TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits β3, β2, β5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in β2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression.

Endocannabinoids are endogenous lipid mediators involved in various biological processes, and have immunomodulatory and anti-inflammatory activities. Anandamide (arachidonoyl ethanolamine, AEA) and 2-arachidonoyl glycerol (2-AG) are the main representatives of this group. The endocannabinoid receptors CB1 and CB2 with AEA have been found in human HaCaT keratinocytes and fibroblasts, but the metabolic pathway leading to endocannabinoid production in the skin has not been fully elucidated. This study aimed to investigate the profile of endocannabinoids and their main metabolizing enzymes in human skin cells and assess whether omega-3 polyunsaturated fatty acids (n-3 PUFA) altered these profiles. In addition, an investigation was carried out to check whether UV radiation could stimulate the production of endocannabinoids and N-acylethanolamines (NAE) in human skin cells. For this purpose HaCaT keratinocytes and 46RB.1N fibroblast cells were treated with 10 and 50µM of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or oleic acid (OA) in the presence or absence of UVR (15mJ/cm2). Data suggest that n-3 PUFA may both directly (by up-regulating NAPE-PLD levels) and indirectly (by decreasing FAAH levels) increased endocannabinoid and NAE levels in HaCaT keratinocytes and 46BR.IN fibroblasts. DHA treatment significantly decreased COX-2 expression in the absence of UVR and inhibited UVR-induced COX-2 overexpression in 46BR.IN fibroblasts. In contrast, DHA appeared to induce COX-2 up-regulation in the absence of UVR and did not prevent UVR induced COX-2 up-regulation in HaCaT keratinocytes. EPA appeared to induce COX-2 down-regulation in the absence of UVR and did not prevent UVR induced COX-2 up-regulation in both HaCaT keratinocytes and 46BR.IN fibroblasts. UVR did not have any significant effect on endocannabinoid and NAE biosynthesis. However, UVR induced endocannabinoid production in some experiments of this study. A clinical study was carried on 16 volunteers from two different ethnic groups and two different skin types. The purpose was to assess the effect of UVR on the serum endocannabinoids and NAE, therefore, the volunteers were subjected to multiple doses (1.3, SED/ 6 min) of UVR for 6 weeks. Data showed that UVR did not have major effect on human serum NAE in both skin phototypes II and V but increased 2-AG in human serum in both skin types but the more pronounced effect was evident in skin phototypes V rather than in skin phototypes II. Human serum docosahxaenoylethanolamide levels were found to be higher in White Caucasians group (skin phototypes II). Based on these it can be concluded that n-3 PUFA and UVR alter the endocannabinoids and NAE profile in HaCaT keratinocytes and 46BR.IN fibroblasts. In addition, results of the clinical study indicated that UVR has no major effects on serum endocannabinoids or NAE therefore, further studies are required to address this question in vivo.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits α3, α2, α5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in α2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression. / EPSRC and DTA grant

Arsenic is a universal environmental toxicant associated mostly with skin related diseases in people exposed to low doses over a long term. Low dose arsenic trioxide (ATO) with long exposure will lead to chronic exposure. Experiments were performed to provide new knowledge on the incompletely understood mechanisms of action of chronic low dose inorganic arsenic in keratinocytes. Cytotoxicity patterns of ATO on long-term cultures of HaCaT cells on collagen IV was studied over a time course of 14 days. DNA damage was also assessed. The percentages of viable cells after exposure were measured on Day 2, Day 5, Day 8, and Day 14. Statistical and visual analytics approaches were used for data analysis. In the result, a biphasic toxicity response was observed at a 5 μg/ml dose with cell viability peaking on Day 8 in both chronic and acute exposures. Furthermore, a low dose of 1 μg/ml ATO enhanced HaCaT keratinocyte proliferation but also caused DNA damage. Global gene expression study using microarray technique demonstrated differential expressions of genes in HaCaT cell exposed to 0.5 μg/ml dose of ATO up to 22 passages. Four of the up-regulated and 1 down-regulated genes were selected and confirmed with qRT-PCR technique. These include; Aldo-Keto Reductase family 1, member C3 (AKR1C3), Insulin Growth Factor-Like family member 1 (IGFL1), Interleukin 1 Receptor, type 2 (IL1R2) and Tumour Necrosis Factor [ligand] Super-Family, member 18 (TNFSF18), and down-regulated Regulator of G-protein Signalling 2 (RGS2). The decline in growth inhibiting gene (RGS2) and increase in AKR1C3 may be the contributory path to chronic inflammation leading to metaplasia. This pathway is proposed to be a mechanism leading to carcinogenesis in skin keratinocytes. The observed over expression of IGFL1 may be a means of triggering carcinogenesis in HaCaT keratinocytes. In conclusion, it was established that at very low doses, arsenic is genotoxic and induces aberrations in gene expression though it may appear to enhance cell proliferation. The expression of two genes encoding membrane proteins IL1R2 and TNFSF18 may serve as possible biomarkers of skin keratinocytes intoxication due to arsenic exposure. This research provides insights into previously unknown gene markers that may explain the mechanisms of arsenic-induced dermal disorders including skin cancer / Environmental Sciences / D. Phil. (Environmental science)

Arsenic is a universal environmental toxicant associated mostly with skin related diseases in people exposed to low doses over a long term. Low dose arsenic trioxide (ATO) with long exposure will lead to chronic exposure. Experiments were performed to provide new knowledge on the incompletely understood mechanisms of action of chronic low dose inorganic arsenic in keratinocytes. Cytotoxicity patterns of ATO on long-term cultures of HaCaT cells on collagen IV was studied over a time course of 14 days. DNA damage was also assessed. The percentages of viable cells after exposure were measured on Day 2, Day 5, Day 8, and Day 14. Statistical and visual analytics approaches were used for data analysis. In the result, a biphasic toxicity response was observed at a 5 μg/ml dose with cell viability peaking on Day 8 in both chronic and acute exposures. Furthermore, a low dose of 1 μg/ml ATO enhanced HaCaT keratinocyte proliferation but also caused DNA damage. Global gene expression study using microarray technique demonstrated differential expressions of genes in HaCaT cell exposed to 0.5 μg/ml dose of ATO up to 22 passages. Four of the up-regulated and 1 down-regulated genes were selected and confirmed with qRT-PCR technique. These include; Aldo-Keto Reductase family 1, member C3 (AKR1C3), Insulin Growth Factor-Like family member 1 (IGFL1), Interleukin 1 Receptor, type 2 (IL1R2) and Tumour Necrosis Factor [ligand] Super-Family, member 18 (TNFSF18), and down-regulated Regulator of G-protein Signalling 2 (RGS2). The decline in growth inhibiting gene (RGS2) and increase in AKR1C3 may be the contributory path to chronic inflammation leading to metaplasia. This pathway is proposed to be a mechanism leading to carcinogenesis in skin keratinocytes. The observed over expression of IGFL1 may be a means of triggering carcinogenesis in HaCaT keratinocytes. In conclusion, it was established that at very low doses, arsenic is genotoxic and induces aberrations in gene expression though it may appear to enhance cell proliferation. The expression of two genes encoding membrane proteins IL1R2 and TNFSF18 may serve as possible biomarkers of skin keratinocytes intoxication due to arsenic exposure. This research provides insights into previously unknown gene markers that may explain the mechanisms of arsenic-induced dermal disorders including skin cancer / Environmental Sciences / D. Phil. (Environmental science)

L’objectif de ce projet est d’étudier l’interaction de nanoparticules polymères avec les membranes, soit directement sur des cellules entières ou grâce à des modèles membranaires biomimétiques, dans l’optique de valider leur utilisation dans le cadre d’applications biologiques. Des nanoparticules (NPs) polymères cœur/enveloppe avec un diamètre inférieur à 100 nm ont été synthétisés. Cette taille a été choisie afin de leur permettre de pénétrer à travers les membranes plasmiques. Des nanoparticules ayant la même composition chimique mais avec un diamètre hydrodynamique supérieur, de l’ordre de 250 nm, ont été également préparées afin de mettre en évidence l’effet de la taille des particules sur le processus d’internalisation cellulaire. Dans cette thèse, une méthode innovante de synthèse monotope a été développée pour obtenir des NPs coeur-enveloppe, compatibles en milieu aqueux et présentant à leur surface des résidus iniferter. Le coeur est composé de polystyrène avec une taille d’environ 30 nm. Un large éventail de fonctionnalités peut être greffé sur la surface du coeur par polymérisation radicalaire contrôlée en faisant varier différents types de monomères. L’épaisseur de l’enveloppe peut être ajustée en fonction de la concentration en monomère et du temps de polymérisation. Les nanoparticules synthétisées ont été caractérisées par diffusion dynamique de la lumière, par spectroscopie infrarouge à transformée de Fourier, par analyse micro-élémentaire et par microcopie à transmission électronique. Les interactions des NPs à coeur polystyrène et avec des enveloppes de charge neutre et négative ont été étudiées avec des cellules kératinocytes épidermiques humaines néonatales (NHEK), des fibroblastes primaires humains et les cellules HACaT de kératinocytes humains. Les études de cytotoxicité réalisées en utilisant un marquage à l’iodure de propidium et un test à la lactate déshydrogénase n’ont relevé aucune toxicité sur les lignées testées. Cependant, le suivi de la prolifération cellulaire par impédance électrique de substrats cellulaires a indiqué que les nanoparticules anioniques induisent une forte diminution de la prolifération des kératinocytes. L’internalisation cellulaire des NPs a été confirmée par microscopie confocale qui n’indique pas leur colocalisation avec les endosomes précoces, les lysosomes et l’actine. De plus, les données obtenues par triage cellulaire par cytofluorométrie soutiennent qu’un mécanisme énergétiquement-dépendant est mis en œuvre pour l’internalisation des NP neutres, ce qui semble être moins le cas pour les nanoparticules négatives. Les membranes biomimétiques ont été employées afin d’étudier les spécificités des interactions entre nanoparticules et lipides dans des conditions contrôlées. L’étude sur des modèles de vésicules géantes couplée à de la spectroscopie de fluorescence a révélé que les nanoparticules coeur/enveloppe sont capables d’interagir profondément dans la région hydrophobe de la membrane, mais uniquement quand la bicouche lipide est en phase fluide désordonnée. Le mode de pénétration des NPs au travers de la bicouche des vésicules semblent engendrer la formation de pores. Un effet plus prononcé de rigidification de la bicouche a pu être observé lors de l’interaction de nanoparticules chargées négativement avec les bicouches de phosphatidycholines. Cet effet pourrait être attribué à un changement de l’orientation des têtes phosphocholines du à des interactions électrostatiques. En conclusion, les nanoparticules polymère que nous avons synthétisées apparaissent être des outils polyvalents pour les études d’interaction cellulaire et d’imagerie. Ces nanomatériaux peuvent être éventuellement être employés pour la délivrance de médicaments en incorporant les molécules actives dans une enveloppe polymère thermosensible par exemple. / This project’s aim was to study polymeric nanoparticle-membrane interactions using both live cells and biomimetic models with the idea to validate such nanoparticles for use in bio-applications. Core-shell polymeric nanoparticles below 100 nm, as this small size is capable of penetrating plasma membranes, were synthesised. Nanoparticles (NPs) with the same chemical composition but with hydrodynamic diameters of ~250 nm, were also prepared in an effort to highlight any effect of NP size on cell internalisation. In this thesis, an innovative method is presented for the synthesis of water-compatible, iniferter-bound polystyrene core shell NPs (~30 nm) using a one-pot synthetic method. A plethora of functionalities could be added to the nanoparticles via shell grafting from the surface of the polystyrene core in the presence of additional monomers via controlled living radical polymerisation. Shell thickness could be tuned as a function of monomer’s concentration and polymerisation time. The nanoparticles were fully characterised by dynamic light scattering, Fourier transform infra-red spectroscopy, microelemental analysis and transmission electron microscopy. Further, the interactions of polystyrene core NPs possessing neutral and anionic shells were investigated using neonatal human epidermal keratinocytes (NHEK), human primary fibroblasts and HaCaT cells. Cytotoxicity studies performed using propidium iodide and lactate dehydrogenase indicated no evidence of cytotoxicity in either cell line. However, cell proliferation monitored by electric cell substrate impedance sensing (ECIS) protocols indicated that anionic nanoparticles induced a dramatic decrease in cell proliferation in keratinocytes. The cellular internalisation of NPs was confirmed by confocal microscopy and no co-localisation was found with early endosomes, lysosomes or actin. Additionally, fluorescence activated cell sorting (FACS) data support the theory that an energy-dependent mechanism is employed for neutral NP internalisation but less so for negatively charged NPs. Biomimetic membrane models were used to investigate specific nanoparticle-lipid interactions under controlled conditions. Employing giant vesicles coupled with fluorescent spectroscopy techniques revealed that core-shell nanoparticles interact deep in the hydrophobic region of bilayers only when the membrane is in the fluid phase. Their mode of entering artificial cells (i.e giant vesicles) appears to cause the formation of pores. Anionic nanoparticles interact with the choline moiety of phosphatidylcholine and confer a rigidifying effect on phosphocholine containing bilayers. Therefore we conclude that the polymeric nanoparticles that we synthesized are versatile tools for cell interaction and imaging studies. These nanomaterials could eventually be applied to drug delivery studies by incorporation of the drug in for instance a thermoresponsive polymeric shell. Furthermore, it is clear that NPs coated with anionic and neutral polymeric shells present a lower toxicity profile than previously reported cationic nanoparticles. Both nanoparticles increase the order lipid bilayer vesicles composed of POPC (the most common glycerophospholipid) in animal and plants. Anionic nanoparticles in particular exhibit a rigidifying effect on POPC lipid bilayers and their mode of entry into cells may be due to the formation of pores which was determined to not induce cell death.

No description available.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Στην παρούσα εργασία πραγματοποιήθηκε γονιδιωματική ανάλυση της επίδρασης αντιψωριασικών φαρμάκων σε καλλιέργειες ανθρώπινων κερατινοκυττάρων με τη χρήση μικροσυστοιχιών DNA. Σε καλλιέργειες κυττάρων HaCaT χορηγήθηκαν τα παράγωγα του ρετινοϊκού οξέος all-trans retinoic acid (ATRA) και acitretin, σε διαβαθμισμένες δόσεις, προκειμένου να διαπιστωθεί η επίδραση τους στα κύτταρα. Μελετήθηκε η βιωσιμότητα των κυττάρων με τις δοκιμασίες της χρωστικής Trypan Blue και ΜΤΤ. Επιλέχθηκαν δύο συγκεντρώσεις (10^-6 και 10^-8 Μ) των φαρμάκων που αντιστοιχούσαν σε βιωσιμότητα κυττάρων περίπου 80%, οι οποίες χορηγήθηκαν εκ νέου σε καλλιέργειες κυττάρων HaCaT. Τα κύτταρα συλλέχθηκαν, έγινε εκχύλιση του RNA και έλεγχος της ποιότητας του με ηλεκτροφόρηση (Bioanalyzer). Το RNA χρησιμοποιήθηκε για την in vitro μεταγραφή cDNA που σημάνθηκε με φθοριοχρώματα και άμεσα ακολούθησε υβριδισμός σε πλακίδιο μικροσυστοιχιών (OneArray) το οποίο περιείχε ανιχνευτές για όλο το ανθρώπινο γονιδίωμα μαζί με τους κατάλληλους μάρτυρες. Σε κάθε πλακίδιο υβριδίστηκαν ταυτόχρονα cDNA από κύτταρα στα οποία είχε χορηγηθεί ρετινοειδές και κύτταρα στα οποία δεν είχε χορηγηθεί. Η επεξεργασία των δεδομένων της σαρώσεως με ειδικό λογισμικό ανέδειξε 700 περίπου γονίδια που ρυθμίζονται θετικά ή αρνητικά σε στατιστικά σημαντικό βαθμό. Για την επαλήθευση των αποτελεσμάτων που προέκυψαν από τις μικροσυστοιχίες, επιλέχθηκαν 34 γονίδια τα οποία συμμετέχουν σε βασικές βιολογικές διεργασίες όπως πρωτεϊνοσύνθεση, κυτταρική σηματοδότηση, πολλαπλασιασμός, κυτταρική διαφοροποίηση, κυτταρικός θάνατος, φλεγμονή. Επιπρόσθετα, επιλέχθηκαν 22 γονίδια τα οποία έχουν επίσης κομβικό ρόλο σε σηματοδοτικά μονοπάτια και κυτταρικές λειτουργίες. Η επιλογή αυτή έγινε για να μελετηθεί πιο σφαιρικά η επίδραση των φαρμάκων σε βασικούς κυτταρικούς μοριακούς μηχανισμούς. Στο σύνολο των επιλεγμένων γονιδίων έγινε ποσοτική Real-Time PCR και για το σκοπό αυτό έγινε σχεδιασμός ειδικών εκκινητών. Η qRT-PCR εν τέλει, επιβεβαίωσε τα αρ-χικά αποτελέσματα από τα microarrays. Διαπιστώθηκε ότι η κυτταρική απόκριση στη χορήγηση των ρετινοειδών, εξειδικευμένα για κάθε δραστική ουσία και για κάθε δόση δεν είναι μονοσήμαντη, αλλά ότι ταυτόχρονα επάγονται λειτουργικά μονοπάτια με διαφορετικούς ρόλους. Επίσης διαπιστώθηκε ότι η μεταβολή κατά δύο τάξεις μεγέθους της δόσης που προσλαμβάνουν τα κύτταρα επάγει αντίρροπες κυτταρικές αποκρίσεις. Συγκεκριμένα, η ολιστική προσέγγιση της μεταβολής της γονιδιακής έκφρασης ανέδειξε ότι η χορήγηση ATRA σε συγκέντρωση 10^-6Μ στις κυτταροκαλλιέργειες ευνοεί την πρωτεϊνοσύνθεση και την διαφοροποίηση των κυττάρων ενώ ασκεί αντιφλεγμονώδη δράση. Η χορήγηση της δραστικής ουσίας ATRA στη δόση 10^-8Μ ευνοεί τη διαφοροποίηση των κυττάρων HaCaT σε μεγαλύτερο βαθμό από τον πολλαπλασιασμό τους. Επιπλέον, φαίνεται ότι σε αντίθεση με τη μεγαλύτερη δόση, ευνοείται η σύνθεση μορίων που επάγουν την φλεγμονή. Παρόμοια, η ασιτρετίνη στη δόση 10^-6Μ ευνοεί τη διαφοροποίηση των κυττάρων και την σύνθεση μορίων που επάγουν τη φλεγμονή. Η ασιτρετίνη στην μικρότερη δόση (10^-8 Μ) ευνοεί κύρια την διαφοροποίηση, λιγότερο τον πολλαπλασιασμό των κυττάρων και φαίνεται ότι προάγει σε σημαντικό βαθμό την απόπτωση. Οι μεγαλύτερες δόσεις των ρετινοειδών που μελετήθηκαν φαίνεται ότι είναι απαγορευτικές για τον πολλαπλασιασμό των κυττάρων σε αντίθεση με τις μικρότερες δόσεις. Επισημαίνεται ότι για τη θεραπεία της ψωρίασης όπου χρησιμοποιούνται, επιθυμητές δράσεις είναι η παραγωγή μορίων με αντιφλεγμονώδη δράση, ο περιορισμός του αυξημένου κυτταρικού πολλαπλασιασμού, αύξηση της κυτταρικής απόπτωσης και τέλος πολύ ση-μαντικό είναι η επιτυχής περάτωση της διαφοροποίησης των κυττάρων. Συμπερασματικά, η χρήση τεχνικών υψηλής απόδοσης, κύρια των μικροσυστοιχιών cDNA που επιτρέπουν την εκτεταμένη μελέτη του γονιδιώματος και της qRT-PCR για πιο στοχευμένη μελέτη, μπορούν να διαδραματίσουν σημαντικό ρόλο στην εξακρίβωση μοριακών μηχανισμών. / In the current project we performed gene expression profiling, using cDNA microarrays, when specific doses of derivatives of retinoic acid were applied in HaCaT cell culture. These specific drugs are used in the treatment of psoriasis but their exact effect in molecular level remains elusive. All-trans retinoic acid and acitretin were applied in gradient doses. Cell viability was monitored using MTT and Trypan blue assays. Two specific doses (10^-6 & 10^-8 M), in which cell viability was approximately 80%, were chosen for the treatment of HaCaT cells. Subsequently, the treated cells were collected and RNA was extracted using standard methods. At a next step, RNA quality was examined by electrophoresis (Bioanalyzer) and spectrometry. High quality RNA showing no traces of degradation was used as template for in vitro transcription. Finally, synthesis of fluoro-labeled cDNA was performed from RNA derived from both treated and untreated samples and was immediately hybridized to DNA microarray slides (OneArray). Analysis of the hybridization data was performed using specific software. As a result, 700 genes (both up-regulated and down-regulated) were chosen for further analysis. Among them, 34 genes were chosen to validate the microarrays results by the use of quantitative Real-Time PCR. These genes appeared to play crucial role in basic cellular functions like protein synthesis, signal transduction, cell death, cell differentiation and proliferation. Furthermore, 22 additional essential genes that are related to the above processes were chosen in aim to examine drugs effects. The data processing revealed that the 10^-6 M dose of ATRA has a positive effect in protein synthesis, cell differentiation and anti-inflammatory action. Moreover ATRA at a concentration of 10^-8 M promotes differentiation more than proliferation and it has inflammatory effect as well as acitretin has in 10^-6 M dose. On the other, hand acitretin in 10^-8 M dose facilitates differentiation more than proliferation but mainly induces cell death. Generally, high doses (10-6 M) of the drugs inhibit cell proliferation more efficient than low doses (10-8 M). In fact, during psoriasis treatment, the anti-inflammatory action, inhibition of cell proliferation, induction of cell differentiation and cell death are considered desirable drug effects. Our study shows that cDNA microarray analysis represents a powerful tool that can be used for extended genomic studies and the results that are obtained can be validated and used for the elucidation of several molecular mechanisms.