Atopic dermatitis (eczema) is a psychosomatic chronic skin condition. Onset of the condition usually occurs in early life. Some people however, do develop it much later. Even though studies have demonstrated high levels of psychological disturbance and maladjustment among most people with AD, some others still do not, suggesting that a certain level of variability exists in the way people adjust to their skin condition. The goal of the present study was to identify the factors that determine adjustment to AD and account for this variability. Existing studies on this topic have considered factors such as: age of onset of the condition, demographic characteristics, disease severity and visibility, cognitive representations of illness and condition appraisals. Most of these studies however are limited in the explanations that they offer. Drawing upon the schema theory model of personality development and psychopathology, the present study proposes that adjustment to AD is mediated by personality-level structures (self-schemas) that have their origins in early experiences. Design: A cross-sectional design was employed with three groups: a) patients with an early onset of atopic dermatitis (n = 130), b) patients with a late onset (n = 76) and c) people with no chronic medical condition (n = 74). Method: All groups completed the Young Parenting Inventory- Revised, and the Young Schema Questionnaire-Short Form 3. The two atopic dermatitis groups also completed the Adjustment to Chronic Skin Disease Questionnaire. Results suggest that: a) people with an early and a late onset of atopic dermatitis were presented with a certain schematic profile that differentiated them from participants in the control groups; b) a certain pattern of early parenting experiences was linked to the development of this schematic profile and c) this schematic profile predicted high levels of dysfunctional coping and difficulties in adjustment. Findings have a clear relevance to the practice of Counselling Psychology. Limitations of the study and clinical implications are discussed.

Human skin colour has significant aesthetic and cultural implications. Cutaneous injuries can result in dys-pigmented scars which are more noticeable, aesthetically unpleasant, and can lead to patient distress and social isolation. Management of dys-pigmented scars has been challenging with variable success. There is a limited understanding of the timing, progression and mechanisms of skin re-pigmentation following wounding. This thesis is a detailed sequential study, which describes and quantifies scar colour changes in pigs of different pigmented strains.The first result chapter describes the observational pigmentary changes in scars of four different pigmented pig strains (Hampshire, Yucatan, Tamworth and Duroc) over time. Two scar re-pigmentation progression patterns, specific to the darkly and the lightly pigmented pigs, are identified and all scar photographs of all pigs at all time-points are scored during non-invasive wound/ scar monitoring using a semi-quantitative scale. In the second result chapter, histo-chemical (DOPA-oxidase) staining was combined with immuno-histochemistry (HMB45) to establish the spatial and temporal distribution and activities of melanocytes in the regenerated epithelium of darkly pigmented pig strains. Results suggest a rise in both inactive and active melanocyte numbers in re-pigmenting scars at early time-points and by late time-points, scars achieved ‘complete re-pigmentation’ and melanocyte numbers were lowest. Late melanocyte proliferation was observed in two scars from two different pigs; one of which manifested this as hyper-pigmentation, macroscopically. In addition, histological analysis of the epidermal melanin staining (Warkel-Luna-Helwig) pattern showed good correlation with the macroscopic appearance of the scars. The effect of changes in scar basement membrane undulation on melanocyte packing density was investigated: changes were small and unlikely to impact melanocyte packing density; hence macroscopic scar colour. Macroscopic and microscopic observations of the pattern of re-pigmentation following creation of partial thickness wounds across the white and black belts of three Hampshire pigs were investigated.The final result chapter describes how colour changes were quantified for scars and normal skin of each pig, at all time-points during non-invasive scar monitoring; using a reflectance spectrophotometer. In addition, the sensitivity of objective colour measurements was investigated. Results using two statistical clustering techniques suggest that colour measurements differentiate scars from the surrounding normal skin and the tristimulus L*a*b* values of scars correlate well with their macroscopic colour appearances. Time-dependent colour changes in scars and normal skin were quantified independently, using polynomial analysis. The results suggest systematic colour changes in most scars of all pig groups, except Yucatans’, which on the other hand, showed systematic colour changes to their normal skin. These findings highlight the importance of independent analysis of scar and normal skin colour measurements with time post wounding. In conclusion, this thesis has investigated timing and progression patterns of scar re-pigmentation in pigs of different pigmented strains.

Introducción: La piel es el órgano más extenso del cuerpo humano. Las heridas cutáneas son producto de la ruptura de la integridad de la piel. Las células mesenquimales (MSCs) están emergiendo como fuertes candidatas para ser usadas como terapia celular en el tratamiento de heridas crónicas, dado su enorme potencial para mejorar la reparación y regeneración de los tejidos posterior a una lesión. Constructos biosintéticos fabricado con la fibroína de la seda celularizados con MSCs de diferentes fuentes han demostrado ser eficaces en la curación de heridas experimentales de la piel. Objetivos: investigar el comportamiento de las células mesenquimales de la gelatina de Wharton del cordón umbilical humano (hWj-MSCs) tanto in vitro como in vivo tras ser sembradas en un constructo electrohilado compuesto de fibroína de la seda y posteriormente implantado en ratones inmunocompetentes. Determinar si las hWj-MSCs aplicadas mediante constructos de fibroína de seda en heridas creadas en el dorso de ratones SKH1 contribuyen a mejorar la cicatrización de la lesión. Metodología: inicialmente hemos puesto a punto el protocolo para el aislamiento y caracterización de las hWj-MSCs. Posteriormente, estas MSCs en combinación con un constructo fabricado a partir de la fibroína de la seda fueron utilizados para probar sus propiedades terapéuticas en un modelo murino experimental de heridas. Resultados: Las hWj-MSCs mostraron una morfología fibroblástica, y presentaron en su superficie la expresión de marcadores mesenquimales que fueron positivos para CD90, CD105, CD73, y sin presencia detectable de marcadores de células hematopoyéticas. Las hWj-MSCs mostraron una capacidad limitada para diferenciarse hacia adipocitos, condrocitos, y osteoblastos. Las hWj-MSCs inhibieron de forma significativa la proliferación de linfocitos T tras ser estimulados con esferas anti-CD3/CD28, así como con células dendríticas maduras alogénicas. Las hWj-MSCs disminuyeron la producción in vitro de la citoquina pro-inflamatoria IFN- por los linfocitos T activados. Este efecto inmunosupresor también estuvo mediado por la producción de los factores anti-inflamatorios TGF-, IDO, y PGE2. Los experimentos con cultivos mixtos de linfocitos en presencia de diferentes inhibidores específicos de la biosíntesis o la señalización de estos factores anti-inflamatorios tales como SB-431542, indometacina (IDM), o 1-metil-triptófano (1-MT) respectivamente, recuperaron casi en su totalidad el nivel de proliferación de las células T estimuladas con células dendríticas maduras. El tratamiento de las heridas de la piel en ratones SKH1 demostró que la combinación de hWj-MSCs y el constructo de fibroína de la seda mejoró significativamente la capacidad de cicatrización de heridas. Conclusiones: Las hWj-MSCs son poco inmunogénicas, no expresan moléculas coestimuladoras, y expresan citoquinas que pueden modular la función inmune. La combinación de hWj-MSCs con los constructos de fibroína de seda contribuyó a la generación de un tejido de granulación de alta calidad, y escasa formación de tejido cicatrizal. La gelatina de Wharton del cordón umbilical humano puede ser una fuente adecuada para la obtención de MSCs para ser utilizadas en la cicatrización de heridas debido a sus diversas ventajas y junto con los beneficios sinérgicos de un constructo pudieran conformar una combinación ideal como apósitos para heridas crónicas de lenta y difícil curación. / Introduction: the skin is the largest organ in the human body. Cutaneous wounds are the result of disrupted skin integrity. Mesenchymal stem cells (MSCs) are emerging as a promising candidate for cell-based therapy for the treatment of chronic wounds because of their enormous potential for enhancing tissue repair and regeneration following injury. Silk fibroin cellularized with MSCs from different sources has been shown to be effective in repairing experimental wounds of the skin. Objectives: to investigate the behavior of Wharton’s jelly mesenchymal stem cells (Wj-MSCs) both in vitro and in vivo when cultivated on electrospun silk fibroin scaffolds and implanted in immunocompetent mice. To determine whether Wj-MSCs delivered in silk fibroin scaffolds into skin defects in SKH1 mice would contribute to dermal wound healing. Methodology: we have first standardized the protocol for the isolation and characterization of Wj-MSCs. Further, these MSCs along with the combination of silk fibroin scaffolds were used to test their wound healing properties by creating skin wounds in an experimental murine model. Results: Wj-MSCs exhibited a fibroblastic morphology and displayed MSC surface markers positive for CD90, CD105, CD73 with no detectable presence of hematopoietic cells markers. Wj-MSCs were limited on their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. Wj-MSCs suppressed T cell proliferation after stimulation with anti-CD3/CD28 coated beads and allogeneic mDCs. Wj-MSCs downmodulated the in vitro production of the pro-inflammatory cytokine IFN- by activated T lymphocytes. This immunosuppressive effect was also mediated by the production of the anti-inflammatory cytokines TGF-, IDO, and PGE2. MLCs experiments in the presence of different specific inhibitors of the biosynthesis or signaling of these anti-inflammatory factors such as SB-431542, indomethacin (IDM) or 1-methyl-tryptophan (1-MT), respectively, recovered almost entirely the proliferation rate of mDCs-stimulated T cells. Treatment of skin injury of SKH1 mice model demonstrated that combination of Wj-MSCs and silk fibroin scaffold exhibited significantly better wound-healing capabilities. Conclusions: Wj-MSCs have reduced immunogenicity, did not express costimulatory molecules, and express cytokines that may modulate immune function. Wharton's jelly MSCs combined with silk fibroin scaffolds in the wound bed contributed to the generation of a high-quality, well-vascularized granulation tissue, enhanced re-epithelialization of the wound, and attenuated the formation of fibrotic scar tissue. Wharton's jelly from the human umbilical cord may be an adequate source for obtaining MSCs for wound healing given its several advantages and together with the synergistic benefits of a nanoscaffold they make ideal combinations as wound dressings for slow healing and hard-to-heal chronic wounds.

El melanoma cutáneo es una neoplasia maligna de los melanocitos que se caracteriza por un curso clínico a menudo indeseable. Aunque actualmente la detección precoz y la cirugía ofrecen una alta tasa de curación, este tumor puede eludir la vigilancia inmunológica del huésped favoreciendo su evolución. Esto puede deberse ciertas interacciones entre los antígenos leucocitarios humanos (HLA) y células T citolíticas específicas de antígeno (CTL) o las células asesinas naturales (NK), las cuales son importantes por su acción anti-tumoral frente al melanoma. En contraste con otras células tumorales, las células de melanoma se consideran altamente inmunogénicas debido a que expresan antígenos que pueden desempeñar un papel decisivo en el reconocimiento de células tumorales y el rechazo por el sistema inmunológico del huésped. OBJETIVOS: Hasta la fecha no han sido completamente dilucidados los mecanismos inmunológicos implicados en el melanoma cutáneo. Se han hecho esfuerzos para encontrar marcadores de susceptibilidad o pronóstico y posibles dianas para las terapias inmunológicas, pero no han dado frutos aún. Por lo tanto, los objetivos de este estudio han sido investigar si: 1. La susceptibilidad o evolución del melanoma están influenciadas o no por los loci HLA A, HLA B, HLA C, HLA E, HLA-DRB1 o HLA-DQB1. 2. Si los cambios tempranos de las células T CD8+ y CD56+ NK que expresan receptores NK participan en la susceptibilidad o curso del melanoma en diferentes grupos de pacientes definidos por su dimorfismo HLA-C (Lys80/Asn80). DISEÑO EXPERIMENTAL: 1. El tipaje de HLA-A y HLA-B con el método de microlinfocitotoxicidad estándar. El tipaje HLA C para analizar el dimorfismo en la posición 80 de la hélice α1 (Asn80/Lys80) capaz de mediar con las células que expresan KIR, se realizó PCR-SSO. 2. El tipaje HLA-DRB1 y HLA-DQB1 y las confirmaciones de HLA de clase I se realizó mediante PCR-SSP. 3. Las células T CD8+ y CD56+ NK de pacientes y controles con diferentes genotipos HLA-C (Asn80/Lys80) se analizaron por citometría de flujo. RESULTADOS: 1. En este estudio, no se ha encontrado una asociación negativa o positiva de los alelos HLA-A, HLA-B, HLA-DRB1, HLA-DQB1 y HLA E con la susceptibilidad o evolución del melanoma melanoma. 2. Este estudio demuestra, por primera vez, que la presencia del epítopo Asn80 podría contribuir a la protección de la enfermedad, en particular el alelo HLA C*07. 3. Este estudio muestra, también por primera vez, que la presencia de HLA-C portadores de Lys80 en homocigosis puede ser considerado como un factor predisponente para el desarrollo de metástasis linfáticas tempranas. 4. Según nuestro conocimiento, describimos por primera vez, una evidencia “in vivo” de la existencia de un estado de activación en pacientes con melanoma en etapas no metastásicas de la enfermedad, determinada por un aumento del número absoluto de linfocitos T de sangre periférica con fenotipo CD8+CD28+DR+ o CD8+CD28-CD161+, que podría estar asociada con una estimulación de la respuesta inmune en las primeras etapas de melanoma cutáneo. 5. Se detecta, por primera vez también, una expansión selectiva de una subpoblación de células T que expresan receptores KIR2DL1/S1 (CD8+CD28CD158a+) junto con un aumento de las células CD56+ NK KIR2DL1/S1 en pacientes de melanoma, mucho más pronunciado en los pacientes portadores del ligando HLA-CLys80 en homocigosis, lo que indica un particular papel regulador inmunológico de las células que expresan receptores KIR2DL1/S1 en el melanoma. 6. Por lo tanto, es tentador especular sobre la posibilidad de la apertura de nuevas vías terapéuticas para el control de la progresión del melanoma, tomando en consideración a estas células como posibles dianas para la intervención inmunológica mediante el bloqueo o la estimulación de sus receptores inhibidores/activadores. / Cutaneous melanoma is a malignant neoplasm of melanocytes characterized by an often undesirable clinical outcome. Although current early detection and surgery offer a high cure rate, this tumor can be elusive for the host immune surveillance, especially if a state of tolerance against tumor is induced. The clinical outcome may be influenced by complex interactions that take place between the different human leukocyte antigens (HLA) and antigen-specific cytolytic T cells (CTL) or natural killer (NK) cells, which are believed to be important in eliciting an effective immune protection against melanoma. Nonetheless, in contrast to other tumor cells, melanoma cells are usually considered highly immunogenic because of the expression of antigens that may play a decisive role in tumor cell recognition and rejection by the host immune system. PURPOSE: To date, immune mechanisms involved in cutaneous melanoma have not been fully elucidated. Efforts have been made to find susceptibility or prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful so far. Therefore, the goals of this study were to investigate: 1. If the susceptibility or outcome of the melanoma is influenced by HLA A, HLA B, HLA C, HLA E, HLA-DRB1 or HLA-DQB1 loci. 2. The influence of early changes in CD8 T and CD56 NK cells expressing NK receptors in different HLA-C dimorphism (Lys80/Asn80) groups in the susceptibility or outcome of the melanoma. EXPERIMENTAL DESIGN: All experiments were made in patients and sex- and age-matched controls. 1. HLA-A and HLA-B typing were performed by the standard microlymphocytotoxicity test. 2. Typing of HLA C dimorphism (Lys/Asn) at position 80 in the α1 helix to examine whether it could mediate in the emergence of cells expressing killer-cell immunoglobulin-like receptors was tested by PCR-SSO. 3. HLA class II typing HLA-DRB1 and HLA-DQB1 typing and resolution of HLA class I ambiguities were performed by PCR-SSP 4. CD8 T and CD56 NK cells were analyzed in patients and sex- and age-matched controls with different HLA-C genotypes (Asn80/Lys80) by flow cytometry. RESULTS: 1. In this study, none of the HLA class I antigens (HLA-A and HLA-B), HLA class II alleles (HLA-DRB1 and HLA-DQB1) and HLA E showed positive or negative association with the overall population of melanoma patients. 2. This study shows, for the first time, that the presence of the Asn80 epitope could contribute to the protection against the disease, particularly the HLA-Cw*07 allele. 3. This study shows, also for the first time, that the presence of Lys80 epitope of the HLA-C molecules in homozygosis may be considered as a predictive factor for the development of early LN metastasis. 4. To our knowledge, we have described for the first time an in vivo evidence of the existence of a status of activation in melanoma patients at the nonmetastatic stages of the disease, determined by an increase of the absolute number of T lymphocytes from peripheral blood with phenotype CD8+CD28+DR+ or CD8+CD28-CD161+, which could be associated with a stimulation of the immune response at the early stages of cutaneous melanoma. 5. We have detected, for the first time too, a selective expansion of T cells subpopulation expressing KIR2DL1/S1 (CD8+CD28-CD158a+) together with an increase of CD56+ NK KIR2DL1/S1 cells in patients of melanoma, much more pronounced in patients carrying the corresponding HLA-CLys80 ligand in homozygosis, which point to a particular immune regulatory role of cells expressing KIR2DL1/S1 in melanoma. 6. Thus, it is tempting to speculate about the possibility of opening new therapeutic ways for the control of melanoma progression, taking into consideration these cells as a target for immune intervention by blocking or enhancing their inhibitory/activatory receptors.

El carcinoma basocelular localmente avanzado (CBCla) se define de forma general, como aquel CBC en el que hay confirmación radiológica de invasión de determinadas estructuras vecinas en profundidad y también probablemente aquel CBC de un tamaño e invasión suficientes (aunque no exista demostración radiológica de invasión en profundidad) en el que la cirugía y RT fueran inadecuadas, insuficientes o contraindicadas para lograr la curación del tumor, ya sea por características del propio tumor (por ej localización, tumores múltiples) o del paciente. En la iniciación y desarrollo de este tipo de tumor, la vía de señalización Hedgehog (Hh) desempeña un papel fundamental. Vismodegib es el primer inhibidor selectivo de la vía de señalización Hh que ha sido aprobado por la FDA (Erivedge, Genentech, Enero 2012) y por la Agencia Europea del Medicamento (EMA) en Julio 2013 para el tratamiento del CBCla y del carcinoma basocelular metastásico ( CBCm). Los estudios publicados hasta la fecha evalúan la eficacia de vismodegib basándose en criterios clínicos y radiológicos, pero disponemos de escasa información respecto a las bases moleculares que justifican la eficacia clínica probada del fármaco. Mediante la realización de este estudio en 15 pacientes con CBCla, se pretende ampliar la información científica disponible e investigar nuevas bases moleculares que expliquen la eficacia, los diferentes niveles de respuesta clínica y fenómenos de resistencia durante el tratamiento con vismodegib. Los objetivos de este trabajo han sido los siguientes: 1- Evaluar la eficacia del tratamiento con vismodegib en nuestra cohorte de pacientes con CBCla (n=15). 2- Estudiar las moléculas/variables que puedan ayudar a determinar la eficacia de vismodegib y establecer posibles fenómenos de resistencia al fármaco. 3- Intentar identificar bases moleculares adicionales que ayuden a la comprensión de los nive¬les de respuesta (completa, parcial y no respuesta), así como fenómenos de resistencia en pacientes con carcinoma basocelular localmente avanzado tratados con vismodegib. 4- Intentar definir y aclarar algunas de las bases moleculares implicadas en el desarrollo de diferenciación escamosa en las biopsias de seguimiento realizadas en el curso del tratamiento. Como resultados del estudio se ha obtenido: 1- Aquellos pacientes diagnosticados de CBCla extenso en tratamiento con vismodegib han ex-perimentado una aparente reducción clínica y/o radiológica tumoral, completa en el 20% de los pacientes y parcial en el 73.3% pudiendo lograr la resolución completa del mismo mediante tratamiento adyuvante con cirugía y/o RT sólos o en combinación, tratamientos que una gran mayoría de pacientes había recibido pero que no habían sido suficientes para obtener la cura¬ción tumoral. 2- Si que se ha observado una tendencia mayoritaria al descenso de los niveles en respuesta al tratamiento para los biomarcadores directamente relaccionados con la vía Hh como son Gli1, HES1, PRKCI, DISP1 y SOX2 con aumento de los marcadores de apoptosis tumoral, coherente con el grado de respuesta al fármaco, pero con incremento o estabilidad de otros correspon¬dientes a otras vías diferentes pero directamente relaccionadas con Hh. Con los datos obteni¬dos en este estudio no se pueden hacer inferencias claras y concretas con respecto a cuales de estos últimos marcadores podrían estar implicados en una menor respuesta y/o fenómenos de resistencia al fármaco. / Locally advanced basal cell carcinoma (laBCC) is defined generally as the BCC where there are radiological confirmation of invasiveness of certain neighboring structures and also the BCC with big size and sufficient invasion (although it do not exist radiological demonstration of invasion in depth) where the surgery and radiotherapy were inadequate, insufficient or contraindicated to achieve tumor cure, either by the tumor characteristics (eg location, multiple tumors) or patient. Hedgehog (Hh)signaling pathway plays a key role in the initiation and development of this type of tumor. Vismodegib is the first selective inhibitor of Hh signaling pathway that has been approved by the FDA (Erivedge, Genentech, January 2012) and the European Medicines Agency (EMA) in July 2013 for the treatment of laBCC and metastatic basal cell carcinoma (mBCC). The published studies in relation to vismodegib, evaluate the efficacy of this drug based on clinical and radiological criteria, but we have little information on the molecular basis justifying the proven clinical efficacy of the drug. Our study was conducted in 15 patients with CBCla diagnosis, and had the objective of increasing the available scientific information and investigate new molecular basis to explain the effectiveness, different levels of clinical response and resistance phenomena during treatment with vismodegib. The objectives of this study were as follows: 1- To evaluate the efficacy of treatment with vismodegib in our cohort of patients with CBCla (n = 15). 2- To study the molecules / variables that may help to determine the effectiveness of vismodegib and establish possible phenomena of drug resistance. 3- Try to identify additional molecular basis that help understanding the response levels (complete, partial and no response) and resistance phenomena in patients with laBCC treated with vismodegib. 4- Try to define and clarify some of the molecular basis involved in the development of squamous differentiation in the follow-up biopsies performed in the course of treatment. The results of our study were as follows: 1- The 20% of patients with extensive CBCla treated with vismodegib experienced an apparent complete clinical and / or radiological tumor reduction and 73.3% achieved a partial response; these patients achieved a complete tumor resolution through adjuvant therapy ( surgery or radiotherapy alone or in combination) 2- We have seen a major trend to lower levels, in response to treatment, for biomarkers directly related with the Hh pathway such as Gli1, HES1, PRKCI, DISP1 and SOX2 with increased apoptosis tumor markers, consistent with the degree of drug response, but with increased stability of other biomarkers belonging to other pathways but Hh related. We have not obtained statistical significance in the analysis.

Consideration of broader outcomes of disease, especially those exclusively experienced and reported by the patient, such as HRQOL, is not only consistent with the ‘whole person’ view of health contained in the 1948 WHO definition, but is also a prerequisite to building health-care systems that are responsive to the needs of the patients. For chronic skin diseases, such as hyperhidrosis, these provide a useful indicator of how a patient feels and functions disease for both practical and methodological reasons. The aims of this study therefore were to investigate the impact of hyperhidrosis on patients’ HRQoL using a mix of qualitative and quantitative methods. In addition, a further aim was to develop and validate a disease-specific instrument for assessing HRQoL in hyperhidrosis. In pursuing the above aims, the feasibility of applying online social networking sites for outcomes research in dermatology was assessed. Patients were recruited through online social networking communities related to hyperhidrosis for all stages of the study. Interviews, focus groups and surveys were used for collecting qualitative data from patients (n = 71) to understand quality of life issues of patients, and to provide the content of the new instrument. Dermatologists (n= 5) and patients (n=7) took part in the content validation of the HidroQoL©. Item reduction and the development of the scale’s structure was carried out through several field-testing studies (n: USA, 559; UK, 115), using the item response theory (IRT) Rasch model and factor analyses. Further psychometric testing was performed in a separate study (n = 241). Distribution-based methods were applied in establishing minimum clinically important difference (MCID). A thematic analysis of the qualitative data collected produced 29 quality of life themes and 102 sub-themes, forming the content for the initial 49-item HidroQoL©. The two expert panels judged the instrument as content valid, with a few suggestions. The Rasch analysis modelling led to the collapsing of response categories (from five to three) and the reduction in number of items (from 49 to 18), to ensure a perfect model fit. Factor analyses supported both a single- and a two-factor structure. In subsequent construct validation study the HidroQoL correlated with the DLQI (rs = 0.572, p < 0.01) and the Skindex-17 (rs = 0.551, p < 0.01). Reliability was high (Cronbach alpha = 0.9; test-retest ICC = 0.93). The scores were sensitive to change in patients’ disease severity (standard response mean = 0.8, 95% C.I: 0.34-1.27). The scale banding proposed for the HidroQoL score is as follows: 0 – 1, no effect at all; 2 – 11, small effect; 12 – 22, moderate effect; 23 – 32, large effect; 33 – 36, very large effect. The MCID values were 1.94 – 3.07, for generalised v hyperhidrosis, 2.16 – 4.36, for axillary hyperhidrosis, 2.15 – 3.39, for palmo-plantar hyperhidrosis. An MCID of three is currently being proposed for all types of hyperhidrosis. This study has provided the initial evidence supporting the appropriateness of the content of the HidroQoL and validity of inferences from its scores for assessing HRQoL in hyperhidrosis. In addition, the availability of MCID estimates for the HidroQoL will facilitate its clinical interpretation in both research and routine clinical practice. This study has also demonstrated how CTT and IRT can be integrated in the development and validation of a new generation of HRQoL instruments, using social network for patient recruitment.

INTRODUCCIÓN: La enfermedad de Schönlein-Henoch es una enfermedad multisitémica que afecta principalmente a vasos de pequeño calibre de la piel, articulaciones, sistema digestivo y riñón.MOTIVO Y JUSTIFICACIÓN DE LA TESIS: El estrés oxidativo puede ser determinante en la aparición del síndrome de Schönlein-Henoch, en su evolución o en la presentación de la nefropatía. Los radicales libres en exceso o un déficit de los mecanismos antioxidantes podría favorecer la aparición de la enfermedad.OBJETIVOS: 1. Estudiar el papel del daño oxidativo en la patogénesis y desarrollo de la púrpura de Schönlein-Henoch.2. Estudiar la implicación de los agentes antioxidantes (glutation peroxidasa, glutation reductasa, superóxido dismutasa, catalasa, ubiquinona, coenzima Q, malondialdehido, vitaminas A, C y E) en el desarrollo de la púrpura de Schönlein-Henoch en sus fases de actividad y remisión.3. Estudiar la implicación de los agentes antioxidantes en la nefropatía del Schönlein-Henoch.MATERIAL Y MÉTODOS: Se estudian 23 niños afectos de púrpura de Schönlein-Henoch. Se han dividido en dos grupos según su afectación renal. El grupo A comprende 12 pacientes con complicación renal y el grupo B 11 sin complicación renal. A todos ellos se les efectúa determinación de enzimas antioxidantes, sustancias antioxidantes y marcador de la peroxidación lipídica.Resultados: En la glutation reductasa se han obtenido valores significativamente inferiores en los niños que presentan enfermedad activa comparados con el grupo control (p<0,05). No hemos encontrado diferencias en el resto de agentes antioxidantes estudiados.CONCLUSIONES: 1. Los pacientes que tenían la púrpura de Schönlein-Henoch en actividad presentaban valores de glutation reductasa significativamente más bajos que el grupo control.2. Los enzimas antioxidantes estudiados: glutation peroxidasa, glutation reductasa, catalasa y superóxido dismutasa, no presntaron diferencias estadísticamente significativas entre el grupo control y los pacientes con púrpura de Schönlein-Henoch en actividad, en remisión o los que presentaron nefropatía.3. Las sustancias antioxidantes estudiadas: ubiquinona, coenzima Q, y vitaminas A, C y E, no presentaron diferencias estadísticamente significativas entre el grupo control y los pacientes con púrpura de Schönlein-Henoch en actividad, en remisión o los que presentaron nefropatía.4. La peroxidación lipídica estudiada mediante la determinación de malondialdehido no ha mostrado diferencias significativas entre el grupo control y los pacientes con púrpura de Schönlein-Henoch en actividad, en remisión o los que presentaron nefropatía. / INTRODUCTION: The Schönlein-Henoch purpura is a multisystemic disease that involves mainly small size vessels from the skin, joints, digestive tract and kidneys.AIM AND JUSTIFICATION OF DISSERTATION: Oxidative stress may be determinant in the occurrence of the Schönlein-Henoch syndrome, in its evolution or in the presentation of nephropathy. An excess of free radicals or a deficiency in the antioxidative mechanisms may favour the occurrence of this disease.OBJECTIVES: 1. To study the role of oxidative damage in the pathogenesis and development of the Schönlein-Henoch purpura.2. To study the involvement of antioxidative agents (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, ubiquinone, co-enzyme Q, malondialdehide, vitamins A, C and E) in the development of the Schönlein-Henoch purpura in its active and remission phases.3. To study the involvement of antioxidative agents in the Schönlein-Henoch nephropathy.Material and methods: Twenty-three children affected by Schönlein-Henoch purpura were studied. They were divided in two groups according to renal involvement. Group A comprised 12 patients with renal complications, and group B, 11 without renal complications. In all of them the antioxidative enzymes, the antioxidative substances and the lipid peroxidation marker were determined.RESULTS: For the enzyme glutathione reductase we obtained significantly lower values in children with active disease compared to the control group (p<0.05). We did not find any differences in the other antioxidative agents studied.CONCLUSIONS:1. Patients with Schönlein-Henoch purpura in active phase showed significantly lower levels of glutathione reductase than the control group.2. The antioxidative enzymes studied: glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase did not show statistically significant differences between the control group and patients with Schönlein-Henoch purpura in active phase, in remission or in the cases with nephropathy.3. The studied antioxidative substances: ubiquinone, co-enzyme Q and vitamins A, C and E) did not show statistically significant differences between the control group and patients with Schönlein-Henoch purpura in active phase, in remission or in the cases with nephropathy.4. The lipid peroxidation studied by the determination of malondialdehide did not show significant differences between the control group and patients with Schönlein-Henoch purpura in active phase, in remission or in the cases with nephropathy.

Keratoderma – pathological hyperkeratosis of palms and soles - is a cause of disability in many clinical situations, including the rare and heterogeneous group of inherited palmoplantar keratodermas (PPKs). The aim of this study was to work towards better understanding of molecular mechanisms active in the pathogenesis of PPK by the creation of a cell and tissue culture resource and its initial application to laboratory studies. My study was based on a diverse group of autosomal dominant disorders, previously ascertained in families from Scotland, in whom the precise genetic aetiology was known. I established a tissue and cell culture resource of inherited keratodermas of known single-gene aetiology from patients with proven keratin 1, 9, 17, loricrin and mitochondrial mutations. An additional pedigree with striate keratoderma with an unknown mutation was recruited, and the causative mutation identified as a novel heterozygous A-to-T transversion in exon 5 (c.430A>T) of the desmoglein 1 gene, converting an arginine residue to a premature termination codon (p. Arg144stop). The keratinocyte culture resource was established from patients with keratin 1, 9, 17 and loricrin mutations, as well as controls. Due to the pain associated with direct infiltration of plantar skin, biopsies were obtained using peripheral nerve block for plantar biopsy. The effectiveness of this approach, which may be useful for future administration of treatment, was made the subject of an open clinical trial. Histological and immunocytochemical studies were carried out on affected plantar skin obtained from PPK patients and compared to control tissue, in an attempt to identify common and distinct pathways resulting in hyperkeratosis. Histological changes, e.g. hypergranulosis, extent of hyperkeratosis, acanthosis or acantholysis, were not uniform across different subtypes of inherited PPK and varied even between individuals within subtypes. Prominent eosin staining of spinous cells was a common feature in inherited PPK due to underlying K1 and K17 mutations. Electron microscopy showed abnormal keratin filaments in PPK with underlying keratin mutations only but was not a uniform finding within subtypes, and other electron microscopic features also varied between individuals. Immunocytochemical study did not demonstrate significant differences in expression of a selection of markers of differentiation (keratins 1, 9, 14 and 17), and cornified envelope protein filaggrin. Abnormal involucrin expression was observed, with premature expression in basal and lower spinous layers in all PPK subtypes raising the possibility of a common underlying mechanism in the development of hyperkeratosis. Prominent loricrin staining was noted in areas of acantholysis in K1 and K9 subtypes, but was uniform across other subtypes. Markers of proliferation and apoptosis demonstrated no overt change in epidermal turnover, although it is possible that only small changes in proliferative index are required to produce plantar hyperkeratosis. Overall, using morphological criteria, plantar hyperkeratosis was not readily distinguishable between inherited PPK of different underlying genetic causes. This raises the possibility that many of the reported structural features of inherited PPK are secondary phenomena as opposed to critical steps in the pathogenesis of hyperkeratosis. Initial attempts at RNA extraction using laser and manual microdissection have to date been unsuccessful in generating RNA of the quality and concentration to run a pilot microarray experiment, using standard RNA extraction kits. Plans for future projects include the further development of a possible microarray experiment in the Pachyonychia Congenita type 2 pedigree with the McLean laboratory in Dundee. The tissue resource has been made available for collaborative study via the GENESKIN project, as well as through the McLean and Lane laboratories, Dundee for both functional studies and immortalisation of cell lines.

Examining surface shape appearance by touching and observing a lesion from different points of view is a part of the clinical process for skin lesion diagnosis. Motivated by this, we hypothesise that surface shape embodies important information that serves to represent lesion identity and status. A new sensor, Dense Stereo Imaging System (DSIS) allows us to capture 1:1 aligned 3D surface data and 2D colour images simultaneously. This thesis investigates whether the extra surface shape appearance information, represented by features derived from the captured 3D data benefits skin lesion analysis, particularly on the tasks of segmentation and classification. In order to validate the contribution of 3D data to lesion identification, we compare the segmentations resulting from various combinations of images cues (e.g., colour, depth and texture) embedded in a region-based level set segmentation method. The experiments indicate that depth is complementary to colour. Adding the 3D information reduces the error rate from 7:8% to 6:6%. For the purpose of evaluating the segmentation results, we propose a novel ground truth estimation approach that incorporates a prior pattern analysis of a set of manual segmentations. The experiments on both synthetic and real data show that this method performs favourably compared to the state of the art approach STAPLE [1] on ground truth estimation. Finally, we explore the usefulness of 3D information to non-melanoma lesion diagnosis by tests on both human and computer based classifications of five lesion types. The results provide evidence for the benefit of the additional 3D information, i.e., adding the 3D-based features gives a significantly improved classification rate of 80:7% compared to only using colour features (75:3%). The three main contributions of the thesis are improved methods for lesion segmentation, non-melanoma lesion classification and lesion boundary ground-truth estimation.

Atopic dermatitis, AD (synonym eczema) is a chronic inflammatory skin disease. It affects between 10 to 20% of children and 1 to 3% of adults worldwide. It is an important cause of morbidity and is estimated to cost £465 million per annum to the UK. AD is part of a family of Th-2 driven diseases and is often the first of these atopic diseases to manifest. The development of AD is often followed by asthma and allergic rhinitis later in life (a phenomenon known as the ‘atopic march’). Up to 50% of moderate to severe AD cases have been associated with genetic mutations affecting the epidermal barrier protein filaggrin. Filaggrin aggregates keratin filaments during terminal keratinocyte differentiation, allowing normal epidermal stratification. The role of filaggrin in maintaining a functional skin barrier is further supported by a clinical study conducted by ourselves. This is the first clinical study on a European cohort (58 participants) which showed that FLG mutations were associated with experimentally demonstrable defects of skin barrier function (increased baseline transepidermal water loss), more so following exposure to a chemical irritant. However, the majority of patients with AD, especially the milder cases, do not have FLG mutations. Some of the wild-type patients in our study were noticed to have accumulation of the large filaggrin proprotein and a lack of filaggrin monomers, indicating defective proteolysis of profilaggrin into the functional monomers. Our study also found disproportionately raised protease inhibitory activities amongst the AD participants. This inappropriately raised protease inhibition may interfere with profilaggrin proteolysis, leading to the development of AD in some wild-type patients. Having demonstrated that deficiency of filaggrin monomers is associated with a defective skin barrier, we focused on the function of filaggrin in the skin and attempted to improve the skin barrier function. In addition to keratin aggregation, filaggrin constitutes the natural moisturizing factors in the epidermis following its natural breakdown into amino acids. We note that filaggrin is disproportionately rich in amino acid histidine, implying that this amino acid may have a particular significance in maintaining a functional epidermal barrier. Using an in-house skin-equivalent model, we have shown that by increasing the histidine content in the cell culture media, we could increase the expression of filaggrin monomers and reduce the penetration of a fluorescent dye into the skin-equivalents. The latter indicates improved barrier function. Finally, we conducted a pilot human study which showed that histidine, when applied to mechanically damaged skin in AD and healthy participants, was associated with a faster recovery of the skin barrier function. These studies suggest that histidine is of therapeutic benefits in AD. A histidine-based treatment may be developed as an alternative to current anti-inflammatory and immunosuppressive agents used to treat AD.