatment. By having a holistic perspective and conducting person centred care, side effects can become more manageable, and women's suffering can be alleviated.

Indiana University-Purdue University Indianapolis (IUPUI) / Breast cancer is the second most common cancer among women in the US after skin cancer. While early detection and improved therapy has led to an overall decline in breast cancer mortality, metastatic disease remains largely incurable, indicating a need for improved therapeutic options for patients. Telomeres are repetitive (TTAGGG)n DNA sequences found at the end of chromosomes that protect the ends from recombination, end to end fusions, and recognition as damaged DNA. The enzyme telomerase acts to stabilize short telomeres, preventing apoptosis or senescence due to genomic instability. Telomerase is active in 85-90% of cancers, and inactive in most normal cells, making telomerase an attractive target for cancer therapy. Use of the telomerase-specific, lipidated oligonucleotide GRN163L can antagonize telomerase activity and telomere maintenance in cancer cells by preventing telomerase from binding to telomeres. GRN163L has been shown by our laboratory to inhibit breast cancer cell growth and metastasis in animal models. However, the mechanisms of cancer cell growth and metastatic inhibition via GRN163L are not completely understood. The overall goal of this research project was to further elucidate the role of telomerase in breast cancer cell survival by: 1) determining the effects of combining telomere dysfunction induced by GRN163L with a DNA damage inducer (irradiation); 2) elucidating the mechanisms underlying the cellular response to GRN163L and the effect of combination therapy with the mitotic inhibitor paclitaxel; and 3) testing the hypothesis that a telomerase inhibitor can augment the effects of trastuzumab in breast cancer cells with HER2 amplification. Results support the central hypothesis that the telomere dysfunction, structural and proliferative changes in breast cancer cells induced by GRN163L can synergize with irradiation, paclitaxel, and trastuzumab to inhibit the tumorigenicity of breast cancer cells both in vitro and in vivo. Furthermore, GRN163L can restore sensitivity of therapeutically resistant breast cancer cells to trastuzumab. These results provide insight into the role of telomerase in cancer cell growth. Additionally, implications of this research support GRN163L as an important part of therapeutic regimens for primary tumors, recurrence, and metastatic disease.

Tamoxifen is an effective anti-estrogen for treatment of women with hormonedependent breast cancer but acquired drug resistance limits its therapeutic benefit. We have previously reported that expression of active Akt3 in MCF-7 breast cancer cells results in estrogen-independent tumors that are actually stimulated to grow after tamoxifen treatment. We hypothesize that this tamoxifen resistance may be attributed to binding of different co-regulator proteins and/or different binding affinity of these proteins to the estrogen receptor in M CF-7 cells overexpressing active Akt3 as compared to parental MCF-7 cells. We have immuno-precipitated the estrogen receptor along with bound co-regulator proteins in both cells lines after tamoxifen, estradiol, or vehicle treatment. After 2-D gel electrophoresis separation of these immuno-precipitated proteins and comparing them using PDQuest 2-D analysis software, we identified protein spots that were statistically different under the treatment conditions between the two cell lines. The isolated protein spots were subjected to MALDI-TOF mass spectrometry. By searching protein databases through the MASCOT website for protein identification, we have identified estrogen receptor co-regulator proteins that may play a potential role in tamoxifen resistance. Current studies are focused on addressing the role of differential protein binding as a possible mechanism of tamoxifen resistance in Akt3 over-expressing breast cancer cells.