Adult Stem Cells (SCs) have been found in almost every organ. They are responsible for<p>homeostasis and tissue repair after injury. SCs reside and self-renew in the adult body<p>throughout the life of the organism. In rapid self-renewing organs, such as the skin, the<p>intestine and the blood, SCs divide many times during the life of the animal in order to sustain<p>the homeostatic needs of the tissue.<p>All cells of the body, including SCs, are constantly subjected to DNA assaults arising from<p>endogenous sources, such as reactive oxygen species (ROS) generated by cellular<p>metabolism, or exogenous assaults arising from the environment. The DNA damage response<p>(DDR) and DNA repair mechanisms protect cells from accumulating DNA damage by<p>inducing transient cell cycle arrest allowing DNA repair, triggering senescence or apoptosis.<p>DNA damages trigger the activation of the effectors of the DDR inducing a transient cell<p>cycle arrest, allowing DNA repair, or triggering a permanent arrest of the cell cycle or<p>apoptosis if damages are too extensive.<p>As skin is the outermost barrier of the body, epidermal cells, including SCs, are<p>continuously subjected to genotoxic stress, such as UV rays, ionizing radiation (IR) and<p>chemicals. The skin epidermis is composed of hair follicles (HFs), its associated sebaceous<p>gland (SG) and the surrounding inter-follicular epidermis (IFE). Different types of SCs<p>maintain the homeostasis of the skin; multipotent adult bulge SCs ensure the cyclic<p>regeneration of the HF and the repair of the epidermis after injury, while individual unipotent<p>SCs ensure homeostasis of the SG and the IFE.<p>In tissues with high cellular turnover, such as the epidermis, the numerous divisions that a<p>SC undergoes could result in the accumulation of replication-associated DNA damage. It has<p>been suggested that adult SCs may undergo asymmetric divisions in which the daughter SC<p>retains the older (thus “immortal”) DNA strand, while the daughter cell committed to<p>differentiation inherits the newly synthesized strand that may have incorporated replicationderived<p>mutations. The in vivo relevance of this mechanism is still a matter of intense debate.<p>We used multiple in vivo experimental approaches to investigate precisely how bulge SCssegregate their chromosomes during HF morphogenesis, SC activation and skin homeostasis.<p>Using pulse-chase experiments with two different uridine analogs together with DNAindependent<p>chromatin labelling, we showed that multipotent HF SCs segregate their<p>chromosomes randomly, and that the label-retention observed in the skin epidermis derives<p>solely from relative quiescence of skin SCs 1.<p>We investigated the in vivo response of multipotent adult HF bulge SCs to DNA damage<p>induced by IR. We showed that bulge SCs are profoundly resistant to DNA damage-induced<p>cell death compared to their more mature counterparts. Interestingly, we demonstrated that<p>resistance of bulge SCs to IR-induced apoptosis does not rely on their relative quiescence.<p>Moreover, we showed that DDR in SCs does not lead to premature senescence. We found that<p>two intrinsic cellular mechanisms participate in the resistance of bulge SCs to DNA damageinduced<p>cell death. Bulge SCs express higher level of the anti-apoptotic Bcl-2 and present<p>more transient activation of p53 due to a faster DNA repair activity mediated by a nonhomologous<p>end joining (NHEJ) mechanism. Since NHEJ is not error free, this property<p>might be a double-edged sword, supporting short-term survival of bulge SCs but impairing<p>long-term genomic integrity 2.<p>While we unveiled the relevance of DSBs repair by NHEJ in the skin epidermis, little is<p>known about the role of homologous recombination (HR) during the morphogenesis of the<p>skin epidermis. Brca1 is an essential protein for HR. Conditional deletion of Brca1 in the<p>developing epidermis leads to congenital alopecia accompanied by a decreased density of hair<p>placodes. The remaining HFs never produce mature hair and progressively degenerate due to<p>high levels of apoptosis. Multipotent adult HF bulge SCs cannot be detected in adult HF in<p>the Brca1 cKO epidermis. Brca1 deletion in the epidermis triggers p53 activation throughout<p>the epidermis, which activates apoptosis. Interestingly, IFE and the isthmus region of the HF<p>do not present any pathological phenotype by constitutive deletion of Brca1. Our results<p>demonstrated the critical role of Brca1 during HF morphogenesis. Future studies will be<p>required to understand the molecular mechanisms controlling this phenotype / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The epidermis is the first barrier of protection of living organisms against external attacks. It is constantly renewed throughout life, through a process called "homeostasis", which ensures that every cell lost on its surface is replaced by new ones. Recent studies have shown that this balance is ensured by a hierarchy of stem cells (SC) and progenitors that perform 3 types of cell divisions, each having a fixed probability. Although the epidermis has been extensively studied during homeostasis, little is known about the cellular dynamics taking place when the epidermis must expand its surface. Are these probabilities of division immutable or can they change? In this project, we focused on two conditions of epidermal expansion: postnatal growth and wound healing. Using the mouse tail epidermis as a model, we show that the re-epithelialization after a wound is achieved via the formation of two transient compartments that are spatially and molecularly distinct :a leading edge and a proliferative hub. We show that the leading edge cells have a specific transcriptional signature that is independent of their quiescent state and we propose new markers not previously described. Using the technique of "lineage tracing", coupled with clonal analysis and mathematical modeling, we highlight the proliferation dynamics of SCs and progenitors during healing. We show that different populations of cells residing in different compartments, the hair follicle infundibulum and the interfollicular epidermis, acquire a similar dynamics and re-activate their SC while the progenitors increase their rate of proliferation without changing their division probabilities. This similar proliferation dynamics in two compartments of the epidermis suggests that division probabilities are not dictated by the cell of origin. Interestingly, cell dynamics is different during postnatal growth. Using lineage tracing, clonal analysis and single-cell transcriptional analysis, we demonstrate that the post-natal epidermis is composed of a homogeneous population of equipotent progenitors which ensure a harmonious tissue growth through a constant imbalance towards self-renewing divisions and an ever decreasing proliferation rate. On the other hand, we show that basal cells in the adult epidermis display a greater molecular heterogeneity and that this heterogeneity is acquired progressively at the end of growth. Finally, by coupling in vivo measurements and in vitro micro-patterning experiments, we show that the orientation of cell division of equipotent progenitors is locally influenced by the alignment of the collagen fibers of the underlying dermis. These data suggest that SC specification occurs late in postnatal development and that proliferation dynamics are not immutable and could therefore be influenced by extrinsic factors. / L’épiderme est la première barrière de protection des organismes vivants contre des attaques extérieures. Il est constamment renouvelé au cours de la vie, via un processus appelé « homeostasie », qui assure que chaque cellule perdue à sa surface soit remplacée par de nouvelles. Des études récentes ont montré que cet équilibre était assuré par une hiérarchie de cellules souches (CS) et de progéniteurs qui réalisent 3 types de divisions cellulaires, chaque type de division ayant une probabilité fixe. Bien que l’épiderme ait été intensivement étudié durant l’homeostasie, peu de choses sont connues concernant la dynamique cellulaire prenant place lors de phénomènes où l’épiderme doit grandir. Ces probabilités de division sont-elles immuables ou peuvent-elles au contraire changer ?Dans ce projet, nous nous sommes intéressés à deux conditions d’expansion de l’épiderme :la croissance post-natale et la cicatrisation des plaies. En utilisant l’épiderme de la queue de souris comme modèle, nous montrons que la ré-épithélialisation d’une plaie est réalisée via la formation de deux compartiments cellulaires transitoires distincts spatialement et du point de vue moléculaire :un front de migration et un centre prolifératif. Nous montrons que les cellules du front de migration ont une signature transcriptionnelle spécifique qui est indépendante de leur état de quiescence et proposons de nouveaux marqueurs non décrits auparavant. En utilisant la technique du « lineage tracing », couplée à une analyse clonale et à de la modélisation mathématique, nous mettons en évidence la dynamique de prolifération des CS et des progéniteurs lors de la cicatrisation. Nous montrons que différentes populations de cellules résidant dans des compartiments différents, l’infundibulum du follicule pileux et l’épiderme interfolliculaire, acquièrent une dynamique similaire et ré-activent leur CS tandis que les progéniteurs augmentent leur taux de prolifération sans changer leur probabilité de division. Cette dynamique de prolifération similaire dans deux compartiments de l’épiderme suggère que les probabilités de divisions ne sont pas dictées par la cellule d’origine. De façon intéressante, la dynamique cellulaire est par contre différente durant la croissance post-natale. En utilisant le lineage tracing, l’analyse clonale et des analyses transcriptionnelles sur cellule unique, nous démontrons que l’épiderme post-natal est composé d’une population homogène de progéniteurs équipotents qui présentent un constant déséquilibre envers des divisions d’auto-renouvèlement et un taux de prolifération décroissant, assurant une croissance harmonieuse de l’épiderme. En revanche, les cellules basales de l’épiderme adulte montrent une plus grande hétérogénéité moléculaire et cet hétérogénéité est acquise progressivement à la fin de la croissance. Enfin, en couplant des mesures in vivo et des expériences de micro-patterning in vitro, nous montrons que l’orientation de la division cellulaire des progéniteurs équipotents est localement influencée par l’alignement des fibres de collagène du derme sous-jacent. Ces données suggèrent que la spécification des CS survient tardivement au cours du développement post-natal et que la dynamique de prolifération n’est pas immuable et pourraient donc être influencée par des facteurs extrinsèques. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Kallikrein-related peptidases (KLKs) constitute a highly conserved serine protease family. Based on in vitro experiments, KLKs are predicted to play an important role in a number of physiolog- ical and pathophysiological processes. However, their role in vivo remains not fully understood, partially due to a lack of suitable animal models. In this work, we aim to prepare a KLK5 and KLK14 double-deficient mouse model. Both KLK5 and KLK14 were proposed to be involved in epidermal proteolytic networks critical for maintaining skin homeostasis. However, both KLK5 and KLK14 single-deficient mouse models show minimal or no phenotype, likely due to similar substrate specificity resulting in functional compensation. Double-deficient mice cannot be easily obtained by crossing due to localization of the Klk5 and Klk14 genes within the same locus on chromosome 7. We report that KLK5 and KLK14 double-deficient mice were success- fully generated, mediated by transcription activator-like effector nucleases (TALENs) targeting Klk14 by microinjection of TALEN mRNA into KLK5-deficient zygotes. Furthermore, we show that KLK5 and KLK14 double-deficient mice are viable and fertile. We believe that these novel mouse models may serve as a useful experimental tool to study KLK5 and KLK14 in vivo.

No description available.

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[ES] En las últimas décadas, la aprobación de los inhibidores de la tirosina quinasa (del inglés TKI) como una nueva clase de terapia dirigida ha mejorado la calidad de vida y las tasas de supervivencia de los pacientes con cáncer. Sin embargo, los efectos adversos asociados a éstos, como son las reacciones cutáneas, siguen siendo un desafío para la terapia controlada. De acuerdo con anteriores estudios fotofísicos y fotobiológicos de TKI realizados por el grupo de investigación, esta tesis sigue un enfoque multidisciplinar para investigar nuevos fármacos fotoactivos dentro de esta familia. En la etapa inicial, se seleccionaron cuatro TKI, gefitinib, axitinib, dasatinib y avapritinib, por su capacidad para absorber luz UVA y por su potencial fototóxico. Los estudios, tanto fotofísicos como fotobiológicos, se llevaron a cabo en estos fármacos. Gefitinib (GFT), un TKI con un cromóforo quinazolina, reveló cambios significativos en la fototoxicidad debido a modificaciones metabólicas en su estructura. Así, la desalquilación de la cadena lateral propoxi-morfolina (DMOR-GFT) presentó el valor más alto de factor de fotoirritación (PIF), aprox. 48, mientras que el metabolito desmetilado (DMT-GFT) mostró un valor de PIF mucho menor (~7), casi la mitad del valor de PIF del fármaco inalterado (~13). Por el contrario, el metabolito que presenta un grupo hidroxilo en lugar de flúor (DF-GFT) resultó no ser fototóxico. Notablemente, solo se confirmó que DMOR-GFT induce fotoperoxidación lipídica mediante un mecanismo oxidativo de Tipo I, basado en la escasa producción de oxígeno singlete y la eficiente desactivación del estado excitado triplete por un modelo lipídico. La fotooxidación de proteínas se evidenció en el caso de GFT y, en menor medida, en DMOR-GFT, pero resultó insignificante para DMT-GFT. Sin embargo, a diferencia de GFT, el daño al ADN inducido por el metabolito desmetilado no se reparó incluso después de varias horas. Axitinib (AXT), comercialmente disponible como el isómero (E)-AXT, tiende a fotoisomerizar a (Z)-AXT, especialmente en presencia de proteínas. Así, se revelaron dos mecanismos de fototoxicidad. En primer lugar, la conversión del (E)-AXT (no citotóxico) en el (Z)-AXT (citotóxico) tras irradiación. En segundo lugar, la fototoxicidad intrínseca exhibida por (Z)-AXT. Además, la fotooxidación de proteínas se atribuyó al isómero Z debido a la similitud en el contenido de carbonilo entre ambos isómeros y la alta afinidad del isómero Z por las proteínas. Finalmente, la fotogenotoxicidad solo se reveló mediante la detección de histonas ¿-H2AX. Dasatinib (DAS) es un TKI propuesto para el uso tópico en enfermedades cutáneas. Tras establecer un PIF inicial de 5, se confirmó la fototoxicidad de DAS en una emulsión oleo-acuosa en epidermis humana reconstruida (RhE), la cual se redujo sustancialmente al incorporar un filtro solar de amplio espectro. DAS presenta capacidad de generar tanto oxígeno singlete como radicales, desencadenando fotooxidación tanto en lípidos como en proteínas. Asimismo, se evidenció daño fotoinducido al ADN tanto mediante el ensayo cometa como la detección de ¿-H2AX. Avapritinib (AVP), un TKI de nueva aprobación, demostró ser un fármaco fototóxico con un valor de PIF de aproximadamente 11. Además, fue capaz de inducir tanto fotooxidación a las proteínas como daño en el ADN. En definitiva, el estudio de la toxicidad cutánea de los TKI en combinación con la luz solar se llevó a cabo mediante una exhaustiva evaluación de su fotocomportamiento tanto en disolución como en células de piel. El objetivo es proporcionar a los profesionales de la salud información actualizada sobre la foto(geno)toxicidad y alentarlos a evaluar e implementar estrategias de fotoprotección para los pacientes sometidos a la terapia basada en TKI. / [CA] En les últimes dècades, l'aparició d'inhibidors de la tirosina cinasa (de l'anglès TKI) com una nova classe de teràpia dirigida ha millorat la qualitat de vida i les taxes de supervivència dels pacients amb càncer. No obstant això, els efectes adversos associats a aquests, com les reaccions cutànies, continuen sent un desafiament per a la teràpia controlada. D'acord amb estudis fotofísics i fotobiològics prèvis de TKI realitzats pel grup de recerca, esta tesi segueix un enfocament multidisciplinari per a investigar nous fàrmacs fotoactius dins d¿aquesta familia. En l'etapa inicial, es van seleccionar quatre TKI, gefitinib, axitinib, dasatinib i avapritinib, per la seua capacitat per absorbir llum en la regió UVA i el seu potencial fototòxic. Gefitinib (GFT), un TKI amb un cromòfor quinazolina, va experimentar canvis significatius en la fototoxicitat a causa de modificacions metabòliques en la seua estructura. La desalquilació de la cadena lateral propoxi-morfolina (DMOR-GFT) va presentar el valor més alt de factor de fotoirritació (PIF), aprox. 48, mentre que el metabòlit desmetilat (DMT-GFT) va mostrar un valor de PIF molt menor (~7), quasi la meitat del valor de PIF del fàrmac inalterat (aprox. 13). Al contrari, el metabòlit que presenta un grup hidroxil en lloc de fluor (DF-GFT) va resultar no ser fototòxic. Notablement, només es va confirmar que DMOR-GFT induïx fotoperoxidació lipídica mitjançant un mecanisme oxidatiu de Tipus I, basat en l'escassa producció d'oxigen singlet i l'eficient desactivació de l'estat excitat triplet per un model lipídic. La fotooxidació de proteïnes va ser evident per a GFT i, en menor mesura, per a DMOR-GFT, però va resultar insignificant per a DMT-GFT. No obstant això, a diferència de GFT, el dany a l'ADN induït pel metabòlit desmetilat no es va reparar fins i tot després de diverses hores. Axitinib (AXT), comercialment disponible com a (E)-AXT, tendeix a fotoisomeritzar a (Z)-AXT, especialment en presència de proteïnes. Així, es van revelar dos mecanismes de fototoxicitat. En primer lloc, la conversió de l'(E)-AXT (no citotòxic) en el (Z)-AXT (citotòxic) després d'irradiació. En segon lloc, la fototoxicitat intrínseca exhibida per (Z)-AXT. A més, la fotooxidació de proteïnes es va atribuir a l'isòmer Z a causa de la similitud en el contingut de carbonil entre ambdós isòmers i l'alta afinitat de l'isòmer Z per les proteïnes. Finalment, la fotogenotoxicitat només es va revelar mitjançant la detecció de histones ¿-H2AX. Dasatinib (DAS) és un TKI proposat per a l'ús tòpic en malalties cutànies. Després d'establir un PIF inicial de 5, es va confirmar la fototoxicitat de DAS en una emulsió oli-aquosa en epidermis humana reconstituïda (RhE), la qual es va reduir substancialment en incorporar un filtre solar d'ample espectre. DAS presenta capacitat de generar tant oxigen singlet com radicals, desencadenant la fotooxidació tant en lípids com en proteïnes. Així mateix, es va evidenciar mitjançant l'assaig cometa i la detecció d'H2AX dany fotoinduït a l'ADN. Avapritinib (AVP), un TKI de segona generació, va demostrar ser un fàrmac fototòxic amb un valor PIF d'aproximadament 11. A més, va ser capaç d'induir tant la fotooxidació a les proteïnes com induir dany en l'ADN. En definitiva, l'estudi de la toxicitat cutània dels TKI en combinació amb la radiació solar es va dur a terme mitjançant una exhaustiva avaluació del seu fotocomportament tant en dissolució como en cèl·lules de pell. L'objectiu és proporcionar als professionals de la salut informació actualitzada sobre foto(geno)toxicitat i fomentar l'avaluació e implementació d'estratègies de fotoprotecció per als pacients sotmesos a la teràpia basada en TKI. / [EN] In recent decades, the emerge of tyrosine kinase inhibitors (TKIs) as a new class of targeted therapy has substantially enhanced the quality of life and survival rates for cancer patients. However, associated adverse effects, such as dermatological reactions, remain a challenge to sustained therapy. In light of our research group established insights into the photophysical and photobiological aspects of some TKIs, this thesis follows a similar multidisciplinary approach to investigate other photoactive drugs within the TKI family. In the initial stage, four TKIs, gefitinib, axitinib, dasatinib, and avapritinib, were selected based on their ability to absorb in the UVA region of the solar spectrum and their phototoxic potential. Consequently, photophysical and photobiological studies were conducted on these TKIs. Gefitinib (GFT) is a TKI with a quinazoline moiety, in which modifications resulting from metabolism significantly alter the phototoxicity potential. Dealkylation of the propoxy-morpholine side chain (DMOR-GFT) exhibited the highest photoirritant value (PIF), reaching approximately 48, while the demethylated metabolite (DMT-GFT) displayed much lower phototoxicity (PIF ~7), nearly half the PIF value of the parent drug (ca. 13). In contrast, replacing the fluorine substituent with OH (DF-GFT) resulted in the absence of phototoxic activity. Surprisingly, only DMOR-GFT was confirmed to induce lipid photoperoxidation which occurred through a Type I oxidative mechanism, based on the weak singlet oxygen production and the efficient quenching of the triplet excited state by a lipid model. Furthermore, protein photooxidation was evident for GFT and, to a lesser extent, for DMOR-GFT, but negligible for DMT-GFT. However, unlike the parent drug, DNA photodamage induced by the demethylated metabolite exhibited limited repair even after several hours. Axitinib (AXT), commercially available as (E)-AXT, showed a tendency for photoisomerization to (Z)-AXT, particularly within proteins. Thus, two phototoxicity mechanisms were unveiled. Firstly, the transformation of the initially non-cytotoxic (E)-AXT into the cytotoxic (Z)-AXT upon radiation. Secondly, the intrinsic phototoxicity exhibited by (Z)-AXT. Moreover, protein photooxidation was unequivocally attributed to the (Z)-isomer due to the similarity in carbonyl content between E/Z-isomers and the high protein affinity of the (Z)-isomer. Finally, the photogenotoxicity was only revealed through the detection of ¿-H2AX histone foci. Dasatinib (DAS) is a TKI suggested for topical treatment of dermatological diseases. Given this context and having determined a PIF value ca. 5, an evaluation of DAS phototoxicity in reconstructed human epidermis (RhE) was conducted. DAS formulated in an oil-in-water emulsion exhibited high phototoxicity, which was substantially reduced upon incorporating a broad-spectrum sunscreen. DAS, capable to generate both singlet oxygen and radicals, triggered photooxidation in both lipids and proteins. Similarly, DNA photodamage was evidenced through comet assay and H2AX foci detection. Avapritinib (AVP), a newly approved TKI, was proven to be a phototoxic drug with a PIF value ca. 11, which was highly photooxidative toward proteins and capable to induce DNA photodamage. All in all, the study of skin toxicity of TKIs in combination with sunlight was achieved through a comprehensive evaluation of their photobehavior both in solution and within skin cells. The aim is to provide healthcare professionals with updated information on photo(geno)toxicity and encourage them to assess and implement photoprotection strategies for patients undergoing TKI-based therapy. / Agradezco a la Universitat Politècnica de València por la ayuda para la formación de doctores dentro del subprograma 1 (PAID-1- 2019) y al Ministerio de Ciencia, Innovación y Universidades por la ayuda para la formación del profesorado universitario (FPU19/00048). Ambas subvenciones resultaron fundamentales para la elaboración de mi tesis doctoral. / Ouardi El Hamidy, ME. (2024). In Vitro Photobehavior of Tyrosine Kinase Inhibitors in Solution and within Skin Cells [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/206167

The nucleus of epidermal keratinocytes (KCs) is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multicolor confocal microscopy, followed by computational image analysis and mathematical modeling, we demonstrate that in normal mouse footpad epidermis, transition of KCs from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and microenvironment including the following: (i) decrease in the nuclear volume; (ii) decrease in expression of the markers of transcriptionally active chromatin; (iii) internalization and decrease in the number of nucleoli; (iv) increase in the number of pericentromeric heterochromatic clusters; and (v) increase in the frequency of associations between the pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear microenvironment required for proper execution of gene expression programs in differentiating KCs, and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions, including disorders of epidermal differentiation and epidermal tumors.

Les aquaporines (AQPs) sont des petites protéines formant des canaux hydriques àtravers les membranes cellulaires. Les AQPs 0, 1, 2 ,4 ,5 ,6 et 8 assurent le transport sélectif de l’eautandis que les AQPs 3, 7, 9 et 10 permettent également le passage du glycérol. Nous avons étudié leurexpression dans l’épiderme, la couche supérieure de notre peau supposée imperméable. Dans lesmélanocytes, des cellules dendritiques responsables de la pigmentation, seule l’AQP1 est exprimée.Nous avons montré que les kératinocytes, les cellules majoritaires de l’épiderme, expriment enprolifération les AQPs 3 et 10, alors que les kératinocytes différenciés expriment les AQPs 3 et 9. Lalocalisation de l'AQP3 a été précédemment rapportée à la membrane plasmique des kératinocytes, de lacouche basales à la couche épineuse. Nous avons localisé l'AQP9 dans les kératinocytes différenciés dela couche granuleuse au contenu riche en glycérol et réduit en eau. De fait nous pensons que l'AQP9 ysert de transporteur de glycérol. Enfin contrairement à d'autres auteurs, nous n’avons pu mettre enévidence de lien entre prolifération tumorale et expression des aquaporines. / Aquaporins (AQPS) are a family of small proteins forming water channels across cell membranes.AQPs 0, 1, 2, 4, 5, 6 and 8 are strictly water channel whereas AQPs 3, 7, 9 and 10 allow transport ofwater and glycerol. We have studied their expression in the epidermis, the outer-most water-impermeablelayer of the skin. In melanocytes, dendritic cells responsible for pigmentation, only AQP1 is expressed, invitro and ex vivo. We have shown that keratinocytes, principal cells of the epidermis, express AQPs 3 and10 in proliferation, whereas differentiated keratinocytes express AQPs 3 and 9. The localisation of AQP3to plasma membrane of keratinocytes was previously reported from the basal layer to the spinous layer ofthe skin. We localised AQP9 in the fully differentiated keratinocytes of the granular layer, where there is ahigh glycerol and low water content. So we think that AQP9 likely functions as a glycerol transporter.Unlike other authors, we were unable to identify a link between tumorous proliferation and the expressionof aquaporins.

The structure, function and accessibility of epidermal skin provide aunique opportunity to study the DNA damage response (DDR) of a normaltissue. The in vivo response can be examined in detail, at a molecularlevel, and further associated to the structural changes, observed at atissue level. We collected an extensive skin biopsy material frompatients undergoing fractionated radiotherapy for 5 to 7 weeks. Several end-points inthe DDR pathways were examined before, during and after the treatment. Quantification of DNA double strand break (DSB) signalling focirevealed a hypersensitivity to doses below 0.3Gy. Furthermore, aconsiderable amount of foci persisted between fractions. The low dosehypersensitivity was observed throughout the treatment and was alsoobserved for several key parameters further downstream in the DDR-pathway, such as p21-associated checkpoint activation, apoptosisinduction and reduction in basal keratinocyte density (BKD).Furthermore, for dose fractions above 1.0 Gy, a distinct acceleration inDDR was observed half way into treatment. This was manifested as anaccelerated loss of basal keratinocytes, mirrored by a simultaneousincrease in DSBs and p21 expression. Quantifications of mitotic events revealed a pronounced suppression ofmitosis throughout the treatment which was clearly low dosehypersensitive. Thus, no evidence of accelerated repopulation could beobserved for fraction doses ranging from 0.05 to 2Gy. Our results suggest that the keratinocyte response primarily isdetermined by checkpoints, which leads to pre-mitotic cell elimination by permanent growth arrest and apoptosis. A comparison between the epidermal and dermal sub-compartments revealsa consistent up-regulation of the DDR response during treatment. Adifference was however observed in the recovery phase after treatment,where miR-34a and p21 remain up-regulated in dermis more persistentlythan in epidermis. Our observations suggest that the recovery phaseafter treatment can provide important clues to understand clinicalobservations such as the early and late effects observed in normaltissues during fractionated radiotherapy.

Human epidermis relies on a population of adult stem cells to maintain its homeostasis. Stem cells transit from a dormant to an active state and undergo a tightly regulated process of differentiation that replenishes the tissue according to its needs. This process either replaces cells that get shed away, or contributes to tissue healing upon injuries, such as wounding. Distinct molecular mechanisms are required to keep human epidermal stem cells localized in their niche and for their active proliferation and mobilization, while others regulate their differentiation status. However, little is known about the proper global chromatin modifications that ensure the correct transition between these stem cell states. This work shows that Cbx4, a Polycomb Repressive Complex-1 (PRC1)-associated protein, maintains human epidermal stem cells slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its anti-senescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors, such as Ezh2, Dnmt1, and Bmi1, to prevent the active stem cell state. Interestingly, Cbx4 also represses genes required for neuronal fate repression, suggesting that it might have a role in ectoderm patterning during development. Cbx proteins are differently expressed during epidermal differentiation and the activity of Cbx4 towards promoting human epidermal stem cell quiescence is unique among the Cbx proteins. This suggests that different Polycomb complexes are assembled, based on the availability of its core member, and balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. / La homeostasis de la epidermis humana depende de una población de células troncales adultas (CTAs). Las CTAs alternan ciclos de quiescencia y actividad, seguidos por una regulación estricta de su diferenciación, según las necesidades celulares del tejido. Este proceso es esencial para repoblar el tejido de células envejecidas o dañadas. Cada estadío por el que transita una CTA está regulado por procesos moleculares específicos. Sin embargo, aún sabemos poco sobre los procesos que regulan la reorganización de la cromatina necesarios para mediar dichas transiciones en la población de las CTAs. Estos resultados demuestran que la proteina Cbx4, pertenciente al complejo Polycomb Repressive Complex-1 (PRC1), es necesaria para mantener a las CTAs de la epidermis humana quiescentes, indiferenciadas, y protegidas de la senescencia. A nivel molecular, la actividad polycomb de Cbx4 es únicamente necesaria para su función antisenescente, pero es dispensable para la regulación de la proliferación y diferenciación de las CTAs. La inhibición de la proliferación y diferenciación celular sin embargo depende de la activdad E3 SUMO ligasa de Cbx4. Analisis del transcriptoma global y de unión a la cromatina (ChIP), demuestran que Cbx4 regula la expresión de moduladores esenciales de la homeostasis de la epidermis, y reprime la expresión de factores necesarios para la activación de las CTAs, tales como Ezh2, Dnmt1 y Bmi1. Cabe destacar que Cbx4 también reprime la expresión de genes que determinam el linage neuronal, lo que sugiere que Cbx4 pueda ser importante para separar el neuroectodermo entre ectodermo y neuronas, durante el desarrollo embrionario. Cbx4 es la única proteina Cbx capaz de inducir entrada en quiescencia de las CTAs, y el resto de proteinas Cbx se expresa de forma diferente durante la diferenciación en la epidermis. Por lo tanto, nuestros estudios sugieren que la actividad de distintos complejos Polycomb actúa en los sucesivos estadíos de quiescencia, proliferación y diferenciación de las CTAs, a la vez que impiden su senescencia de forma constante.