No / Red deer stags produce an androgen-dependent mane of long hairs only in the breeding season; in the non-breeding season, when circulating androgen levels are low, the neck hair resembles the rest of the coat. This study was designed to determine whether androgen receptors are present in deer follicles throughout the year or only in the mane (neck) follicles when circulating testosterone levels are high in the breeding season. Although androgens regulate much human hair growth the mechanisms are not well understood; they are believed to act on the hair follicle epithelium via the mesenchyme-derived dermal papilla. The location of androgen receptors in the follicle was investigated by immunohistochemistry and androgen binding was measured biochemically in cultured dermal papilla cells derived from mane and flank follicles during the breeding season and from neck follicles during the non-breeding season. Immunohistochemistry of frozen skin sections using a polyclonal antibody to the androgen receptor localised nuclear staining only in the dermal papilla cells of mane follicles. Saturation analysis assays of 14 primary dermal papilla cell lines using [(3)H]-mibolerone demonstrated high-affinity, low-capacity androgen receptors were present only in mane (breeding season neck) cells; competition studies with other steroids confirmed the specificity of the receptors. Androgen receptors were not detectable in cells from either the breeding season flank nor the non-breeding season neck follicles. The unusual biological model offered by red deer of androgen-dependent hair being produced on the neck in the breeding, but not the non-breeding season, has allowed confirmation that androgen receptors are required in follicle dermal papilla cells for an androgen response; this concurs with previous human studies. In addition, the absence of receptors in the non-breeding season follicles demonstrates that receptors are not expressed unless the follicle is responding to androgens. Androgen receptors may be induced in mane follicles by seasonal changes in circulating hormone(s).

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle's, less in Huxley's/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (β-catenin/Lef1) markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways.

Os tecidos da polpa dentária e vibrissa são dois microambientes celulares que compartilham a mesma origem embrionária. Ambos possuem o seu nicho especifico pós-natal que abrigam células tronco adultas (CTA). O objetivo do trabalho foi investigar a expressão diferencial dos marcadores de células pluripotentes, mesenquimais e neuroepiteliais nas populações de células tronco isoladas das vibrissas (CTV) e polpa de dente (CTPD) de camundongos C57BL-6. Resultados obtidos no presente trabalho, utilizando o método de imunofluorescência, revelaram que as CTA de ambos tecidos expressam um amplo painel de marcadores de pluripotência (Oct4, Nanog e Sox2), mesenquimais (CD73, CD90 e CD105), hematopoiético (CD34), crista neural (CKit), neuronal (Nestina) e epitelial (Integrina α6, LGR5 e LGR6) e indica possível potencial destas células em diversas linhagens celulares. Desta forma, células isoladas destes tecidos podem ser interessantes para serem aplicadas em diversos tratamentos na medicina regenerativa. Portanto, o estudo comparativo da expressão de um amplo painel de marcadores de células tronco em CTV e CTPD pode vir a aumentar o leque de possibilidades de sua utilização na terapia celular. Com isso, as células isoladas de polpa dentária foram submetidas à análise de expressão de marcadores já citados e a outros conhecidamente positivos e específicos para os folículos piloso como Citoqueratina 15 (CK15), LRig1 e Blimp1. Foram realizados ensaios de imunofluorescência, imunohistoquímica, citometria de fluxo e RT-PCR. Os dados obtidos nos permitiram concluir, que as CTA isoladas das ambas as fontes são bastante semelhantes em relação ao seu imunofenótipo, porém as características da sua diferenciação precisam ainda ser analisadas. / The tissues of the dental pulp and vibrissae are two cellular microenvironments that share the same embryonic origin. Both have their postnatal specific niche that keep adult stem cells (ASC). The objective of this study was to investigate the differential expression of pluripotent markers, mesenchymal and neuroepithelial in populations of stem cells isolated from whiskers (WSC) and dental pulp (DPSC) C57BL-6 mice. Results obtained in this study, using immunofluorescence, revealed that the ASC both tissues express a broad panel of pluripotency markers (Oct4, Nanog and Sox2), mesenchymal cells (CD73, CD90 and CD105), hematopoietic (CD34), crest neural (cKIT), neuronal (Nestin) and epithelial (Integrin α6, LGR5 and LGR6) and indicates possible potential of these cells in several cell lines. Thus, isolated cells of these tissues may be interesting for application in various treatments in regenerative medicine. Therefore, the comparative study of the expression of a broad panel of stem cell markers in WSC and DPSC could increase the range of possibilities for their use in cell therapy. Thus, the isolated cells were subjected to the dental pulp marker expression analysis cited above and others known to be positive and specific to hair follicles as Cytokeratin 15 (CK15), and LRig1 Blimp1. Immunofluorescence assays were performed, immunohistochemistry, flow cytometry and RT-PCR. The data allowed us to conclude that the ASC isolated from both sources are quite similar with respect to their immunophenotype, but the characteristics of differentiation remain to be analyzed.

Zielsetzung dieser Arbeit war es, das Verhalten der in modernen Sonnenschutzmitteln eingesetzten Titandioxid-Mikropartikel mit quantitativen Methoden zu bestimmen, um Aussagen über ihre Eignung zu erhalten. Dabei stand die Frage nach der Verteilung der Substanz innerhalb des Stratum corneum im Mittelpunkt der Untersuchungen. Insbesondere war wegen der bekannten photokatalytischen Aktivität von Titandioxid zu klären, ob ein Kontakt mit den lebenden Bereichen der Haut ausgeschlossen werden kann. Für die Messungen wurden zwei kommerziell genutzte, unterschiedlich beschichtete Titandioxid-Mikropartikel eingesetzt. Eine wesentliche Voraussetzung für diese Untersuchungen war die Anwendung der Abrissmethode (Tape stripping) in Kombination mit der spektroskopischen Bestimmung der Extinktion im sichtbaren Bereich zur Berechnung des Hornschichtprofils. Die Konzentration der Titandioxid-Partikel wurde mit Hilfe von Röntgenfluoreszenz-Messungen bestimmt. So war es erstmals möglich, in vivo standardisierte und reproduzierbare Untersuchungen zum Penetrationsverhalten von beschichteten Titandioxid-Mikropartikeln in die Hornschicht der menschlichen Haut durchzuführen. Durch Langzeitapplikation der Substanzen und die Beobachtung der Titandioxid-konzentrationen in der Hornschicht über mehrere Tage konnten auch Aussagen zum Penetrationsverhalten der applizierten Mikropartikel gemacht werden. Es wurde eindeutig gezeigt, dass die untersuchten Substanzen unabhängig von ihrer Struktur, von ihrer Beschichtung und vom Probanden hauptsächlich in den obersten Schichten des Stratum corneum lokalisiert sind. Nach Klärung dieser grundsätzlichen Fragen war es notwendig, die Ursache für das Auftreten extrem geringer Titandioxid-Konzentrationen auf Abrissen zu bestimmen, die aus tieferen Schichten des Stratum corneum entnommen wurden. Durch die Kombination der Abrissmethode mit einem speziellen Färbeverfahren und der Laser-Scan-Mikroskopie ergaben sich deutliche Hinweise auf die Bedeutung der Follikelkanäle für das beobachtete Phänomen. Röntgenspektroskopische Untersuchungen an Biopsien zeigten, dass diese Mikropartikel in die Haarfollikel eindringen und damit Bereiche unterhalb des Stratum corneum erreichen. Hierbei wurden die Mikropartikel in dieser Region nur in Follikelkanälen, nicht aber im Bereich der lebenden Zellen nachgewiesen. Diese Ergebnisse belegen, dass Titandioxid nur in einzelne Follikelkanäle penetriert, eine Aussage, die im Rahmen der vorliegenden Untersuchungen erstmals beschrieben wurde. In Übereinstimmung mit der Zielsetzung der Arbeit konnte unter Einsatz unterschiedlicher Untersuchungsmethoden gezeigt werden, dass die beschichteten Titandioxid-Mikropartikel im oberen Bereich der Hornschicht lokalisiert sind und damit als hocheffiziente Lichtschutzfilter den Schutz der darunter liegenden lebenden Bereiche der Haut garantieren. Der sichere Nachweis der Titandioxid-Mikropartikel innerhalb einzelner Follikelkanäle besitzt grundsätzliche Bedeutung für das Verständnis von Penetrationswegen. / The objective of this work was to determine the behaviour of titanium dioxide microparticles, used in modern sunscreen agents, with quantitative methods in order to receive statements about their suitability. The focal point of the investigations was the question on the distribution of the substance within the stratum corneum. Because of the well-known photocatalytic activity of titanium dioxide, our aim was to clarify whether a contact with the living cells of the skin could be excluded. For these measurements, two commercial titanium dioxide micro particles were used, covered with different coating materials. The calculation of the stratum corneum profile was a substantial prerequisite for these investigations. This was done by application of the tape stripping method in combination with the spectroscopic determination of the absorption in the visible range. The concentration of the titanium dioxide particles on the tape strips was determined by X-ray fluorescence measurements. In this manner it was possible for the first time, to perform standardized and reproducible in vivo measurements to investigate the penetration behaviour of coated titanium dioxide micro particles into the horny layer of the human skin. Predications could be made about the penetration behaviour of the applied micro particles by observation of the titanium dioxide concentrations in the stratum corneum during several days after long-term application of the substances. It could be shown that the examined substances were localized mainly in the upper layers of the stratum corneum, independently of their structure, coating and the volunteers. After clarifying these basic questions, it was necessary to explain the occurrence of extremely small concentrations of titanium-dioxide on tape strips, which were taken from deeper layers of the stratum corneum. This observed phenomenon was investigated by combining tape stripping with a selective staining of the tapes and with laser scanning microscopy. Investigations of a biopsy with x ray fluorescence measurements showed that the micro particles penetrate into the hair follicles, and in this manner reached areas below the stratum corneum. In this region, the micro particles were found only in follicle channels, but not within the area of the living cells. These results prove that titanium dioxide micro particles only penetrate into single follicle channels, a statement, which has been described here for the first time. In agreement with the objective of this thesis, it was shown, that the coated titanium dioxide micro particles were localized within the upper area of the horny layer and as high efficient sunscreen filters consequently guarantee, the protection of the living areas of the skin below. The proof of the titanium dioxide micro particles within individual follicle channels has basic importance for the understanding of the penetration pathways.

INTRODUÇÃO: A alopecia frontal fibrosante (AFF) é uma alopecia cicatricial primária linfocítica descrita em 1994, cuja prevalência vem aumentando rapidamente em todo mundo. A participação de um fator desencadeante ambiental na patogênese da doença é aventada e uma pesquisa recente encontrou uma associação da doença com o uso de cosméticos faciais. Alterações da pele e dos pelos da face e do corpo têm sido descritas em pacientes com AFF nos últimos anos e alguns estudos sugerem que essas alterações possam preceder a perda dos cabelos, indicando o início da doença fora do couro cabeludo. OBJETIVO: Estudar o acometimento da pele e dos pelos na face, pescoço e região anterossuperior do tórax em uma série de pacientes com AFF. MATERIAL E MÉTODOS: A pesquisa constou de três partes. Na primeira parte foram investigadas evidências clínicas e dermatoscópicas do acometimento da pele e dos pelos fora do couro cabeludo em 37 pacientes. A segunda parte do estudo constou da avaliação da espessura epidérmica em biópsias realizadas na face, pescoço e região anterossuperior do tórax de 20 pacientes com AFF e 20 controles. Na terceira parte do estudo foi utilizada microscopia confocal de reflectância a laser \"in vivo\" para comparar a espessura epidérmica e a densidade folicular da pele da linha de implantação frontal de 21 pacientes a de 21 controles. RESULTADOS: O acometimento dos pelos velos da face não se restringiu a linha de implantação fronto-temporal e variou de 30 a 97% dependendo da região estudada, sendo mais frequente quanto mais próximo da linha de implantação frontal do couro cabeludo. Pápulas da face foram encontradas em 60% dos pacientes estudados, localizadas principalmente na região temporal (11/37 casos), seguida pela região malar (10/37 casos) e mento (6/37 casos). Metade dos pacientes (51%) apresentaram lesões hipercrômicas compatíveis com o diagnóstico de líquen plano pigmentoso associado a AFF, acometendo face (18/19 casos), pescoço (7/19 casos) e região anterossuperior do tórax (4/19 casos). As lesões hipercrômicas mostraram-se mais raras em pacientes com fototipo baixo (p=0,022). A espessura da epiderme dos pacientes de AFF não apresentou diferença quando comparada com a dos controles independente da metodologia utilizada. Densidade folicular menor que 3,56 folículos/mm2 na linha de implantação frontal ao exame de microscopia confocal apresentou 90,5% de sensibilidade e 90,5% de especificidade para o diagnóstico de AFF e implicou num risco 90,24 (IC95% 9,5-1132; p < 0,001) vezes maior de ter a doença. CONCLUSÕES: O acometimento dos pelos velos da face é frequente e pode ser detectado de forma rápida e não invasiva pela dermatoscopia. As pápulas da face estão presentes em 60% dos pacientes. As lesões de liquen plano pigmentoso são menos frequentes em pacientes com fototipos baixos. A epiderme dos pacientes de AFF não apresenta uma menor espessura quando comparada com controles pareados por gênero, idade, fototipo e local examinado. A densidade folicular da linha de implantação frontal \"in vivo\" medida através do exame de MCRL apresenta ótima acurácia para o diagnóstico de AFF / INTRODUCTION: Frontal fibrosing alopecia (FFA) is a lymphocytic primary cicatricial alopecia first described in 1994. Its incidence has been rapidly rising worldwide, possibly related to an environmental trigger. The use of facial leave-on creams has been associated with the disease in a recent publication. Vellus follicles involvement and epidermal changes outside the scalp region have been described in FFA patients in the past few years and seem to be an early event in the disease course. OBJECTIVES: To evaluate vellus follicle and epidermal involvement over the facial, neck and upper chest skin in a series of FFA patients. METHODS: This study consisted of three parts. In the first part, prevalence of clinical and dermoscopic findings related to vellus follicle and epidermal involvement in 37 FFA patients was investigated. In part two, epidermal thickness in skin biopsies from 20 FFA patients was compared with 20 control biopsies from the same body site. In the last part, epidermal thickness and follicular density over the frontal hairline were investigated in a group of 21 FFA patients and 21 gender, age and phototype matched controls through \"in vivo\" reflectance confocal microscopy. RESULTS: Vellus follicle involvement in FFA is not restricted to frontal hairline and varies from 30 to 97% according to facial region, with greater frequencies observed on the upper face region. Facial papules were detected in 60% of our patients, most frequently over the temples (11/37 patients), malar (10/37 patients) or chin (6/37 patients) area. Half of our patients (51%) presented hyperchromic lesions compatible with FFA associated lichen planus pigmentosus. Hyperchromic lesions were observed over the face (18/19 patients), but also over the neck (7/19 patients) and upper chest (4/19 patients) skin. Hyperchromic lesions were less frequent in patients with lighter phototypes (p=0.022). Epidermal thickness of FFA patients did not differ from controls both in histology and \"in vivo\" evaluation. Frontal hairline follicular density lower than 3.56 follicles/mm2 on confocal microscopy examination presented 90.5% sensitivity, 90.5% specificity and OR = 90.24 (CI95% 9.5-1132; p < 0.001) for FFA diagnosis. CONCLUSIONS: Facial vellus follicle involvement is frequent and can be easily detected through dermoscopy in most patients. Facial papules are observed in 60% of our patients. Lichen planus pigmentosus lesions are less frequently observed in fair skin patients. Epidermal thinning is not observed in FFA patients when adequate control group is included. Frontal hairline follicular density measured by confocal microscopy has high accuracy for FFA diagnosis

INTRODUÇÃO: A alopecia frontal fibrosante (AFF) é uma alopecia cicatricial primária linfocítica descrita em 1994, cuja prevalência vem aumentando rapidamente em todo mundo. A participação de um fator desencadeante ambiental na patogênese da doença é aventada e uma pesquisa recente encontrou uma associação da doença com o uso de cosméticos faciais. Alterações da pele e dos pelos da face e do corpo têm sido descritas em pacientes com AFF nos últimos anos e alguns estudos sugerem que essas alterações possam preceder a perda dos cabelos, indicando o início da doença fora do couro cabeludo. OBJETIVO: Estudar o acometimento da pele e dos pelos na face, pescoço e região anterossuperior do tórax em uma série de pacientes com AFF. MATERIAL E MÉTODOS: A pesquisa constou de três partes. Na primeira parte foram investigadas evidências clínicas e dermatoscópicas do acometimento da pele e dos pelos fora do couro cabeludo em 37 pacientes. A segunda parte do estudo constou da avaliação da espessura epidérmica em biópsias realizadas na face, pescoço e região anterossuperior do tórax de 20 pacientes com AFF e 20 controles. Na terceira parte do estudo foi utilizada microscopia confocal de reflectância a laser \"in vivo\" para comparar a espessura epidérmica e a densidade folicular da pele da linha de implantação frontal de 21 pacientes a de 21 controles. RESULTADOS: O acometimento dos pelos velos da face não se restringiu a linha de implantação fronto-temporal e variou de 30 a 97% dependendo da região estudada, sendo mais frequente quanto mais próximo da linha de implantação frontal do couro cabeludo. Pápulas da face foram encontradas em 60% dos pacientes estudados, localizadas principalmente na região temporal (11/37 casos), seguida pela região malar (10/37 casos) e mento (6/37 casos). Metade dos pacientes (51%) apresentaram lesões hipercrômicas compatíveis com o diagnóstico de líquen plano pigmentoso associado a AFF, acometendo face (18/19 casos), pescoço (7/19 casos) e região anterossuperior do tórax (4/19 casos). As lesões hipercrômicas mostraram-se mais raras em pacientes com fototipo baixo (p=0,022). A espessura da epiderme dos pacientes de AFF não apresentou diferença quando comparada com a dos controles independente da metodologia utilizada. Densidade folicular menor que 3,56 folículos/mm2 na linha de implantação frontal ao exame de microscopia confocal apresentou 90,5% de sensibilidade e 90,5% de especificidade para o diagnóstico de AFF e implicou num risco 90,24 (IC95% 9,5-1132; p < 0,001) vezes maior de ter a doença. CONCLUSÕES: O acometimento dos pelos velos da face é frequente e pode ser detectado de forma rápida e não invasiva pela dermatoscopia. As pápulas da face estão presentes em 60% dos pacientes. As lesões de liquen plano pigmentoso são menos frequentes em pacientes com fototipos baixos. A epiderme dos pacientes de AFF não apresenta uma menor espessura quando comparada com controles pareados por gênero, idade, fototipo e local examinado. A densidade folicular da linha de implantação frontal \"in vivo\" medida através do exame de MCRL apresenta ótima acurácia para o diagnóstico de AFF / INTRODUCTION: Frontal fibrosing alopecia (FFA) is a lymphocytic primary cicatricial alopecia first described in 1994. Its incidence has been rapidly rising worldwide, possibly related to an environmental trigger. The use of facial leave-on creams has been associated with the disease in a recent publication. Vellus follicles involvement and epidermal changes outside the scalp region have been described in FFA patients in the past few years and seem to be an early event in the disease course. OBJECTIVES: To evaluate vellus follicle and epidermal involvement over the facial, neck and upper chest skin in a series of FFA patients. METHODS: This study consisted of three parts. In the first part, prevalence of clinical and dermoscopic findings related to vellus follicle and epidermal involvement in 37 FFA patients was investigated. In part two, epidermal thickness in skin biopsies from 20 FFA patients was compared with 20 control biopsies from the same body site. In the last part, epidermal thickness and follicular density over the frontal hairline were investigated in a group of 21 FFA patients and 21 gender, age and phototype matched controls through \"in vivo\" reflectance confocal microscopy. RESULTS: Vellus follicle involvement in FFA is not restricted to frontal hairline and varies from 30 to 97% according to facial region, with greater frequencies observed on the upper face region. Facial papules were detected in 60% of our patients, most frequently over the temples (11/37 patients), malar (10/37 patients) or chin (6/37 patients) area. Half of our patients (51%) presented hyperchromic lesions compatible with FFA associated lichen planus pigmentosus. Hyperchromic lesions were observed over the face (18/19 patients), but also over the neck (7/19 patients) and upper chest (4/19 patients) skin. Hyperchromic lesions were less frequent in patients with lighter phototypes (p=0.022). Epidermal thickness of FFA patients did not differ from controls both in histology and \"in vivo\" evaluation. Frontal hairline follicular density lower than 3.56 follicles/mm2 on confocal microscopy examination presented 90.5% sensitivity, 90.5% specificity and OR = 90.24 (CI95% 9.5-1132; p < 0.001) for FFA diagnosis. CONCLUSIONS: Facial vellus follicle involvement is frequent and can be easily detected through dermoscopy in most patients. Facial papules are observed in 60% of our patients. Lichen planus pigmentosus lesions are less frequently observed in fair skin patients. Epidermal thinning is not observed in FFA patients when adequate control group is included. Frontal hairline follicular density measured by confocal microscopy has high accuracy for FFA diagnosis

The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to ii hair follicles, suggesting substantial differences in global genome rearrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factormediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin.

Abstract Skin stem cells are essential for maintaining epidermal homeostasis by providing new cells to replace those that are lost during normal tissue turnover and repair after injury. Adult epidermal stem cells serve also as the cells of origin for different types of skin cancers. This PhD study investigates the hair follicle (HF) stem cells and skin cancer, and the microenviromental factors that regulate the initiation of tumors and their progression to malignancy. Collagen XVIII is a common basement membrane multidomain proteoglycan highly expressed in HF stem cells (HFSCs) and in malignant skin squamous carcinoma cells. It is known to be essential for the development of the eye, and mutations in the COL18A1 gene cause a rare disease, Knobloch syndrome, with severe eye defects. However, the roles of collagen XVIII in other tissues and diseases are not well understood. Using genetically modified mice, we show here that collagen XVIII is important for the structure and formation of hemidesmosomes, the junctional complexes between epidermal cells and basement membrane. We propose that the disturbed adhesion of the HFSCs to the underlying BM in mice with collagen XVIII ablation leads to defects in stem cell viability and HF cycling. Overexpression of the N-terminal noncollageous sequences of collagen XVIII were sufficient to rescue the abnormal HF phenotype observed in the absence of collagen XVIII. We also found that mice lacking this collagen develop fewer chemical-induced skin tumors, which we suggest is due to increased apoptosis in skin stem cells upon carcinogen-induced DNA damage, and to reduced stemness of tumor cells. In another project we studied the functions of Bmx, a cytoplasmic protein tyrosine kinase, which is upregulated in different types of cancer. However, little is known about the pathways that Bmx regulates in tumors. Therefore, we investigated its roles in cancer using syngeneic tumor assays, and chemical and genetic experimental tumor models. We show here that Bmx promotes tumor growth and progression and induces tumor angiogenesis by contributing to the transduction of VEGF signals. / Tiivistelmä Ihon kantasoluilla on tärkeä tehtävä ihon epidermiksen tasapainon (homeostaasin) ylläpitämisessä. Kantasolut jakautuvat tarvittaessa ja tuottavat uusia erilaistuneita soluja sekä ihon pintasolukon normaalin uusiutumisen että vahingoittuneen kudoksen korjaamisen yhteydessä. Ihon kantasoluilla uskotaan olevan myös tärkeä rooli erilaisten ihosyöpien synnyssä. Tässä tutkimuksessa tutkittiin ihon karvatupen kantasoluja ja ihosyöpää, ja selvitettiin kantasoluja ympäröivän kudoksen, mikroympäristön (kantasolulokero), merkitystä ihosyövän synnyssä ja etenemisessä. Kollageeni XVIII on useista toiminnallisista osista koostuva tyvikalvojen proteoglykaani, joka ilmenee voimakkaasti ihon karvatupen kantasoluissa, sekä ihon pahanlaatuisissa levyepiteelikarsinoomasoluissa. Vaikka kollageeni XVIII:n biologiset tehtävät elimistössä ovat vielä jokseenkin epäselviä, sen tiedetään olevan välttämätön silmän kehittymiselle. Kollageeni XVIII:n geenimutaatioiden on osoitettu aiheuttavan Knoblochin oireyhtymän, jota sairastavilla potilailla on muutoksia silmän rakenteessa. Tässä väitöskirjatyössä hyödynnettiin useita muuntogeenisiä hiirimalleja ja osoitettiin, että kollageeni XVIII säätelee merkittävästi ihon karvatupen erilaistuvien solujen elinkykyä ja karvojen kasvukiertoa. Kollageeni XVIII:n puutos aiheutti muutoksia karvatupen pullistuma-alueen (bulge) liitoskomplekseissa (hemidesmosomi), jotka ankkuroivat alueen tyvisolut karvatuppea ympäröivään tyvikalvoon. Tulokset viittaavat siihen, että puutteelliset liitokset solujen ja tyvikalvon välillä ja niistä johtuvat signaalinvälityksen häiriöt voivat aiheuttaa muutoksia karvatupen solujen ominaisuuksissa. Kollageenin XVIII:n aminoterminaalisen osan tuottaminen poistogeenisen hiiren ihossa riitti palauttamaan hemidesmosomit, karvatupen solujen elinkyvyn ja karvasyklin normaaliksi. Työssä havaittiin myös, että kollageeni XVIII puute vähensi kemikaalikäsittelyillä aiheutettujen ihokasvainten määrää hiirillä, mitä voidaan osaltaan selittää sillä, että kollageeni XVIII:n puutos lisäsi DNA-vaurioista kärsivien erilaistuvien solujen ohjelmoitua solukuolemaa (apoptoosi) ja vähensi kasvainsolujen kantasoluominaisuuksia. Tässä työssä tutkittiin myös monissa syövissä voimakkaasti ilmenevän solunsisäisen signaalinvälittäjän tyrosiinikinaasi Bmx:n tehtäviä syövässä hyödyntäen istutettavia kasvainsoluja sekä kemiallista ja geneettisiä kokeellisia syöpämalleja muuntogeenisillä hiirillä. Tutkimuksessa ositettiin Bmx:n osallistuvan verisuonikasvutekijän signaalinvälitykseen ja täten edistävän syöpäkasvainten verisuonten uudismuodostusta sekä syövän kasvua ja etenemistä.

Abstract Collagen XVIII is a multidomain basement membrane proteoglycan with three tissue-specific isoforms. Endostatin, the C-terminal part of collagen XVIII, has antiangiogenic properties, while the frizzled-like domain of the longest isoform is suggested to be capable of inhibiting the Wnt/β-catenin signaling network. This study utilized several genetically modified mouse lines and electron microscopy to achieve new information on the biological role of collagen XVIII, its different isoforms, and the frizzled domain. Lack of collagen XVIII was found to affect the integrity of basement membranes of various tissues, leading to an abnormally loosened network structure. In the choroid plexus, the change in the basement membrane ultrastructure caused alterations in the production of the cerebrospinal fluid and predisposed to the development of hydrocephalus. In the kidney, broadening of the proximal tubular basement membrane was shown to be due specifically to the lack of the short isoform, while the lack of the two longer isoforms led to podocyte foot process effacement. Moreover, lack of collagen XVIII was found to cause softening of the kidney glomeruli and the levels of serum creatinine were elevated in the mutant animals, indicating altered kidney function. The hair follicle cycle was used as a model to study the possible role of the frizzled domain of collagen XVIII in the Wnt/β-catenin signaling cascade. The longer collagen XVIII isoforms were shown to be expressed in the basement membrane facing the dermal papilla and in the hair follicle bulge, containing the follicular stem cells. Lack of the long isoforms led to abnormalities in the progression of the first hair cycle, and the phenotype could be rescued via transgenic delivery of the frizzled domain of the longest isoform, suggesting its involvement in the regulation of the Wnt/β-catening signaling network during the cyclic growth of the hair. / Tiivistelmä Kollageeni XVIII on useista toiminnallisista osista koostuva tyvikalvojen proteoglykaani, jolla on kolme eri kudoksissa esiintyvää isomuotoa. Sen C-terminaalisella endostatiini-osalla on verisuonten kasvua estäviä vaikutuksia, kun taas pisimmän isomuodon frizzled-osan uskotaan estävän Wnt/β-kateniini signalointireitin toimintaa. Tässä tutkimuksessa saatiin uutta tietoa kollageeni XVIII:n, sen eri isomuotojen sekä frizzled-osan biologisesta merkityksestä useiden geenimuunneltujen hiirimallien sekä elektronimikroskopian avulla. Kollageeni XVIII:n puutoksen todettiin vaikuttavan tyvikalvojen rakenteen eheyteen useissa eri kudoksisssa, johtaen epänormaalisti löyhtyvään verkkorakenteeseen. Suonipunoksessa tämä tyvikalvon hienorakenteen muutos vaikutti aivo-selkäydinnesteen tuottumiseen ja altisti vesipään kehittymiselle. Munuaisessa proksimaalisen munuaistiehyen tyvikalvon levenemisen osoitettiin johtuvan lyhyen isomuodon puutoksesta, kun taas kahden pidemmän isomuodon puuttuminen aiheutti podosyyttien jalkalisäkkeiden leviämistä. Lisäksi kollageeni XVIII:n puuttumisen osoitettiin johtavan hiirimallien munuaiskerästen pehmenemiseen sekä veren kreatiniinitason kohoamiseen, viitaten munuaistoiminnan häiriöihin. Karvatuppien syklistä kasvua käytettiin mallina tutkittaessa kollageeni XVIII:n frizzled-osan mahdollisia vaikutuksia Wnt/β-kateniini signalointireittiin. Pidempien kollageeni XVIII isomuotojen osoitettiin tuottuvan karvanystyn tyvikalvossa sekä karvatupin kantasolut sisältävällä pullistuma-alueella. Pitkien isomuotojen puuttuminen johti karvojen ensimmäisen kasvukierron epänormaaliin etenemiseen. Tämä voitiin estää siirtogeenisen frizzled-osan avulla, mikä viittasi sen osallisuuteen Wnt/β-kateniini signalointireitin säätelyyn karvan syklisen kasvun aikana.