Orientador: Luiz Henrique de Araújo Machado / Resumo: A dermatite atópica canina é uma importante alergopatia de ordem genética, inflamatória, pruriginosa e crônica. Os tratamentos existentes atualmente são sintomáticos, algumas vezes insatisfatórios no controle da doença e passíveis de efeitos colaterais. As células-tronco mesenquimais (CTM), pelas suas propriedades imunomoduladores e anti-inflamatórias, podem ser uma alternativa no tratamento da dermatite atópica. Foi realizado um estudo longitudinal, duplo-cego, com período inicial de tratamento placebo com três aplicações quinzenais de soro fisiológico, com posterior aplicação de CTM heterólogas, derivadas de tecido adiposo, em seis cães com dermatite atópica, totalizando três aplicações quinzenais na dose de 5x106 células por animal. A escala visual de prurido esclarecido (EVPE), o escore de lesões cutâneas (CADESI-4), a resposta inflamatória cutânea pela análise das citocinas IL4, IL6, IL10, IL31 e TNF-alfa e da proteína filagrina pela técnica de imunoistoquímica e a avaliação dos lipídeos epidérmicos pela coloração de Sudam III foram comparadas entre o tratamento placebo e com as CTM. O tratamento com CTM foi benéfico para a melhora das manifestações clínicas nos animais com dermatite atópica, diminuindo de forma significativa o escore CADESI-4 e EVPE, com redução consistente de mais de 50% destes dois parâmetros ao final do tratamento com CTM. A melhora clínica evidente nos animais com DA tratados com terapia celular aliadas à baixa frequência de efeitos colaterais, sina... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The atopic canine is an inflammatory allergic dermatitis frequent and important. The distinct treatment available is symptomatic, currently unsatisfactory in the control of the disease and susceptible to collateral effects. The mesenchymal Stem Cells (MSC) by their immunomodulators and anti-inflammatory characteristics can be an alternative in the treatment of the atopic dermatitis. A longitudinal study was performed, blind-double, with an initial period of placebo treatment within saline solution, after the application of 5x106 heterologous CTM, derived from adipose tissue, in six dogs with atopic dermatitis. Each treatment constituted of three applications in by-weekly intervals. The visual analog scale of pruritis, the score of skin lesions (CADESI-4), the skin inflammatory response and filaggrin through the immunohistochemistry technique and the evaluation of the skin lipids by the Sudam III coloration were compared between the placebo treatment and the CTM treatment. The treatment with CTM with the used protocol was beneficial to the improvement of the clinical responses of the animals with atopic dermatitis, decreasing significantly the CADESI-4 and EVDP score, with a significant reduction of more than 50% of these two parameters at the end of the treatment with CTM while compared to the placebo. The clinical improvement was evident in animals with DA treated with cellular therapy allied to the low frequency of the collateral effects, indicates that with CTM can be cons... (Complete abstract click electronic access below) / Doutor

Charles University, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Anna Nováčková Supervisor doc. Mgr. Jarmila Zbytovská, Dr. rer. nat. Title of Doctoral Thesis Study of substances affecting permeability of the skin barrier The skin barrier plays a vital role in protecting the human body and enables mammals' life on dry land. The epidermis has the primary barrier function due to several cells' layers, which gradually differentiate to their final stage, the stratum corneum (SC). SC is formed by stratified keratinocytes (known as corneocytes) surrounded by a lipid matrix. This intercellular matrix consists of an approximately equimolar ratio of ceramides, free fatty acids and cholesterol. These are particular substances formed in the epidermis from their precursors during the keratinocyte's differentiation, and their arrangement into the multilamellar structure is essential for the impermeability of the skin barrier. However, some substances or factors can disrupt the skin barrier. It is usually an undesirable process of lipid disbalance resulting in disorders or diseases of the skin barrier. On the other hand, specific substances have been developed for a reversible disruption of the skin barrier (so-called enhancers) to allow drug...

Introdução: Dermatite atópica (DA) é uma doença cutânea crônica, predominante na infância, cujo sintoma principal é o prurido de intensidade variável, e os sinais são classicamente as lesões de padrão eczematoso. Há anormalidades na formação e função da barreira cutânea, que estão presentes não somente nas lesões cutâneas como na pele clinicamente não afetada. Objetivo: Analisar a correlação entre as medidas biofísicas da função de barreira cutânea e os critérios clínicos e intensidade da dermatite, de acordo com os critérios de Rajka e Langeland. Métodos: 231 doentes do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, com diagnóstico clínico de dermatite atópica segundo os critérios diagnósticos de Rajka e Langeland foram avaliados por exame físico, anamese, medidas biofísicas de grau de hidratação de camada córnea pelo método de capacitância (corneometria) e pelo método de perda de água transepidérmica (TEWL); a medida sérica de IgE também foi solicitada no ato do exame. Resultados: Houve uma relação significativa entre as medidas de corneometria, TEWL e gravidade clínica da dermatite atópica. Os dados demonstraram uma correlação inversamente proporcional entre a corneometria e o TEWL, e houve uma diferença estatisticamente significativa (p<0,001) entre as médias de corneometria e TEWL e grau de DA (leve, moderada ou intensa). Com relação aos níveis séricos de IgE, as medidas de corneometria apresentaram uma correlação negativa significativa; para TEWL, a correlação positiva foi estatisticamente significativa (p<0,001). Conclusão: As medidas biofísicas de barreira cutânea na DA, mesmo em pele aparentemente não lesada, podem funcionar como fator de avaliação do grau clínico da DA e da intensidade do prurido. / Background: Atopic dermatitis (AD) is a chronic dermatosis, predominant in childhood, characterized by pruritus and eczematous type lesions with xerosis as the proeminent clinical sign. Objectives: To analyze the correlation between biophysical measurements of skin barrier function and other assessment criteria of clinical severity according to Rajka and Langelands criteria. Methods: Biophysical measurements (Transepidermal water loss and corneometry) were obtained from 231 patients from the department of dermatology, Hospital das Clinicas FMUSP with the diagnsosis of atopical dermatitis. Serum levels of IgE were also evaluated. Results: A significant correlation between corneometry, TEWL and clinical severity of atopic dermatitis were found. Data showed an inverse correlation between corneometry, TEWL, and AD severity, and a significant difference (p<0,001) between means of corneometry and TEWL and AD severity (mild, moderate and severe). As for IgE levels, corneometry had significant negative correlation, in contrast with TEWL, wich showed a significant positive correlation (p<0,001). Conclusion: Biophysical measurements of skin barrier in non lesional skin of atopic dermatitis may work as an evaluation factor for AD severity and pruritus.

Les modèles actuels de prédiction de la sensation thermique et du confort thermique ainsi que les solutions visant à améliorer l’état de bien-être thermique des occupants d’un bâtiment sont insuffisants. Ils ne prennent pas assez en compte les différences interindividuelles de jugement thermosensoriel. Pourtant, ces différences, souvent associées à la sensibilité thermique de chaque individu, existent mais restent inexpliquées sur le plan physiologique. Ces travaux de thèse, qui se sont déroulés en deux étapes, ont pour objectif d'identifier les causes physiologiques potentielles des différences interindividuelles du ressenti thermique, à travers des expérimentations multiparamétriques basées sur des mesurées cutanées. Toutes les mesures ont été réalisées après 30 minutes d’acclimatation en environnement thermique contrôlé. La première étape, exploratoire, a permis d’analyser à la fois l’activité neurosensorielle, les propriétés thermo-vasculaires et les propriétés du film hydrolipidique cutané de deux groupes présentant des sensibilités au froid distinctes (selon leur sensation thermique déclarée). Ainsi, les expérimentations ont montré qu’il était plus pertinent d’analyser davantage les propriétés cutanées thermiques et hydriques (reliées aux mécanismes de thermorégulation) plutôt que l’activité neurosensorielle des volontaires pour caractériser les différences interindividuelles de jugement thermosensoriel. Elles ont également mis en évidence la nécessité de contrôler les facteurs non thermiques des environnements et de sélectionner rigoureusement les sujets. La deuxième étape s’est focalisée sur l’analyse des propriétés thermo-vasculaires et des propriétés du film hydrolipidique de deux groupes de sensibilité au froid. Pour cela, 13 femmes ont été confrontées à 6 environnements de températures modérées comprises entre 17°C et 30°C (avec 2 transitions chaudes et 2 transitions froides) et les groupes ont été construits à partir du degré de frilosité déclaré par les sujets. Des différences sur les paramètres cutanées ont alors pu être relevées entre les deux groupes. Le résultat le plus significatif est que les individus dits « frileux » présentent une activité microcirculatoire plus intense sur les joues avec une vasoconstriction plus forte au froid et une vasodilatation plus forte au chaud que l’autre groupe « non sensible au froid » (p=0,002 d’après le test de l’ANCOVA pour l’effet des groupes). De plus, il a été montré que l’approche multiparamétrique (introduction de variables non thermiques comme variables prédictives) ainsi que la prise en compte des sensibilités thermiques individuelles améliorent la prédiction du confort thermique surtout pour le groupe « frileux » (+ 6,4 %). / Current models for predicting thermal sensation and thermal comfort as well as the solutions to improve the state of thermal well-being of the occupants of a building are insufficient. They do not sufficiently take into account interindividual differences of thermosensory judgment. However, these differences, often associated with thermal sensitivity of each person, exist but remain unexplained physiologically. This work, divided into two stages, is intended to identify the potential physiological causes of interindividual differences of thermal feeling through multiparametric experiments based on skin measurements. All measurements were performed after 30 minutes of acclimatization in controlled environment. The exploratory phase allowed to analyze both neurosensory activity, thermo-vascular properties and properties of the skin hydrolipidic film of two groups with different cold sensitivities (depending on their declared thermal sensation). For example, experiments have shown that it was more appropriate to analyse thermal and hydric skin properties (related to thermoregulation mechanisms) rather than neurosensory activity of volunteers to characterize interindividual differences of thermosensory judgment. They have also highlighted the need to control the non-thermal factors of environments and rigorously select subjects. The second step focused on the analysis of thermo-vascular properties and properties of the hydrolipidic film of two groups of different cold sensitivity. Thirteen women have faced in 6 environments of moderate temperatures between 17 ° C and 30 ° C (with 2 warm transitions and 2 cold transitions). Groups were built according to their degree of cold sensitivity. Differences in skin parameters have been found between the two groups. The most significant result is that cold-sensitive individuals have a more intense microcirculatory activity on cheeks with a stronger vasoconstriction in cold environments and a stronger vasodilation in warm environement than the non cold-sensitive group (p = 0. 002 according to ANCOVA test for groups effect). In addition, it has been shown that the multi-parametric approach (introduction of non-thermal parameters as predictors) as well as taking into account individual thermal sensitivities improve the prediction of thermal comfort especially for the cold-sensitive group (+ 6.4%).

Introdução: Dermatite atópica (DA) é uma doença cutânea crônica, predominante na infância, cujo sintoma principal é o prurido de intensidade variável, e os sinais são classicamente as lesões de padrão eczematoso. Há anormalidades na formação e função da barreira cutânea, que estão presentes não somente nas lesões cutâneas como na pele clinicamente não afetada. Objetivo: Analisar a correlação entre as medidas biofísicas da função de barreira cutânea e os critérios clínicos e intensidade da dermatite, de acordo com os critérios de Rajka e Langeland. Métodos: 231 doentes do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, com diagnóstico clínico de dermatite atópica segundo os critérios diagnósticos de Rajka e Langeland foram avaliados por exame físico, anamese, medidas biofísicas de grau de hidratação de camada córnea pelo método de capacitância (corneometria) e pelo método de perda de água transepidérmica (TEWL); a medida sérica de IgE também foi solicitada no ato do exame. Resultados: Houve uma relação significativa entre as medidas de corneometria, TEWL e gravidade clínica da dermatite atópica. Os dados demonstraram uma correlação inversamente proporcional entre a corneometria e o TEWL, e houve uma diferença estatisticamente significativa (p<0,001) entre as médias de corneometria e TEWL e grau de DA (leve, moderada ou intensa). Com relação aos níveis séricos de IgE, as medidas de corneometria apresentaram uma correlação negativa significativa; para TEWL, a correlação positiva foi estatisticamente significativa (p<0,001). Conclusão: As medidas biofísicas de barreira cutânea na DA, mesmo em pele aparentemente não lesada, podem funcionar como fator de avaliação do grau clínico da DA e da intensidade do prurido. / Background: Atopic dermatitis (AD) is a chronic dermatosis, predominant in childhood, characterized by pruritus and eczematous type lesions with xerosis as the proeminent clinical sign. Objectives: To analyze the correlation between biophysical measurements of skin barrier function and other assessment criteria of clinical severity according to Rajka and Langelands criteria. Methods: Biophysical measurements (Transepidermal water loss and corneometry) were obtained from 231 patients from the department of dermatology, Hospital das Clinicas FMUSP with the diagnsosis of atopical dermatitis. Serum levels of IgE were also evaluated. Results: A significant correlation between corneometry, TEWL and clinical severity of atopic dermatitis were found. Data showed an inverse correlation between corneometry, TEWL, and AD severity, and a significant difference (p<0,001) between means of corneometry and TEWL and AD severity (mild, moderate and severe). As for IgE levels, corneometry had significant negative correlation, in contrast with TEWL, wich showed a significant positive correlation (p<0,001). Conclusion: Biophysical measurements of skin barrier in non lesional skin of atopic dermatitis may work as an evaluation factor for AD severity and pruritus.

Atopic dermatitis (AD) and other atopic diseases are strongly related to skinbarrier dysfunction, a biomeasure which has limited and unsatisfactory assessmenttechniques. Machine learning (ML) powered electrical impedance spectroscopy(EIS) has been shown to differentiate defective barrier function in mice and adults. Techniques such as principal component analysis (PCA) andsupport vector classifiers (SVC) can be used as ML tools to evaluate EIS measurements. EIS measurements taken on unaffected skin of children aged between 4 monthsand 3 years were collected and analysed using the children’s AD status. Measurements were grouped into one of four groups based on this AD status; No AD (No AD was developed up until 2 years of age), Pre AD (measurements takenbefore the onset of AD), AD remission (measurements taken after the onset ofAD) and AD flare (measurements taken during active AD). A SVC model was trained on the raw EIS measurement data to distinguish measurements from twoof the binarized AD status groups; AD flare and No AD. An additional SVC model was trained on the No AD group, distinguishing measurements based onbinarized age groups: measurements taken at 4 months against measurements taken at 3 years of age. The AD model tested on AD flare against No AD within the test set yielded AUC of 0.92, with a sensitivity and specificity of 89.29% and 88.89% respectively. When testing on all groups of the test set, Pre AD and AD remission groups had group means between the AD flare and No AD groups. No data bias against age was detected in the model. The results of the age model showed that age could be chronologically identified by the age model. The AD model was able to differentiate active AD children from children never experiencing AD symptoms on visually unaffected skin and in turn detecting skin barrier dysfunction. Separate studies would need to be conducted to test the predictive power and external validity of the model. Age is a significant factor to consider when designing ML models using EIS data in children, with proper balance in the training set a data bias within the model can be avoided. EIS is a versatile technique due to its data rich nature. Machine learning powered electrical impedance spectroscopy measurements are able to detect skin barrier dysfunction. Age is a significant factor when measuring EIS on children, but can be managed. / Atopisk dermatit (AD) och andra atopiska sjudomar är starkt kopplade tillhudbarrärdefekter, en hudegenskap där diagnosverktygen är begränsade och otillräckliga. Maskininlärning (ML) i kombination med elektrisk impedansspektroskopi (EIS) har visats att kunna differentiera defekta hudbarrärer i möss och hos vuxna människor. Tekniker såsom principalkomponentanalys (PCA) och stödvektormaskiner (SVM) kan användas som ML verktyg för att utvärdera EIS-mätningar. Statistiska analysverktyg såsom reciever operator characteristics (ROC) tillsammans med arean under kurvan (AUC) och andra precisionsmått kan användas för att utvärdera ML modeller. EIS-mätningar utförda på opåverkad hud hos barn mellan 4 månader och 3 års ålder samlades och analyserades med hjälp av barnens atopiska status. Samtliga mätningarna grupperades i en av totalt fyra grupper baserade på deras atopiska status; Ingen AD (Ingen AD hade utvecklats upp till 2 års ålder), Pre AD (mätningarna togs innan sjukdomsförloppet), AD remission (mätningarna togs efter sjukdomsförloppet) och AD aktiv (mätningarn togs under sjukdomsförloppet). En SVM modell tränades på EIS-rådata för att särskilja mellan två av grupperna binärt; AD aktiv och ingen AD. En ytterliggare SVM modell tränades på endast ingen AD-gruppen för att särskilja mellan mätningarna tagna vid åldrarna 4 månader och 3 år, omgjorta till binära grupper.  AD modellen testades med en jämförelse mellan AD aktiv-gruppen och ingen AD-gruppen med test data och resulterade i en AUC på 0.92, med en respektive sensitivitet och specificitet på 89.29\% samt 88.89\%. Modellen testades även på de ytterliggare grupperna i test datan, Pre AD och AD remission som båda hade gruppmedelvärden som låg mellan AD aktiv-gruppens och ingen AD-gruppens. Inga systematiska fel med avseende på barnens ålder hittades i AD-modellen. Resultaten från ålders-modellen visade på att modellen kunde rangordna mätningarna baserat på ålder. AD-modellen kunde differentiera barn med aktiv AD mot barn som aldrig hade uppvisat atopiska symptom på opåverkad hud, vilket innebär en differentiering av hudbarrärdefekter. Separata studier är nödvändiga för att testa prediktiv prestanda hos modellen samt dess externa validitet. Ålder är en signifikant faktor när ML modeller med EIS-data ska designas, men med tillräcklig balans i träningsdatan kan systematiska fel förhindras. EIS är en versatil teknik med avseense på dess datakomplexitet. EIS tillsammans med maskininlärning kan särskilja defekta hudbarriärer hos barn. Ålder är en signifikant faktor när EIS mäts på barn, men kan hanteras.