OBJETIVOS GENERALES: a) Establecer los tiempos del ciclo folicular en la cara.b) Establecer cambios de comportamiento inducidos por el tratamiento en el ciclo de crecimiento folicular.c) Establecer pautas de tratamiento en fotodepilación.d) Establecer dosis idóneas según fototipos y color del pelo.OBJETIVOS ESPECÍFICOS: a) Establecer la eficacia en términos de porcentaje de pérdida de pelos terminales y permanencia del resultado de un sistema de fotodepilación (Epilight®) en mujeres de fototipos II, III y IV de Fitzpatrick, afectas de hirsutismo idiopático o de origen hormonal, con pelo grueso, negro u oscuro en la cara.b) Evaluar, mediante la técnica del tricograma por unidad de área (modificado) y el folículograma (mediante biopsia), el efecto del sistema sobre el ciclo de crecimiento folicular de dicha región anatómica.c) Establecer el intervalo ideal entre las sesiones de tratamiento en el área mencionada.d) Establecer las alteraciones estructurales y ultraestructurales del folículo piloso, mediante técnicas de microscopía de luz óptica (tinción de hematoxilina-eosina y orceína), microscopía electrónica de transmisión e inmunohistoquímica (técnica del úlex europeus y anticuerpo monoclonal anti-procolágeno I).HIPÓTESIS:La permanencia de los resultados de la fototricólisis depende del ritmo del ciclo de crecimiento folicular de la localización estudiada y ello debe determinar el número total de sesiones de tratamiento, así como el intervalo entre las mismas, de las aplicaciones de una fuente de luz pulsada intensa.El fotocontaje digitalizado permitió medir la progresión del recuento piloso, la tasa depilatoria y establecer que la fotodepilación mediante luz pulsada intensa es efectiva desde la primera sesión de tratamiento y que la estabilidad de los resultados es dos años. Asimismo, este método permitió establecer una correlación significativa entre el número de sesiones de tratamiento y la tasa depilatoria. El tricograma por unidad de área (modificado) evidenció la inversión de la relación pelo anágeno vs pelo no anágeno desde la primera hasta la última sesión de tratamiento y que este efecto telogenizante fue inmediato y se mantuvo durante el período de tratamiento. El foliculograma mostró una inversión de la relación pelo anágeno vs pelo no anágeno desde la primera hasta la última sesión de tratamiento, confirmándose así el efecto telogenizante ya observado en el tricograma. La técnica histológica de la hemoatoxilina-eosina puso de manifiesto daños estructurales inmediatos y tardíos de la unidad folicular, pero sobre todo la persistencia del daño de la vaina radicular, situación que ocasiona una pérdida de la comunicación entre la papila y el sistema de células germinativas del promontorio y otras regiones del folículo piloso que produce finalmente la depilación prolongada. Asimismo, esta técnica mostró un aumento significativo del infiltrado inflamatorio de moderada intensidad alrededor del folículo piloso responsable del proceso inflamatorio que produce alopecia, fibras de colágeno en la dermis media y alrededor del folículo piloso, trayectos fibrosos en la dermis, relacionados al daño térmico inespecífico. Se observó un aumento significativo de las fibras elásticas alrededor del folículo piloso y de las glándulas écrinas del mentón, evidenciado mediante la técnica de la orceína. La técnica inmunohistoquímica del úlex europeus no mostró daño del endotelio vascular específico por la luz pulsada intensa pero sí un aumento significativo del número de vasos permeables en las dos áreas tratadas, hallazgo que refleja el efecto vasodilatador de los pulsos de corta duración (3.5-3.8 ms) de luz pulsada intensa. Se observó un aumento no significativo de la expresión del autoanticuerpo monoclonal anti-procolágeno I en la zona de la membrana basal, alrededor de los folículos pilosos y en los septos fibrosos del tejido adiposo de la dermis profunda en ambas áreas estudiadas. La falta de expresión del autoanticuerpo en la dermis media obedece a que la técnica tiñe sólo células productoras de procolágeno I pero no las fibras colágenas extracelulares maduras. La microscopía electrónica de transmisión confirmó la especificidad y selectividad del daño térmico inducido por la luz pulsada intensa en las estructuras melanizadas: queratinocitos basales pigmentados, epitelio folicular, tallo piloso y la vaina radicular. Por otro lado, esta técnica también confirmó el daño mitocondrial capaz de ocasionar el colapso metabólico y finalmente la muerte celular. Finalmente, la observación clínica permitió establecer que efectos colaterales fueron discretos y transitorios. El efecto paradójico, crecimiento de pelo terminal en áreas próximas al área tratada, observado en pacientes afectas de síndrome del ovario poliquístico podría deberse al paso de un ciclo folicular asincrónico a un ciclo folicular sincrónico en fase anágena.Se ha confirmado el efecto telogenizante de la luz pulsada intensa en el ciclo folicular desde la primera sesión de tratamiento, así como el efecto añadido de las sesiones adicionales de tratamiento y una correlación significativa entre el número de sesiones de tratamiento y la tasa depilatoria. Por otro lado se ha demostrado el daño estructural específico de la unidad folicular, sobretodo de la vaina epitelial, situación que repercute en la estabilidad de los resultados (2 años) debido a una pérdida de la comunicación entre la papila y el sistema germinativo folicular. La presencia significativa de infiltrado inflamatorio perifolicular, fibras de colágeno en la dermis media y alrededor del folículo piloso, trayectos fibrosos en la dermis y, finalmente, fibras elásticas alrededor del folículo piloso confirman el daño térmico inespecífico y el proceso alopécico inducido por la luz pulsada intensa. / BACKGROUND: A few studies on ultrastructural findings associated to intense pulsed light (IPL) photodepilation have been published.OBJECTIVE: To evaluate clinical, histological, immnunohistochemical, and ultrastructural changes in females with facial hirsutism submitted to IPL photodepilation.Patients and Methods: Forty-nine Caucasian females with facial hirsutism were submitted to multiple sessions (n=8) of IPL photodepilation. Clinical evaluation of hirsutism and hormonal status were performed before treatment. Clinical pictures, hair count, trichogram, and skin biopsy were performed before, after treatment, and during the follow-up period. A questionnaire was administered after each treatment session and during the follow-up period to evaluate side-effects. Skin specimens were submitted to hematoxyline-eosin (HE), orcein, ulex europeus, monoclonal autoantibody against procollagen I, and transmission electron microscopy (TEM). Folliclegram was also performed in transverse HE preparations. The treatment protocol included: cut-filter of 695-755 nm, spot size of 10x45 mm, pulse duration of 3.5-3.8 ms, delay of 20-30 ms, triple mode, and a fluence energy range between 40 and 43 J/cm2. A total of 390 treatment sessions (mean 8, range: 3-9) at 8-week intervals were administered between October 1999 and April 2001. Follow-up was done 6, 12, 24, and 36 month after the last treatment session.RESULTS: Post-treatment clinical and photographic evaluation showed progressive decrease in terminal hair. Hair count showed a progressive decrease in terminal hair number. The trichogram and folliclegram showed progressive telogenization after treatment. Microscopy evaluation showed neocolagenogenesis, miniaturization and telogenization of hair follicles.Immunochemistry showed presence of auto-antibody against pro-collagen I in the fibrous septa of deep fat. TEM of the epidermis showed necrosis, inter and intracellular vacuolization, dense mitochondrias, debris among cells, and apoptosis. TEM of the hair follicle showed abundant intracellular glycogen in the external layer, parakeratosis and keratohyalin in Henle layer, and necrosis of the inner shaft/central portion of the hair shaft. Other TEM findings included necrotic debris in dermis. CONCLUSION: Photodepilation with an intense pulse light ( IPL ) source in hirsute women is a safe and effective technique, with some collateral effects that usually are mild and transitory.

En el año 1971 y atraída por la gran personalidad del Prof. J. Piñol Aguadé, inicié el estudio de la Especialidad de Dermatología en la Cátedra por él dirigida. De este modo, me encontré en una Escuela profundamente interesada en las enfermedades producidas por alteraciones en el metabolismo de las porfirinas. Esto era debido a que el profesor Piñol seguía con sumo interés a los pacientes porfíricos y mostraba constantemente su preocupación por los problemas clínicos y patogénicos que presentaban.Aunque los primeros artículos sobre la porfirina de dicha Cátedra se publicaron ya en 1952, el origen del estudio sistemático del tema se encuentra en 1957, cuando los profesores Vilanova y Piñol Aguadé publicaron "La Porfiria Crónica del adulto", monografía donde se recapitulaban los conocimientos que se tenían en aquella época sobre el tema. A partir de entonces se sucedieron periódicamente publicaciones de la Cátedra sobre diversos aspectos de estas enfermedades (incidencia en nuestro ambiente, peculiaridades clínicas e histológicas, tanto de las lesiones cutáneas como complicaciones oculares, hepáticas o articulares).En 1972, el interés del Prof. Piñol Aguadé se centraba en los estudios bioquímicos y genéticos de la Profiria Cutánea Tarda, y fue entonces cuando recibí el encargo de iniciar estos trabajos. Tras una presentación preliminar en el VIII Congreso Hispano Portugués de Dermatología (1973), los trabajos han proseguido con un incremento en el número de pacientes estudiados. Por otra parte, se ha establecido una conexión fluida con otros centros de Investigación de Porfirinas (como el "Welsh National School" de Cardiff) y se ha creado una infraestructura específica para el estudio de las alteraciones bioquímicas de estos pacientes, contando con la valiosa ayuda del Profesor J.M. Mascaró. Todos estos factores ha permitido que la tesis doctoral que presentamos a continuación tenga un gran número de elementos de trabajo; precisamente la combinación y profundización de dichos elementos, la aparición de nuevos problemas y la propuesta de posibles soluciones pueden considerarse nuestra principal aportación al estudio de las porfirias.

La determatitis atópica (DA) es una enfermedad inflamatoria cutánea de curso crónico y recidivante, cuyo aspecto clínico difiere en las distintas edades, por lo que es probable que también intervengan agentes etiológicos distintos siendo este motivo la hipótesis de este trabajo.El presente trabajo se ha planificado persiguiendo los siguientes objetivos:1.Comprobar si los niños afectos de eczema atópico están sensibilizados a neumoalergenos. 2.Comprobar si esos neumoalergenos son responsables de la dermatitis o si son la causa de alguna enfermedad respiratoria de etiología alérgica (rinitis, asma), concomitante con la DA. 3.Comprobar si existe sensibilización a alimentos. 4.Verificar si la sensibilización a uno u otro tipo de alergeno tiene lugar a cualquier edad o si se observan diferencias en distintas edades.El grupo de trabajo consta de 79 pacientes pediátricos divididos en 3 grupos:1º Niños con DA(según Hanifin y Rajka) 64, divididos en 2 subgrupos según presenten alergia respiratoria(AR)-asma y/o rinitis- 37 pacientes, el motivo de incluir este grupo es comprobar si las pruebas positivas podrían relacionarse o no con la enfermedad cutánea, el otro subgrupo esta formado por pacientes con DA con un total de 27. 2º Grupo control: 8 niños con AR, para comprobar si este grupo reacciona a la prueba del parche con neumoalergenos, esta prueba será la base para comprobar la posible responsabilidad de los neumoalergenos en la etiología de la DA, pero su utilidad se vería invalidada si este grupo también respondiese a esta prueba. 3º Grupo control:7 niños sanos para descartar pruebas positivas inespecíficas en la población no atópica. Todos los grupos se han dividido por edades según las fases de la D.A., fase lactante (< 2 años): 21, fase infantil (2-10 años).37, fase adolescente (>10 años):21. A todos los pacientes se les efectuó : IgE sérica total (RIA), IgE alérgeno específica (RAST), Prick-test y Patch-test esta prueba se realizo en la espalda con los mismos extractos que los del Prick-test. Estas 3 pruebas se realizaron con un estándar de neumoalergenos y alergenos alimentarios. En caso de prueba del parche positiva se realizó biopsia con estudio anatomopatológico y con Ac. Monoclonales (CD3,CD4,CD8,CD19,CD54-ICAM,OKT6,HLA-DR, Anti-human IgE,CD57)para ver si se reproduce la lesión de eczema. Entre los resultados destacan diferencias etiológicas entre los pacientes con DA según presenten clínica respiratoria y según la edad, con un claro predominio de sensibilización a alimentos en el grupo de menos de 2 años, existiendo una correlación etiológica de la DA con la sensibilización a alimentos. En la edad intermedia de 2 a 10 años destaca la sensibilización mixta, en gran parte por la afectación respiratoria simultánea, pero es muy probable que los neumoalergenos participen en la etiopatogenia de la DA. En los mayores de 10 años predomina la sensibilización a neumoalergenos por coincidir la mayoría con clínica respiratoria. Un 34,3% de pacientes con DA presentan prueba del parche positiva, siendo prácticamente la mitad de las pruebas positivas a ácaros. En los casos en que la prueba del parche a neumoalergenos fue positiva y se realizó biopsia (48,5%), en todas ellas fue compatible con eczema. La prueba del parche proporciona un importante valor diagnostico en la DA, no encontrándose ninguna prueba del parche positiva en el grupo control. Todas las biopsias de pruebas del parche a neumoalergenos reproducen la lesión típica del eczema lo que demuestra su implicación en la etiopatogenia de la dermatitis. Por último mencionar que en los pacientes con DA sin clínica respiratoria con prueba del parche positiva a neumoalergenos, parece ser que la vía de sensibilización es la cutánea puesto que no presentan clínica respiratoria.

Keloid disease (KD) is a cutaneous fibroproliferative tumour characterised by heterogeneity, locally aggressive invasion and therapeutic resistance. Clinical, histological and molecular differences between the keloid scar centre and margin as well as recent evidence of the importance of epithelial-mesenchymal interactions (EMI) in KD pathobiology contribute to the complexity and diversity of KD, which coupled with the lack of a validated animal model have hindered research and effective management. Despite significant progress in the field of KD research, reliance on conventional monolayer cell culture and whole tissue analysis methods have failed to fully reflect the natural architecture, pathology and complexity of KD in vivo. In order to address these challenges, a site-specific in situ approach was therefore employed here for the first time in KD research. The first aim of this work was to compare the value of this contemporary approach with traditional methods of tissue dissection. The second aim was to compare the genomic expression between well-defined, distinct keloid sites and normal skin (NS). The third aim was to develop and explore hypotheses arising from this site-specific gene expression profiling approach, so as to enhance understanding of KD pathobiology as a basis for improved diagnostic and therapeutic strategies in future KD management. The fourth aim was to probe these hypotheses with relevant functional in vitro studies. The current site-specific in situ approach was achieved through a combination of laser capture microdissection and whole genome microarray, allowing separation of epidermis from dermis for keloid centre, margin and extralesional sites compared with NS. This in situ approach yielded selective, accurate and sensitive data, exposing genes that were overlooked with alternative methods of dissection. Identification of significant upregulation of the aldo-keto reductase enzyme AKR1B10 in all three sites of the keloid epidermis (KE) in situ, implicated dysregulation of the retinoic acid (RA) pathway in KD pathogenesis. This hypothesis was supported by showing that induced AKR1B10 overexpression in NS keratinocytes reproduced the keloid RA pathway expression pattern. Moreover, co-transfection with a luciferase reporter plasmid revealed reduced RA response element activity. Paracrine signals released by AKR1B10-overexpressing keratinocytes into conditioned medium resulted in TGFβ1 and collagen upregulation in keloid fibroblasts, suggesting the disturbed RA metabolism exerts a pro-fibrotic effect through pathological EMI, thus further supporting the hypothesis of RA deficiency in KE. Gene expression profiling further revealed an upregulation of NRG1 and ErbB2 in keloid margin dermis. Exogenous NRG1 led to enhanced keloid fibroblast migration with increased Src and PTK2 expression, which were attenuated with ErbB2 siRNA studies. Together with the observed failure to recover this expression with NRG1 treatment, suggested the novel KD pathobiology hypothesis that NRG1/ErbB2/Src/PTK2 signaling plays a role in migration at the keloid margin. In addition to these hypotheses, LCM methodology with comprehensive analysis of the data permitted the development of additional novel working hypotheses that will inform future KD research, including inflammatory gene dysregulation and cancer-like stem cells that may contribute to the therapeutic resistance characteristic of KD.

Photoaged skin caused by chronic ultraviolet radiation (UVR) is characterised clinically with hyperpigmentation, coarse skin texture and deep wrinkles; the worst outcome is skin cancer. Histological investigation of the alteration within major extracellular matrices (ECM; elastic fibres, fibrillar collagens) is essential study to understand the cellular effect on skin structure from UVR. This thesis used an acute dose of radiation to examine in humans in vivo the effect of UVR on ECM components before assessing whether a dietary intervention could protect skin from UVR damage. Green tea catechins (GTCs) have anti-oxidant properties and may be an interesting option as a systemic photoprotection agent. Hence this thesis assesses: 1) the effect of acute irradiation of skin on dermal ECM damage to see whether it mimics the changes observed in photoageing and; 2) whether dietary supplementation with GTC will provide dermal ECM protection. UV-induced change in elastic fibre network. Initially, the effect of two different UV light sources on elastic fibre protein (elastic fibres, fibrillin-rich microfibrils and fibulin-2 and -5 microfibrils) remodelling was performed. The effect of ultraviolet B vs full-spectrum solar simulated radiation (SSR) were investigated in a small sample of healthy Caucasian volunteers (n = 6 per group). At 24 hour after 3× MED irradiation, Weigert's resorcin–fuchsin stained elastic fibres showed a significant reduction regardless of irradiation protocol (UVB, P<0.01; SSR P<0.05). Specific components were identified by immunohistochemistry; a significant reduction in fibrillin-rich microfibrils (FRM) was observed in UVB-irradiated skin (P<0.05), whilst fibulin-5-positive microfibrils were only affected by SSR (P<0.05). The data revealed, therefore, differential effects on UV wavelength on ECM remodelling. SSR, the more physiologically relevant light source was used in subsequent studies Supplement effect in SSR-induced damage in elastic fibre. Fifty healthy volunteers were recruited to this randomised control trial to investigate whether GTC can protect skin from photodamage. Volunteers were randomized to GTC (1080 mg plus 100 mg vitamin C; n=25) or placebo (maltodextrin; n = 25) daily for 12-weeks with compliance assessed biochemically in urine samples. Of the n = 50 recruited, 44 volunteers completed the study. In baseline, UVR challenge resulted in a significant remodeling of the cutaneous elastic fiber system (P<0.001), particularly fibulin-2 and fibulin-5-positive microfibrils at 24-hr after 3×MED irradiation. In post-supplementation, fibulin-5 positive microfibrils were protected from UVR remodeling (% staining, mean ± SE; no UV, 18.1±0.89; UVR, 17.1±0.61; P=0.30) whilst no protection was seen in the placebo group (no UVR, 19.41±0.79; UVR, 17.69±0.61; P<0.05). Supplement effect in SSR-induced damage in collagenous matrix. In the identical experiment, collagenous matrices including synthesis of procollagen I was also examined as fibrillar collagens are the major ECM components providing strength within dermis. The fibrillar collagen and newly synthesised procollagen I were stained by Picrosirius red and immunohistochemistry respectively. At baseline, acute irradiation significantly reduced papillary dermal fibrillar collagens (P<0.001) and induced deposition of newly synthesised pro-collagen I (P=0.02). In post-supplementation, GTC enhanced the deposition of thin collagen fibres in the dermis. Whilst placebo showed no effect on the altered organisation of fibrillar collagens or deposition of pro-collagen I following the irradiation challenge, GTC protected the organisation of fibrillar collagens in the papillary dermis (P=0.97).This novel in vivo human study may be used to recapitulate elastic fibre and collagen changes associated with photoageing and may be useful for dissecting out the mechanisms underlying extracellular matrix damage in response to chronic sunlight exposure. Furthermore, in a randomized control trial, dietary GTC protected fibulin-5 microfibrils and collagen fibres in the papillary dermis from UV-mediated degradation. The mechanism by which this protection occurs requires further study.

Background: Psoriasis is a complex disease with a genetic component contributing to disease pathogenesis. Chronic plaque psoriasis can be dichotomised into two subtypes according to age of onset; type 1 (early-onset; <40 years) and type 2 (late-onset; ≥40 years). Despite clinical and biological differences between the two subtypes, the genetics underpinning late-onset psoriasis remains poorly characterised compared to early-onset psoriasis. Aims: The aim of this project was to identify genetic loci associated with late-onset psoriasis, to assess the overlap of loci with early-onset psoriasis and to elucidate the functional role of the identified variants. Methods: The study had three parts; the first was a candidate-gene association study of the IL1B gene. A total of 16 SNPs from the region were genotyped in 595 late-onset and 1,137 early-onset psoriasis samples and compared to 4,770 controls from the European population. The second was a large-scale study conducted in 543 late-onset psoriasis and 4,373 controls using the Immunochip array. The third was a functional study using bioinformatics data mining, chromatin immunoprecipitation and electrophoretic mobility shift assay techniques to analyse the role of a disease-associated variant at the biological level. Results: The candidate-gene study replicated a previously reported association at a promoter polymorphism, rs16944 (P<0.05), within the IL1B gene and discovered a novel association at a second variant, rs11687624 (P<3.12x10-3), in late-onset psoriasis. None of the variants analysed were significantly associated with early-onset psoriasis. Bioinformatic eQTL data suggests the two variants and their proxies are associated with the expression of IL1A, IL1B, IL38 and PAX8. The Immunochip study identified 6 non-HLA loci (P<2.3x10-5) previously associated with early-onset psoriasis to also be associated with late-onset psoriasis (IFIH1, IL12B, IL23A, IL23R, TRAF3IP2 and ZNF313). Conditional analysis of the MHC region also identified two loci (HLA-C and HLA-A). A novel locus, IL1R1, was associated with late-onset psoriasis, but not early-onset psoriasis. Bioinformatic data mining found no role for the IL1R1 variants as eQTLs and prioritised the IL1B variant rs2708914 for functional analysis. The transcription factor STAT3 was found to be enriched at rs2708914 in keratinocyte and CD8+ T-lymphocyte cell lines. Allele-specific binding could not be established. Conclusions: This project is the largest genetic study of late-onset psoriasis to date and provides evidence that it shares susceptibility loci with early-onset psoriasis as well as having specific susceptibility loci. These findings provide further evidence for the dichotomisation of chronic plaque psoriasis, firstly to facilitate better understanding of the pathogenesis of the two subtypes and secondly to enable tailored therapy to be developed. Both have potential benefits for patients in the future. The genetic and functional studies conducted have provided a platform from which further studies can be carried out.

The human body consists of a number of very complex, highly specialised organs which perform a variety of functions that are essential to life and health. One of the main functions of the skin, the largest of the human organs, is to maintain the integrity of the body. It does this by acting as a physical barrier, preventing micro-organisms and other potentially harmful substances from entering the body. When the integrity of the skin is damaged through injury, a self-protective mechanism is triggered and the reparative wound healing process begins. Under normal circumstances the wound healing process culminates in the skin recuperating its normal characteristics and functions at the site of the injury, with only a small visible mark being left behind. However, in some cases the wound healing process may become altered leading to the production of abnormal scars, such as keloids. Keloid scars are formed from scar tissue at the site of an injury, as a result of excessive tissue repair that extends beyond the boundaries of the original wound. These scars are characterised by excess collagen deposition produced during the wound healing process. It is estimated that as many as 20% of the black and Hispanic population are affected by keloid scarring. In addition to the aesthetic aspect, keloid scars can also be painful, itchy and prone to become infected. Keloid scar formation can be triggered by skin injuries caused by, for example, acne, wounds, shaving, burns, and surgical incisions. The mechanism by which keloid scars form is currently not well understood and consequently no effective treatments exist to date.This thesis describes an investigation into the mechanical properties of single keloid and normal skin fibroblast cells for the purpose of establishing if there is a quantitative difference between the two types of cells. This information will be of benefit to researchers looking for a better understanding of the keloid formation mechanism and for those seeking improved treatments. An atomic force microscope (AFM) was employed to indent single Keloid and normal skin fibroblast cells taken from five patients. Values for the apparent Young's modulus of the cells were then calculated by fitting the experimental data using Hertz's model. Apparent Young's modulus values were then compared. The findings of the analysis indicate that statistically, there is a significant difference in the Young's modulus values of normal and keloid cells, with keloid cells exhibiting substantially greater stiffness than normal skin fibroblast cells. To enable the keloid and normal skin fibroblast cells to be studied in as close to their natural, physiological environment as is possible the AFM experiments described in this thesis were undertaken in a phosphate buffered saline (PBS) solution. In such cases the use of a fluid medium presents additional complexities, not least of which is the introduction of a hydrodynamic drag force due to viscous friction of the cantilever with the liquid which can affect the experimental data obtained and consequently any material properties calculated as a result of using these data. In order to investigate the effect of dragging force on the experimental data obtained from the AFM a novel integrated finite element based model was developed. The model, described in this thesis, provides quantification of the drag force in AFM measurements of soft specimens in fluids, consequently enabling more accurate interpretation of the data obtained from AFM experimentation. The model is validated using extensive data obtained from AFM experimentation undertaken in a number of fluids of different viscosities, at a variety of tip velocities and platform-tip separations and by comparison with an existing analytical model. The novel model is shown to accurately account for drag forces in AFM in fluid media without the need for extrapolation of experimental data and can be employed over the range of tip geometries and velocities typically utilised in AFM experimentation.The work described in this thesis demonstrates that the AFM is a valuable tool that can be used to successfully investigate the mechanical properties of biological samples in fluids. It was shown that increased accuracy in the interpretation of data obtained from AFM experimentation can be obtained by taking into account the hydrodynamic drag force due to viscous friction of the cantilever with the liquid. The investigation into the mechanical behaviour of keloid cells described in this thesis significantly adds to the yet small body of research undertaken on keloid cells to date. The findings of the investigation will provide valuable information that will be of benefit in the future to researchers looking to develop effective treatments for the prevention, reduction or removal of keloid scars.

People with psoriasis are known to engage in high levels of unhealthy lifestyle behaviours which may lead to poorer psoriasis outcomes and increase the risk of cardiovascular disease (CVD). Thus, helping individuals with psoriasis understand the link between behaviours and health risks, that is health risk communication, and direct support for lifestyle behaviour change (LBC) are important aspects in optimal management of psoriasis, a long-term inflammatory skin condition. There are two aspects of the literature that remain unclear. First, whether adequate support is given to patients to enable them to understand the links between lifestyle behaviours and health outcomes is part of psoriasis patient management strategies. Second, whether there is agreement around effective health risk communication techniques. This programme of research aimed to examine these gaps in the literature using four related studies. The first study used content analysis to examine general and dermatology-specific healthcare professionals’ core training competencies for evidence of skills relating to LBC. An important finding was the lack of explicit skills relating to LBC and changing understanding of health risks. There was little or no reference to recognised LBC techniques that could be used to support and facilitate LBC with patients. The second study used observational techniques to examine messages about the links between behaviour and health outcomes and LBC signposting (such as leaflets or posters about healthy living) for patients with psoriasis in primary and secondary care patient waiting areas. There was little evidence of psoriasis-specific information about healthy living. Generic information (not specifically about psoriasis) was often of poor quality and was poorly displayed, and did not conform to evidence-based recommendations for effective LBC signposting. The third study combined observational and qualitative techniques to examine how healthcare professionals communicate information about CVD risk to patients and the role of LBC in reducing risk in the context of primary care risk assessments with people with psoriasis. A key finding was that interpretation of risk information was not always linked to specific advice about how to modify each risk factor. Discussion was mostly instructional rather than a shared collaborative discussion about behaviour change and risk reductionThe fourth study used experimental methods to examine the effects of message framing theory as a health risk communication strategy on reported behavioural intentions (BIs) in people with psoriasis. An important finding was that for messages about psoriasis symptom reduction, gain-framed (positively-framed) messages were more effective in increasing BIs for alcohol reduction. Conversely, for messages about CVD risk reduction, loss-framed (negatively-framed) messages were more effective for increasing BIs to reduce alcohol consumption. The body of work presented in this thesis demonstrated that much needs to be done to increase the skill sets of healthcare professionals in order to help people with psoriasis recognise the specific links between their own health behaviours and health outcomes. In addition specific recommendations have been suggested as a way of improving risk communication strategies, such as using theory-based personally-relevant health information for people with psoriasis.

The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and β-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of β-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that β-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of β-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of β-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

La piel es el órgano más extenso del organismo y puede sufrir, además del envejecimiento cronológico, el denominado fotoenvejecimiento o envejecimiento cutáneo patológico. Está caracterizado por un envejecimiento cutáneo más extenso y sobre todo, de mayor gravedad, pues engloba la fotocarcinogénesis o presentación de cánceres cutáneos. Desde la Antigüedad, el hombre tuvo conciencia de la importancia del Sol en su vida, pero también de sus efectos indeseables. En la actualidad las sociedades de los países desarrollados invierten sumas millonarias en la investigación de sustancias que eviten o retrasen los efectos del envejecimiento, no sólo por el culto a la belleza, sino por el gran coste económico que tiene la asistencia sanitaria de las lesiones. En los últimos años, los aceites esenciales han recibido gran atención dada su capacidad antioxidante, que podría interferir en los mecanismos de fotoenvejecimiento, evitando la formación de radicales libres y potenciando el subsistema inmunológico cutáneo. Por otro lado, a las dificultades metodológicas que supone la lenta evolución de los tumores, se suman las limitaciones éticas del desarrollo de modelos experimentales en humanos. Por esta razón, el primero de los objetivos de nuestro trabajo fue el establecimiento de un modelo de fotoenvejecimiento cutáneo en ratones SKH-1, mediante la exposición crónica a Radiación Ultravioleta (RUV), para en el segundo objetivo de nuestro estudio, evaluar las propiedades de dos aceites, el de almendras dulces y el de la crisálida del gusano de la seda, como posibles fotoprotectores. Para la realización del estudio, utilizamos 60 ratones SKH1/CRL, que se sometieron a radiación ultravioleta, 60 minutos por sesión, tres veces por semana, durante un total de 80 sesiones, con 1.688 J/cm2 de energía total absorbida por cada animal. Los ratones se dividieron en tres grupos. El primero (Control) sólo recibió RUV y los otros dos fueron tratados tópicamente, previamente a la irradiación, con aceite de almendras dulces (Grupo II) y con aceite de la crisálida del gusano de seda (Grupo III). Todos los animales expuestos a las RUV presentaron las alteraciones características del fotoenvejecimiento y fotocarcinogénesis cutánea, por lo que consideramos este modelo experimental idóneo para el estudio de esta patología, ya que la exposición crónica a las radiaciones provocó todo el espectro de lesiones propias del fotoenvejecimiento, si la exposición era prolongada (entre 65 y 80 sesiones), además de tratarse de un modelo fácilmente reproducible y de bajo coste. No obstante, en nuestro estudio observamos diferencias significativas entre los animales tratados con el aceite de la crisálida del gusano de seda (Grupo III) respecto a los de los otros dos grupos. En los ratones del grupo III se observa un retraso en la presentación de las lesiones cutáneas (6-9 semanas), con respecto a los otros grupos y hubo dos animales sin lesiones tumorales al final del experimento. Además, el área media de las lesiones era menor en el grupo III un 59,14% respecto al grupo control (reducción del 40,86%); el grupo tratado con aceite de almendras dulces, tan sólo redujo el área media en un 10%. Esto sugiere las propiedades fotoprotectoras del aceite de crisálida del gusano de seda, probablemente debidas a la acción de la sericina que contiene, que es un potente antioxidante. Este retraso en la presentación de lesiones neoplásicas y su menor volumen fue confirmado por el estudio inmunohistoquímico, respecto a la metaloproteinasa 9 (MMPs-9) y los inhibidores TIMP. / The skin is the largest organ of the body and may, in addition to chronological aging, suffer some disease called photoaging. It is characterized by a large aging of the skin and above all, what is more dangerous, photocarcinogenesis or presentation of skin cancers . Since antiquity, men became aware of the importance of the sun in our life, but also of its undesirable effects. At present, a lot of companies of developed countries invest a large amount of money in researching, looking for substances that prevent or delay the effects of aging, not only for beauty, but also by the great economic cost of health care. In recent years, essential oils have received great attention due to its antioxidant capacity, which may interfere with the mechanisms of photoaging, preventing the formation of free radicals and boosting the skin 's immune subsystem. Furthermore, to the methodological difficulties of the slow evolution of tumors, ethical constraints on development of experimental models in humans are added . Therefore, the first objective of our work was to establish a model of cutaneous photoaging in SKH -1 mice by chronic exposure to Ultraviolet Radiation (UVR). The second objetive of our study was to evaluate the properties of two oils, sweet almond and chrysalis of silkworm, as possible photoprotective . For the study, we used 60 mice SKH1/CRL, that received ultraviolet radiation, 60 minutes per session, three times per week, for a total of 80 sessions , with 1,688 J/cm2 of total energy absorbed by each animal. The mice were divided into three groups. The first (control) received only UVR and the other two were treated topically prior to irradiation, with sweet almond oil (Group II ) and oil of silkworm chrysalis (Group III). All animals exposed to UVR showed the characteristic alterations of cutaneous photoaging and photocarcinogenesis, so consider this experimental model as ideal for the study of this disease, since chronic exposure to radiation caused the entire spectrum of lesions characteristic of photoaging, if the exposure was prolonged (65 to 80 sessions), besides being an easily reproducible and not expensive model . However, in our study we observed significant differences between animals treated with oil of chrysalis silkworm (Group III) compared to the other two groups. In Group III, mice observed a delay in the presentation of skin lesions (6-9 weeks), with respect to the other groups, and there were three animals without tumor lesions at the end of the experiment. In addition, the mean area of lesions was lower in group III (59.14 %) than in the control group (40.86 % reduction), the group treated with sweet almond oil only reduced the area by 10 %. This suggests the photoprotective properties of oil of silkworm chrysalis, probably due to the action containing sericin, which is a potent antioxidant. This delay in the presentation of neoplastic lesions and their smaller area was confirmed by immunohistochemical study, regarding metalloproteinase 9 ( MMP -9) and TIMP inhibitors.