Hypothesis: Trimethoprim promotes wound healing, decreases lesion counts and improves quality of life of recessive dystrophic epidermolysis bullosa (RDEB) patients. Objectives: Assess feasibility of conducting a large randomized clinical trial. Determine efficacy of trimethoprim in healing of chronic wounds, decreasing lesion counts and improving quality of life of RDEB patients. Methods: Prospective, randomized, double-blinded, placebo-controlled, cross-over pilot study. Results: Ten patients enrolled in the trial, 7 completed both study periods. Despite showing that all patients improved on trimethoprim and that there was a 41% difference in affected area percent change favoring trimethoprim, the cross-over analysis did not show a significant difference between the drug and placebo (p=0.08). Secondary outcome measures did not achieve statistical significance. Limitations: Small sample size, large variation in wound size and unaccounted confounders. Conclusions: Although patients experienced improvement while on trimethoprim, no statistical significant change was showed when compared to placebo.

Magister Chirurgiae Dentium (MChD) / Introduction:Hereditary Epidermolysis bullosa (EB) is a group of rare mechanobullous dermatological disorders in which blisters develop following gene mutations. These genes encode structural proteins that anchor the epidermis to the underlying dermis.There are four main types of Epidermolysis bullosa, with more than 20 subtypes. The medical, physical and psychosocial aspects of Epidermolysis bullosa are well documented (Lucky et al, 2005; Mellerio et al, 2005). Many studies have documented case reports of associated oral lesions (Silva et al, 2004; Pacheco and de Sousa Araugio 2008; Siqueira et al, 2008). However, no assessment of the impact of these oral lesions on the affected person's everyday life has been made. The morbidity of the oral lesions associated with EB is expected to have an impact on the quality of life of these patients.Aim:To assess the prevalence and impact of oral lesions on daily activities in persons with Epidermolysis bullosa in Cape Town, South Africa, utilizing the Oral Impact on Daily Performance (OIDP) measure. Research Design and Methodology A case-controlled, descriptive analysis of the way in which oral lesions impact on quality of life in persons with Epidermolysis bullosa was carried out using semi-structured interviews. Fourteen persons with a confirmed diagnosis of hereditary Epidermolysis bullosa who attended the dermatology clinics at the Red Cross and Groote Schuur hospitals participated in the study. The control group comprised eighteen persons closely matched for gender, age, and dental status. Three persons with EB were unavailable for inclusion in the study.Results and Discussion Fourteen persons with Epidermolysis bullosa and eighteen controls were included in the study. Epidermolysis bullosa Simplex comprised the largest sub-group (n=9). Two persons had Junctional Epidermolysis bullosa, two had recessive Dystrophic Epidermolysis bullosa and one person had Kindler syndrome. The oral manifestations observed were consistent with those reported in the literature(Chimenos et al, 2003; Silva et al, 2004; Pekinar et al, 2005). No significant oral lesions (other than tooth decay) were seen in persons in the Epidermolysis bullosa Simplex group. Oral ulcers, atrophy of the dorsal surface of the tongue and gingival erythema were seen in persons with Junctional Epidermolysis Bullosa. The two individuals with Dystrophic Epidermolysis bullosa had a maximal oral opening of 15mm and 24mm. Ankyloglossia, depapillation of the dorsal tongue, absence of palatal rugae and poor oral hygiene was seen in these two persons. The patient with Kindler syndrome presented with erythematous and inflamed gingiva and cratering in the maxillary anterior interdental area. The gingiva appeared desquamative, fragile and bled with even the slightest provocation. Healing peri-oral blisters and angular cheilitis was also seen. His mouth opening was restricted to a maximal oral aperture of 13mm and his tongue extrusion was limited to only the tip of the tongue passing over the lower anterior incisor teeth.Defects in the tooth enamel was recorded in both participants with Junctional Epidermolysis bullosa and one person with dystrophic Epidermolysis bullosa, as well as excessive occlussal tooth wear (attrition), which may have been secondary to enamel hypoplasia. The dental caries status of the Epidermolysis bullosa and control groups varied according to age. The dmf for persons with Epidermolysis bullosa (all of whom had Epidermolysis bullosa Simplex), was lower than in the control group. The DMF in EB persons (15.3) was higher than in the control group (10.1).Toothache and tooth decay were the most common perceived complaints in both the Epidermolysis bullosa and control participants, accounting for the high overall OIDP score in both groups (87.5%). No statistically significant difference was found between the two groups (85.7% and 88.9% for Epidermolysis bullosa and control group persons respectively).Conclusion:The results of the study show that oral lesions (particularly tooth decay and toothache) in persons with Epidermolysis bullosa do affect their daily activities and the impact thereof is high. Other oral manifestations, irrespective of the subtype, had little impact on the OIDP score. This may be because the EB persons become tolerant of and “learn to cope” with them.Recommendations:Epidermolysis bullosa is a rare condition and not all persons with EB will present with lesions. However, all health personnel (including oral health profession) must be cognizant of this condition, in order to manage these persons safely, without incurring harm inadvertently. Thus, the overall management of persons with Epidermolysis bullosa must encompass ways to minimize and prevent trauma; provide an optimum wound healing environment; provide pain management and judicious checks for the development of premalignant lesions. This necessitates a multidisciplinary and holistic approach, with emphasis on patient involvement. To this end, an oral health care programme should form an integral part of their management because of the risk of dental disease. Periodic recall visits will enable the monitoring of home care and minimize the need for advanced restorative procedures. In this way, one may reduce the impact any oral problems may have, so that they do not further influence the patients well being.

Title: Integration of pupil with epidermolysis bullosa dystrophica disease with special focus on physical education into the primary school education. Objectives: The aim of this thesis is to point out possibilities of pupil's integration with corporal defect, concretely epidermolysis bullosa dystrophica into primary school education with special focus on Physical education through case study. Methods: We have used case study, questionnaire and interview methods because of uniqueness of occurrence this disease in primary school. The main case study is focused on pupil with EBD disease. The partial research is focused on classmates, teachers, teacher's assistant and girl's family. Results: Results have shown us the only partly possibility to integrate pupil with EBD disease into PE. The most suitable period of time was 1 - 3 school year. The research has shown girl's fitting in class and also her good feelings there. In generally we can say, that pupil is well perceived by classmates and teachers. There is very important teacher's and assistant's activity. The class teacher promoted positive, peaceful and friendly atmosphere since first year a thanks to her the class functions as a whole now. According classmates and teachers is schoolgirl fully integrated in class. The important is also teacher's...

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Submitted by Silvana Teresinha Dornelles Studzinski (sstudzinski) on 2016-12-22T11:39:50Z No. of bitstreams: 1 Silvana Mara Janning Prazeres_.pdf: 8729770 bytes, checksum: 3fc520ee401ed119bf5220fd27c4390e (MD5) / Made available in DSpace on 2016-12-22T11:39:50Z (GMT). No. of bitstreams: 1 Silvana Mara Janning Prazeres_.pdf: 8729770 bytes, checksum: 3fc520ee401ed119bf5220fd27c4390e (MD5) Previous issue date: 2016-10-05 / Nenhuma / A epidermólise bolhosa (EB) hereditária é uma genodermatose, ou seja, doença de pele transmitida geneticamente. É uma doença rara, a qual se caracteriza pelo aparecimento de bolhas, ulcerações ou feridas em qualquer local da superfície corporal em resposta a mínimos traumas ou desencadeadas espontaneamente. Os objetivos deste estudo foram elaborar uma cartilha para o cuidado, no âmbito hospitalar, de neonato com epidermólise bolhosa. Investigar como os profissionais de saúde que atuam em UTI neonatal enfrentam o nascimento e os cuidados a serem dispensados com o neonato em EB, visando subsidiar a elaboração da cartilha. Trata-se de um estudo de natureza qualitativa, exploratória e descritiva, realizado em instituições de saúde que já tiveram casos da doença em neonatos hospitalizados na UTI neonatal na cidade de Porto Alegre/RS. A pesquisa seguiu a resolução 466/12 do Conselho Nacional de Saúde, sendo o projeto aprovado pelo Comitê de Ética e Pesquisa da UNISINOS (parecer 780.693 de 08/09/2014) e das instituições coparticipantes (pareceres: 837.816 de 17/09/2014 e 14171 de 22/10/2014). Participaram 14 profissionais da saúde, que durante sua trajetória haviam cuidado de recém-nascidos com EB. Oito são enfermeiros, três técnicos de enfermagem e três médicos. A coleta dos dados foi realizada por meio de entrevista em profundidade, composta por duas questões abertas: descreva suas experiências no cuidado, tratamento e acompanhamento aos pacientes com EB, que estão ou que ficaram sob seus cuidados durante a internação hospitalar? e Diga como foi para você cuidar de uma criança com EB? A análise dos dados foi constituída pela análise temática. Emergiram três categorias: experimentação do cuidado, família, e equipe de saúde. Os resultados apontaram que o nascimento de uma criança com EB acarreta sofrimento tanto pela insegurança na condução do caso, quanto pela gravidade da doença e seu enfrentamento durante a vida do paciente assistido e seus familiares. Considera-se que a elaboração desta cartilha poderá subsidiar o cuidado de neonatos com EB, contribuindo para a qualificação do cuidado também intra-hospitalar, bem como, para qualificar a orientação aos pais de neonatos com EB, acerca do cuidado domiciliar. / The Epidermolysis Bullosa (EB) is a hereditary genodermatosis. In other words, it is a genetically transmitted skin disease. It’s a rare disease, which is characterized by the manifestation of blisters, ulcerations or wounds anywhere on the body surface either in response to the smallest traumas or spontaneously triggered. The objectives of this study were the elaboration of a practical guide for the handling of newborn babies who are of Epidermolysis Bullosa (EB); investigate how health professionals who work in neonatal intensive care units (ICUs) face the birth and the necessary care of newborn EB carriers, aiming to support the elaboration of the practical guide. This study has a qualitative, exploratory and descriptive nature and it was carried out in health institutions, which have already had cases of the disease in ICUs in the city of Porto Alegre/RS. The research followed the 466/12 resolution of the National Health Council. The project was approved by the UNISINOS Ethical and Research Committee (technical opinion 780.693 on September 8th 2014) and by the coparticipating institutions (technical opinions 837.816 on September 9th 2014 and 14171 on October 22nd 2014) Ethical and Research Committees. A total of 14 health professionals who had taken care of newborns with EB during their trajectory participated in this research. Eight of them are nurses, plus three nurse technicians and three physicians. The data collection was carried out by in-depth interviews, composed by two open questions: describe your experience concerning the care, treatment and monitoring of EB patients who are or have been under your care during hospitalization. And they were asked to tell what it was like to them. The data analysis was constituted by thematic analysis. This resulted in three categories: care experimentation, family and health team. The results indicated that the birth of an EB child leads to suffering due to the insecurity in terms of handling the case as much as to the gravity of the disease and its confrontation during the lives of the assisted patient and their family members. It is considered that the elaboration of this guide can support the assistance of EB newborns, contributing to the care qualification, including in-hospital assistance, and to a better home care orientation to the children’s parents.

Epitheliogenesis Imperfecta (EI) is a mechanobullous disease that occurs in newborn American Saddlebred and Belgian Draft foals. Necropsy evaluations of two American Saddlebred foals revealed broad skin lesions, dental abnormalities and oral mucosa lesions. Construction of a partial pedigree showing occurrences of EI in American Saddlebred horses was consistent with a recessive pattern of inheritance. An allelic frequency of 0.04 was estimated for the EI gene. The pathological signs of EI were similar to a disease in humans known as Herlitz Junctional Epidermolysis Bullosa (HJEB). HJEB is caused by a defect in one of the three subunits of the laminin 5 protein (LAM 3, LAM 3 and LAM 2), which leads to a separation of the epidermis from the dermis. Transmission electron microscopy revealed a separation within the lamina lucida at the sites of epidermal/dermal splits in the skin of EI affected foals. This indicated that a defect in the laminin 5 protein was responsible for EI. Linkage disequilibrium (LD) between microsatellite markers and the EI disease locus was tested for in the American Saddlebred and Belgian Draft breeds. Genotyping of microsatellite alleles was used to determine fit to Hardy-Weinberg equilibrium for control and EI populations for both breeds using Chi square analysis. Two microsatellite loci (ASB14 and AHT3) were not in Hardy-Weinberg equilibrium in EI affected American Saddlebred horses. This suggested that the EI disease locus was located on ECA 8, the putative location of LAM 3. No evidence of LD between any of the tested microsatellite loci and the EI locus was observed in the Belgian Draft samples. A cDNA library was built from Thoroughbred horse skin to serve as a resource for sequencing equine skin gene transcripts. 313 ESTs were sequenced, of which 207 were putatively identified (66%) by database search. Examination of the pathology and ultrastructure of EI affected foals and comparison with HJEB indicated that laminin 5 was the responsible defective protein. The LD analysis suggested that LAM 3 was the EI disease locus in American Saddlebred horses.

Les Epidermolyses Bulleuses Dystrophiques (EBD) sont des génodermatoses rares et sévères transmises sur un mode autosomique récessif (EBDR) ou dominant (EBDD), dues à une perte de l’adhésion dermo-épidermique. Les patients atteints d’EBD souffrent de décollements bulleux cutanéo-muqueux qui menacent le pronostic fonctionnel et vital dans les formes les plus graves. Toutes les formes d’EBD sont dues à des mutations du gène COL7A1 codant pour le collagène VII qui est le constituant des fibres d’ancrage assurant l’adhésion de l’épiderme au derme. Il n’existe actuellement pas de thérapie satisfaisante des EBD. La première partie de ma thèse visait à démontrer la faisabilité d’une approche thérapeutique par saut d’exon des EBDR. Cette stratégie consiste à exciser l’exon porteur de la mutation durant le processus d’épissage, afin de restaurer l’expression d’une protéine fonctionnelle. Les exons 73 et 80 de COL7A1 sont particulièrement intéressants car ils sont le siège de mutations récurrentes et que leur excision préserve le cadre ouvert de lecture. Nous avons dans un premier temps démontré le caractère non indispensable des séquences codées par ces exons in vivo en utilisant un modèle de xénogreffe de peau humaine EBDR reconstruite, génétiquement modifiée à l’aide de vecteurs rétroviraux exprimant l'ADNc de COL7A1 délété des séquences des exons 73 ou 80. Puis, j’ai pu établir que la transfection d’oligoribonucléotides antisens (AONs) dirigés contre certaines séquences régulatrices de l’épissage permettait d’induire le saut en phase de ces exons dans des cellules primaires de patients EBDR, avec une efficacité atteignant 90% de saut d’exon. Les analyses par western blot et immunocytofluorescence après transfection ont permis de mettre en évidence une réexpression significative du collagène VII (jusqu’à 25%) dans les cellules de trois patients EBDR. Enfin, j’ai pu démontrer la réexpression du collagène VII in vivo, après injection de différentes doses d’AONs dans des peaux équivalentes générées avec des cellules de patients et greffées sur des souris immunodéficientes. Dans la seconde partie de ma thèse, j’ai étudié une famille EBD particulière, dont les deux enfants atteints présentaient une EBD beaucoup plus sévère que leur mère et leur grand père maternel, atteints d’une forme modérée d’EBDD. Le séquençage des 118 exons de COL7A1 et des régions d’épissage adjacentes a permis d’identifier une seule mutation dominante c.6698G>A (p.Gly2233Asp) dans l’exon 84, à l’état hétérozygote chez les quatre sujets. A partir de l’étude des transcrits paternels, j’ai pu identifier une nouvelle mutation c.2587+40G>A dans l’intron 19 de COL7A1, qui active un site donneur cryptique dans l’intron 19, entraînant sa rétention partielle et la formation d’un codon stop prématuré. La confirmation de la présence de cette seconde mutation, récessive, dans l’ADN des deux enfants a ainsi permis d’expliquer les différences phénotypiques observées, les deux enfants atteints étant hétérozygotes composites pour une mutation dominante et une mutation récessive de COL7A1. Cette mutation récessive constitue la mutation intronique la plus distante des sites consensus d’épissage de COL7A1 et souligne l’importance de l’étude des ARNm pour la recherche de mutations dans le cadre des EBD. Dans une dernière partie de ma thèse, j’ai débuté la caractérisation d’un modèle murin knock-in d’EBDR développé par notre laboratoire, qui mime certaines des caractéristiques phénotypiques des patients EBDR. Mon travail a permis de démontrer in vivo la faisabilité de l’approche par saut d’exons pour COL7A1. Cette première étape importante conduit à développer des études de preuve de principe et de toxicologie dans des modèles animaux, dans la perspective d’une transition vers la clinique. Il illustre également les variations pathologiques d’épissage pouvant faire l’objet d’approches thérapeutiques similaires. / Dystrophic Epidermolysis Bullosa (DEB) is a group of rare and severe genetic skin disorders, inherited in a dominant (DDEB) or recessive (RDEB) manner, and characterised by loss of adhesion between the epidermis and the underlying dermis. DEB patients suffer from severe blistering of the skin and mucosae after mild traumas, and in the most severe forms, DEB can be life-threatening. DEB is caused by mutations in the COL7A1 gene encoding type VII collagen that assembles into anchoring fibrils forming key dermo-epidermal adhesion structures. To date, there is no specific treatment for DEB. The first part of my thesis was to develop exon skipping as a therapeutic approach for RDEB. In this work, exon skipping strategy consists in modulating the splicing of a premessenger RNA to induce the skipping of a mutated exon and lead to the synthesis of a shorter but functional protein. Exons 73 and 80 of COL7A1 are of particular interest since they carry many recurrent mutations and their excision preserves the open reading frame. In first instance, we have demonstrated the dispensability of these exons for type VII collagen function in an in vivo xenograft model using RDEB cells transduced with retroviral vectors containing COL7A1 cDNAs, deleted of the sequences of exon 73 or 80. I have subsequently transfected primary RDEB keratinocytes and fibroblasts with antisense oligoribonucleotides (AONs) targeting key splicing regulatory elements of these exons, and achieved efficient skipping of these exons (up to 90%). Western blot and immunocytofluorescence experiments demonstrated significant type VII collagen re-expression (up to 25% of the normal amount) in cells from three RDEB patients. Finally, I have generated skin equivalents with cells of these patients, grafted them on immunodeficient mice and injected different doses of AONs in the grafts, and I have demonstrated type VII collagen re-expression in vivo. In the second part of my thesis, I have studied the case of a particular DEB family, in which two affected children presented a DEB much more severe than their mother and maternal grandfather, suffering from a mild form of DDEB. Sequencing of the 118 exons ofCOL7A1 and of their flanking splice sites, lead to the identification of a single dominant mutation c.6698G>A (p.Gly2233Asp) in exon 84, at the heterozygous state in the four individuals. By carrying out analyses on the paternal transcripts, I have identified a novel c.2587+40G>A recessive mutation in intron 19, which activates a cryptic donor splice site in this intron, leading to its partial retention and to the formation of a premature termination codon. I confirmed the presence of this second, recessive, mutation in the DNA of the two affected children, thus providing an explanation for the observed intrafamilial phenotypic variability: the two affected offsprings being compound heterozygotes for a dominant mutation and a recessive mutation in COL7A1. This novel mutation is the deepest intronic mutation found in COL7A1 so far, and emphasizes the importance of studying COL7A1 at the transcripts level to unravel intronic mutations, understand their molecular consequences and their involvement in the development of the disease. In the last part of my thesis, I have started the characterisation of a knock-in murine model of RDEB generated by our laboratory, which mimics some of the phenotypic characteristics of RDEB patients. My thesis work provided the in vivo demonstration of the feasibility of an exon skipping therapeutic approach for COL7A1. This first important step leads to development of proof of concept studies and toxicological studies in different animal models, with the aim of a clinical translation. It also illustrates the pathological splicing alterations that could benefit from similar approaches.

Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.

Abstract Transmembrane collagen XVII (BP180) is a structural component of hemidesmosomes that connects the two layers of skin. Collagen XVII is associated with both autoimmune and inherited bullous skin diseases. Mutations in collagen XVII gene cause junctional epidermolysis bullosa, and in the diseases of the pemphigoid group autoantibodies target collagen XVII. In this work, collagen XVII was studied in both junctional epidermolysis bullosa and gestational pemphigoid. Two novel glycine substitution mutations were found in the largest collagenous domain of collagen XVII. Analysis of recombinantly produced mutated proteins showed that these novel mutations and previously described glycine substitution mutations decrease the thermal stability of collagen XVII ectodomain. In addition, these mutations were found to cause intracellular accumulation of the mutated proteins and affect the post-translational modifications of collagen XVII. Meanwhile, an in-frame deletion of nine amino acids had no effect on the thermal stability or secretion of the collagen XVII ectodomain. Gestational pemphigoid autoantigen collagen XVII has been mainly studied in the skin, and its expression and function during pregnancy are so far largely unknown. For the first time, collagen XVII was shown to be expressed by cytotrophoblasts of the first trimester human placenta and by cultured cytotrophoblasts. Transmigration assay of cytotrophoblasts indicated that collagen XVII promotes trophoblast invasion, and may thus have a role in placental formation. In addition, significant amounts of in vivo produced collagen XVII were found in the amniotic fluid throughout pregnancy. Collagen XVII expression was also observed in hemidesmosomes of amniotic membranes and in cells cultured from amniotic fluid. These findings suggest that collagen XVII could have a function, albeit so far unknown, during pregnancy.

The diploma thesis is focused on the topic of Living with patients with rare diseases - Epidermolysis bullosa in the Czech Republic is focused on identifying the role of individual actors involved in financing support for patients and evaluating the significance of their roles. Patients suffering from rare diseases have a very specific situation in terms of financial contributions from the public budget, as their situation is not common. The diploma thesis is focused on the role of public sector actors, the non-profit sector (the role of Debra) and patient families in financing treatment and necessary care. It examines and compares the share of funding support for these individual actors, especially focusing on the share of funding from the public sector and the non-profit sector, where the majority is occupied by Debra, which focuses on supporting patients with butterfly wing disease. The thesis examines whether the role of the public sector in financing is sufficient and how significantly its role is complemented by the non-profit sector. Subsequently, the quality of life and social construction of patients in the Czech Republic is evaluated, as well as the provision of care and financial assistance for their illness. The situation of patients will be compared with the situation in other European...