De part leurs nombreuses activités biologiques et leur propriétés physicochmiques et structurales, les peptides présentent un intérêt considérable pour la conception de molécules actives mais aussi pour l'élaboration de biomatériaux. Pour lutter contre l'alopécie (perte de cheveux) et la canitie (blanchiment des cheveux), nous avons axé nos travaux sur la recherche de peptides bioactifs. Pour cela, nous avons identifié des peptides têtes de série provenant soit de la littérature soit d'un criblage réalisé par l'institut européen de biologie (IEB). Ces peptides têtes de séries ont été modifiés afin d'améliorer leur activité et leur biodisponibilité tout en tenant compte du mode d'administration par voie topique. Lors de ce travail, nous avons également développé deux nouvelles méthodologies permettant la polymérisation de séquences peptidiques. En effet, les polymères à base de peptide présentent un intérêt majeur pour des applications en biotechnologie (tissus artificiels, implants), ou comme systèmes de transport ou de délivrance de principes actifs. Nous avons notamment mis au point la polymérisation de peptides hybrides présentant des fonctions dimethylsilanol ainsi que la polymérisation par ouverture du cycle de N-carboxyanhydrides portant une séquence peptidique. Ces deux stratégies ont permis d'obtenir des polymères linéaires ou en peigne. / Because of their numerous biological activities and their structural and physico-chemical properties, peptides are of considerable interest for the design of active molecules but also for the development of biomaterials. To fight against alopecia (hair loss) and canities (whitning hair), we focused our attention on the research of bioactive peptides. In this context, we have identified leads peptides either from the literature or from a screening conducted by the European Institute of Biology (IEB). These Leads were modified to improve their activity and bioavailability knowing that they will be applied topically. In this work, we have also developed two new methodologies for the polymerization of peptide sequences. Indeed, peptide-based polymers are of major interest for applications in biotechnology (i.e. artificial tissue,implants), or as systems of transport and delivery of drug. The first methodology relies on the polymerization of the hybrid peptides displaying dimethyl hydroxysilane functions. The other one involves the ring opening polymerization of N-carboxyanhydrides bearing a peptide sequence. Both strategies were used to obtain linear or comb peptide-polymers.

Bröstcancer är den vanligaste cancerformen bland kvinnor och den näst vanligaste orsaken till död av cancer hos alla kvinnor. Kemoterapi är den viktigaste behandlingen för många maligna tumörer, men förutom deras fördelaktiga resultat orsakar de också relaterad toxicitet och biverkningar. Syftet med litteraturstudien var att belysa hur kvinnor med bröstcancer upplever alopeci till följd av kemoterapi. Det induktiva tillvägagångssättet användes då fokus låg på att förstå mänsklig erfarenhet inom det utvalda området genom att samla fakta. Med hjälp av innehållsanalys analyserades tio kvalitativa vetenskapliga artiklar. Litteraturstudiens resultat presenteras i fem kategorier: alopeci-synen på sig själv, alopeci-andras reaktioner, vikten av att få information om alopeci, strategier för hantering av alopeci, omgivnings betydelse. I resultat framkom att alopeci är en traumatisk upplevelse för de flesta bröstcancerpatienter. Alopeci orsakade en minskning av självkänslan bland kvinnor och ett ökat missnöje med utseendet. Något som också hade en negativ effekt på kvinnor i förhållande till alopeci var att de upplevde att deras identitet förändrades till ”cancerpatient”. / Breast cancer is the most common form of cancer among women and the second most common cause of death in cancer among women. Chemotherapy is the most important treatment for many malignant tumors, but in addition to their results, they also cause related toxicity and side effects. The purpose of the literature study was to shed light on how women with breast cancer experience alopecia as a result of chemotherapy. The inductive approach was used when the focus was on understanding human experience in the selected area by gathering facts. With the help of content analysis, ten qualitative scientific articles were analyzed. The result of the literature study is presented in five categories: the alopecia view of oneself, the reactions of alopecia by others, the importance of getting information about alopecia, strategies for managing alopecia, the significance of the environment. The results showed that alopecia is a traumatic experience for most breast cancer patients. Alopecia caused a decrease in self-esteem among women and increased dissatisfaction with appearance. Something that also had a negative effect on women in relation to alopecia was that they experienced that their identity was changed to "cancer patient".

Understanding the genetic architecture of complex disorders is important for identifying disease mechanisms and potential molecular targets for therapeutic interventions. Genetic diseases are broadly classified as either Mendelian (monogenic) diseases or complex (polygenic) diseases. Common, polygenic disorders result from inheritance of multiple common variants with low penetrance. In contrast, monogenic, Mendelian disorders are caused by rare variants with high penetrance at a single genetic locus. However, an increasing number of studies support a role for rare variants of moderate effect size in complex diseases. As a result, genetic approaches previously utilized for discovering rare variants in Mendelian diseases, such as next generation sequencing, can now effectively be applied to complex diseases to define the contribution of both rare and common variants to the genetic burden of polygenic traits and diseases. Alopecia Areata (AA) is a complex autoimmune disease characterized by non-scarring hair loss that is due to a combination of both enviornmental and genetic factors. Our previous Genome-wide Association Study (GWAS) identified at least 14 genetic regions contributing to AA disease susceptibility. Although useful in identifying disease-associated loci and surrounding linkage disequilibrium (LD) blocks, GWAS is not sufficiently granular to 1) elucidate causal (association-driving) variants; and 2) discover rare risk variants. This level of resolution can only be achieved by deep sequencing followed by functional validation of variants. The goal of this thesis was to address these challenges in AA using two genetic approaches that have not been previously utilized in the context of this disease. In Chapter 2, I performed a hypothesis-driven analysis of common variants in a GWAS-associated locus using targeted genomic sequencing. In Chapter 3, I utilized whole exome sequencing (WES) in an unbiased approach to assess rare variant contribution in AA disease risk. To conduct these analyses simultaneously, we designed a whole exome sequencing (WES) chip that also included custom capture of 24 Mb of genomic regions covering the 14 genetic loci previously identified using GWAS. I applied these two sequencing approaches in a large AA patient cohort and identified potentially causal variants in several genes. To interrogate the consequences of these variants, I performed functional analyses to determine the effects of disease-causing variants on the target organ of AA attack, the hair follicle (HF). In Chapter 2, I report on the use of targeted genomic sequencing to interrogate the coding and non-coding regions surrounding a previously implicated GWAS locus. This approach provided fine mapping of coding and non-coding common variants in regions that may be contributing to disease risk. In this thesis, I focused on the effect of genetic perturbations on the end-organ HF, and consequentially prioritized my functional analysis using two criteria: 1) genes expressed in the (HF) and; 2) GWAS regions that were not previously implicated in other autoimmune diseases. One of the regions that satisfied these criteria harbored the Syntaxin 17 (STX17) gene, which encodes a SNARE protein involved in autophagy and mitochondrial fission. Targeted genomic sequencing of the STX17 region in 849 AA cases identified 35 non-coding and 0 coding variants in high LD with the GWAS SNP. Thirty-three variants were significantly enriched in cases compared to controls, and the remaining two were nominally significant. Thirty-two of the significantly associated AA variants were confirmed to be AA skin eQTLs that downregulated expression of STX17 in affected scalp skin of AA patients. Downstream analyses incorporated in silico and functional cell assays that uncovered a novel autophagy-independent role for STX17 in melanocyte biology. I discovered that a reduction of STX17 expression was associated with an accumulation of a melanocyte-specific antigen and increased immunogenicity, as seen by CD8+ T cell infiltrates in the skin of AA patients with low levels of STX17 expression. I used a targeted sequencing approach to successfully identify candidate causal variants driving the GWAS association at the STX17 locus, and propose a novel mechanism underlying STX17-dependent melanocyte perturbation and AA disease. In the second section of this thesis, we used the WES feature of the chip to assess the genetic contribution of rare variation in AA, in a genome-wide and unbiased manner. WES data and gene-level burden analyses of 18,653 genes in 849 AA patients was compared to 15,640 controls to identify rare variants associated with AA. Unexpectedly, this analysis identified one gene, encoding a hair-specific keratin, Keratin 82 (KRT82) that harbored significantly more rare damaging mutations in AA cases compared to controls (p=2.68E-06). Eleven rare damaging mutations were found in 51 AA patients in the heterozygous state (6.01%) compared to 2.58% controls. These variants resided in evolutionary conserved amino acid residues, and nine out of the eleven mutations were located in established disease-causing domains in keratin proteins. I determined that KRT82 expression was absent or largely reduced in AA hair follicles, including the bulb region, the site of AA immune attack. Moreover, AA patients with damaging variants and reduced KRT82 expression had increased perifollicular CD8+ T cell infiltrates in comparison to control HFs with intact KRT82 expression remaining. I proposed that damaging mutations in the coding regions of KRT82 resulted in loss of functional protein, thereby weakening the protective HF cuticle and predisposing the HF to immune attack. In summary, I used two genetic approaches (targeted genomic sequencing and WES) to identify common (Chapter 2) and rare variants (Chapter 3) with novel contributions to the complex genetic architecture of AA. I focused my functional studies on genes expressed in the target HF, with the goal of defining the role of unidentified, variant-mediated end-organ disruption in the predisposition of AA patient HFs to aberrant autoimmune attack. Up to now, most efforts in AA mechanistic studies have focused on the aberrant immune response. The work in my thesis uncovered novel roles for perturbations in the HF itself as a participating factor in AA disease risk.

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.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders. / Saudi government

Kurdistan Regional Government/Ministry of Higher Education and Scientific Research

Lack of hair due to alopecia or skin grafting procedures causes significant distress due to hair's role in social and sexual communication. Only limited pharmacological agents are currently available to stimulate hair growth; their development is hampered by inappropriate model systems. Most research involves large terminal scalp follicles rather than the clinical targets of tiny vellus or intermediate follicles. The overall aim of this thesis was to develop a novel model system based on intermediate hair follicles. Initially, intermediate follicles from female pre-auricular skin were characterised and compared to matched terminal follicles. Intermediate follicles were smaller, less pigmented, shorter and possessed a more 'tubular' bulb morphology than their more 'bulbous' terminal counterparts. Significant correlations were demonstrated between various hair follicle measurements and corresponding dermal papilla diameters. Isolated terminal follicles grew significantly more than intermediate hair follicles in organ culture for 9 days. Testosterone (10nM), the major regulator of human hair growth, increased only intermediate follicle growth; the anti-androgen, cyproterone acetate (1¿M), prevented this stimulation, unlike the 5¿-reductase type 2 inhibitor finasteride (40ng/ml). Immunohistochemistry demonstrated androgen receptor and 5¿-reductase type 2 proteins in both follicle types, while quantitative real-time PCR and gene microarray analysis detected their increased gene expression in intermediate follicles. Thus, smaller intermediate follicles showed major morphological and gene expression differences to terminal follicles in vivo and retained significant, biologically-relevant differences in vitro in organ culture including androgen-responsiveness. Therefore, intermediate hair follicles offer a novel, exciting, more clinically relevant, albeit technically difficult, model for future investigations into hair growth.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13497号 / 論農博第2901号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5403(農学部図書室) / (主査)教授 谷 史人, 教授 佐々木 努, 教授 保川 清 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No / Hair diversity, its style, colour, shape and growth pattern is one of our most defining characteristics. The natural versus temporary style is influenced by what happens to our hair during our lifetime, such as genetic hair loss, sudden hair shedding, greying and pathological hair loss in the various forms of alopecia because of genetics, illness or medication. Despite the size and global value of the hair care market, our knowledge of what controls the innate and within-lifetime characteristics of hair diversity remains poorly understood. In the last decade, drivers of knowledge have moved into the arena of genetics where hair traits are obvious and measurable and genetic polymorphisms are being found that raise valuable questions about the biology of hair growth. The recent discovery that the gene for trichohyalin contributes to hair shape comes as no surprise to the hair biologists who have believed for 100 years that hair shape is linked to the structure and function of the inner root sheath. Further conundrums awaiting elucidation include the polymorphisms in the androgen receptor (AR) described in male pattern alopecia whose location on the X chromosome places this genetic contributor into the female line. The genetics of female hair loss is less clear with polymorphisms in the AR not associated with female pattern hair loss. Lifestyle choices are also implicated in hair diversity. Greying, which also has a strong genetic component, is often suggested to have a lifestyle (stress) influence and hair follicle melanocytes show declining antioxidant protection with age and lowered resistance to stress. It is likely that hair research will undergo a renaissance on the back of the rising information from genetic studies as well as the latest contributions from the field of epigenetics.

No / Although we have made significant progress in understanding the regulation of the UVR-exposed epidermal-melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR-shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis-follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub-populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species-specific issues involved which the general reader of the pigmentation literature (with its substantial mouse-based data) may not fully appreciate.