No / Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications. / (Science Fund Vot No.: 0201-01-13-SF0104 or S024) awarded by Malaysia Ministry of Science and Technology (MOSTI) and IGSP Grant Vot No. U679 awarded by Universiti Tun Hussein Onn Malaysia.

No / The human epidermis holds the full machinery for cholinergic signal transduction. However, the presence of the vesicular transporter (vesicular acetylcholine (ACh) transporter (VAChT)) for both choline and ACh has never been shown in this compartment. The results of this study confirm the presence of VAChT in cutaneous nerves and in both epidermal melanocytes and keratinocytes as well as in their nuclei using immunofluorescence labelling in situ and in vitro, Western blot analysis of cellular and nuclear extracts and reverse transcription-PCR. These results underline that ACh/choline transport in the non-neuronal epidermis is no different from the neuronal pathway. However, the function of VAChT in the nucleus remains to be shown.

No description available.

Certains virus respiratoires comme rhinovirus semblent favoriser la colonisation par staphylococcus aureus. Cependant, les détails des mécanismes impliqués dans cette synergie n’ont pas été suffisamment élucidés. Le but de cette thèse a été de développer et valider un modèle in vitro mimant la colonisation du vestibule nasal par s. aureus en utilisant les kératinocytes humains hacat. Ce modèle a permis d’étudier (i) les pouvoirs d’adhésion et d’internalisation d’une collection de souche clinique de s. aureus, (ii) l’efficacité intracellulaire des molécules antimicrobiennes utilisées dans le cadre de la décolonisation nasale de s. aureus, (iii) l’effet de la clarithromycine sur l’infection par rhinovirus et (iv) l’impact de l’infection par rhinovirus ou de l’inflammation non spécifique sur la colonisation par s. aureus. ce travail a principalement permis d’identifier un nouveau mécanisme alternatif de l’internalisation de s. aureus à travers la liaison entre la protéine bactérienne eap (extracellular adherence protein) et le récepteur cellulaire icam-1 (intracellular adhesion molecule 1). Cette voie alternative est favorisée en cas d’infection par rhinovirus ou d’inflammation, ce qui pourrait expliquer les observations cliniques de l’augmentation de la charge de s. aureus ou du risque d’infection par cette bactérie lors des infections virales respiratoires ou d’inflammation post-traumatique. Les résultats de cette thèse illustrent la complexité des interactions entre les cellules épithéliales de la muqueuse, s. aureus et les pathogènes viraux et ouvrent les perspectives sur d’autres études nécessaires afin de proposer des stratégies préventives ou thérapeutiques adaptées. / Some respiratory viruses such as rhinoviruses seem to promote staphylococcus aureus colonization. However, the details of the bacterial and cellular mechanisms involved in this synergy have not been sufficiently elucidated. The aim of this thesis was to develop and validate an in vitro model mimicking s. aureus colonization of the nasal vestibule by using hacat human keratinocytes. This model allowed to study (i) the adhesion and internalization capacities of various clinical s. aureus strains, (ii) the intracellular efficiency of the antimicrobial molecules used for s. aureus nasal decolonization, (iii) the effect of clarithromycin on rhinovirus infection, and (iv) the impact of rhinovirus infection and non-specific inflammation on s. aureus colonization. This work has mainly identified a new alternative mechanism for the internalization of s. aureus through the binding between the bacterial protein eap (extracellular adherence protein) and the cell receptor icam-1 (intracellular adhesion molecule 1). This alternative pathway is favored in case of rhinovirus infection or inflammation; which could explain the clinical observations of the increase of the load of s. aureus or the risk of infection by this bacterium during respiratory viral infections or post-traumatic inflammations. The results of this thesis illustrate the complexity of the interactions between the mucosal epithelial cells, s. aureus and viral pathogens and suggest that other studies are needed to propose appropriate preventive or therapeutic strategies.

The signaling mechanisms involved in UVB-induced skin cancer are complex and although the scope of this work is inherently limited in focus, the findings may provide insight into how estrogen receptor signaling impacts cell growth, senescence, and apoptosis to protect keratinocytes. Additional signaling due to E2-activation of the estrogen receptor may provide back-up or redundant pathways in response to UVB.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

Les ultraviolets A (UVA) sont carcinogènes et produisent des espèces réactives de l'oxygène (ERO). Le récepteur à la mélanocortine de type 1 (MC1R) est un récepteur couplé aux protéines G (RCPG) qui est impliqué dans la mélanogénèse et dans l'inflammation cutanée. Certains variants du gène sont associés à un risque accru de mélanomes et de carcinomes cutanés. Le MC1R est exprimé surtout dans les mélanocytes mais son expression peut être induite par les UV in vitro dans les kératinocytes et in vivo dans la peau. Le récepteur MC1R est activé par l'α-MSH. L'objectif de ce travail de thèse a été d'étudier les effets du récepteur MC1R sur le stress oxydatif induit par les UVA dans des lignées kératinocytaires humaines HaCaT exprimant le récepteur MC1R ou son variant non fonctionnel Arg151Cys. Nous avons montré que la production d'ERO intracellulaire induite par les UVA est fortement inhibée dans les cellules HaCaT-MC1R et que cette inhibition est renforcée en présence d'α-MSH. L'inhibition du stress oxydatif induit par les UVA dans les cellules transfectées par le MC1R est en partie dépendante de la phosphorylation de la sous-unité activatrice, NoxA1 de la NADPH oxydase. Le traitement des cellules HaCaT-MC1R par un inhibiteur du récepteur au facteur de croissance épidermique (EGFR) restaure l'habilité de ces cellules à induire un stress oxydatif après irradiation UVA. Ces résultats montrent que l'activité constitutive du récepteur MC1R dans des kératinocytes pourrait inhiber le stress oxydatif induit par les UVA via des mécanismes dépendants de l'AMPc et de l'EGFR. / Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to Melanocortin-1 Receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R151C) non- functional variant (HaCaT-R151C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R151C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by α-MSH treatment of HaCaT-MC1R cells before UVA exposure; (3) after UVA irradiation, NoxA1 phosphorylation was increased i n HaCaT-MC1R compared to HaCaT and HaCaT-R151C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of UVA-ROS production; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.

Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

In vivo, l'épiderme se régénère en continu à travers la prolifération des cellules souches basales. Celles-ci obtenues à partir d'une biopsie cutanée, peuvent être greffées pour régénérer la peau. En vue de la transplantation kératinocytaire, nous avons étudié in vitro leur capacité à régénérer un épithélium en fonction de leur origine anatomique, et précisé leur phénotype. Par rapport aux différents sites étudiés, nous montrons que les kératinocytes issus de prépuce ont la plus grande capacité proliférative et régénérative, associée à une expression exacerbée de marqueurs d'immaturité, la p63 et la kératine 19 et une faible expression des marqueurs de la différenciation (involucrine et filagrine). Ces résultats nous ont amené à utiliser ces kératinocytes préputiaux en suspension pour transplanter les enfants brûlés, ce qui évite l'utilisation d'une membrane de transfert, source potentielle d'inefficacité. La transplantation des brûlures avec ces cellules autologues conduit à une épithélialisation accélérée avec une cicatrice de grande qualité. Ces résultats et la facilité de la procédure nous permettent de proposer la greffe autologue kératinocytaire de prépuce comme une procédure standard, qui peut être ajoutée à l'arsenal thérapeutique des équipes prenant en charge les enfants brûlés. / In vivo, the skin regenerates continuously through the proliferation of basal stem cells. That can be obtained from a skin biopsy and may be grafted to regenerate skin. In vitro, we studied the regenerative keratinocyte capacities according to site donors, and we noted that keratinocytes from foreskin have the greatest proliferative and regenerative capacities, combined with expression of progenitor cell markers (p63 and keratin 19) and low expression of epidermal differentiation markers (involucrin and filaggrin). These results led us to use these preputial keratinocytes in suspension for graft in burned children. Apply directly cells with a suspension device, eliminates the use of a transfer membrane and a potential source of inefficiency. In our study, boys grafted with autologous keratinocytes isolated from foreskin leads to epithelialization with a high quality of scar. This technique is easily applicable to a large number of pediatric surgery teams. Our clinical success and the safety of the procedure in the operating room enable us to propose the kératinocyataire autologous transplantation as a standard procedure, which can be added to the therapeutic armamentarium in burns.