Le xeroderma pigmentosum (XP) est une affection autosomique récessive avec déficience dans un système de réparation de l'ADN appelé réparation de l'excision des nucléotides (NER). Le XP comprend 7 groupes de complémentation, de XPA à XPG. Le xeroderma pigmentosum type C (XPC) est l'une des formes les plus courantes en Europe, aux Etats-Unis et au Japon. Les hétérozygotes ne sont pas affectés, alors que les homozygotes ont une sensibilité au soleil précoce et sévère qui endommage la peau et les yeux. La maladie commence au début de la vie avec la première exposition à la lumière du soleil. L'âge médian d'apparition est de 1 à 2 ans, la peau présentant rapidement les signes associés habituellement à des années d'exposition au soleil. Le terme XP fait référence au phénotype qui inclut la xérose et l'hyperpigmentation. Cependant, l'altération du phénotype pigmentaire de la peau associée aux effets du rayonnement UV, qui est parmi les premières manifestations cliniques dans la plupart des troubles NER, a été relativement négligée jusqu'à présent, sans étude systématique. En conséquence, l'objectif principal de cette thèse est d'étudier le mécanisme par lequel la déficience en protéine XPC affecte la pigmentation. Nos données cliniques ont révélé que les manifestations pigmentaires chez les patients XP-C apparaissent progressivement pour être manifestes vers l'âge de deux ans sous forme, dans les zones exposées au soleil, de nombreuses taches hypo- et hyper-pigmentées. De plus, le phénotype pigmentaire (c'est-à-dire la distribution des taches hypo- et hyper-pigmentées) des patients XP-C examiné sur une longue période de temps a révélé un profil relativement stable dans l'espace. L'analyse des zones hypo- et hyper-pigmentées de la peau chez les patients XP-C a montré que le nombre de mélanocytes et la quantité de mélanine sont significativement diminués dans la zone hypo pigmentée. L'analyse protéomique sur les taches hypo- et hyper-pigmentées a été réalisée et a permis d’identifier des voies de signalsation d’intérêt. Pour étudier en détail le mécanisme moléculaire soustendant l'effet de la déficience en XPC sur la pigmentation, nous avons utilisé la peau reconstruite humaine, qui reproduit de nombreuses caractéristiques morphologiques et moléculaires de la peau humaine. Les résultats montrent que l'extinction du gène XPC dans les mélanocytes en utilisant la technologie CRISPR/ Cas9 pourrait être utilisé comme un modèle pour reproduire la maladie en raison de la rareté des patients. / Xeroderma pigmentosum (XP) is an autosomal recessive disorder with deficiency in aDNA repair system called nucleotide excision repair (NER). XP comes into 7 complementationgroups, XPA-XPG. Xeroderma pigmentosum type C (XPC) is one of the morecommon forms in Europe, USA and Japan. Heterozygotes are unaffected, while homozygoteshave an early and severe sun sensitivity which damages the skin and eyes.The disease begins in early life with the first exposure to sunlight. The median age ofonset is 1–2 years of age, with skin rapidly exhibiting the signs associated with years ofsun exposure. The term XP refers to the phenotype that includes xerosis and hyperpigmentation.However, alteration in the skin pigmentary phenotype associated with theeffects of UV radiation which is among the first clinical manifestations in most of theNER disorders, has been neglected up to now and it has not studied in detail. Accordingly,the main objective of this thesis is to investigate the mechanism by which XPC deficiencyaffects pigmentation.Our clinical data revealed that pigmentary manifestations in XP-C patients often appearprogressively and are characteristic by two years of age as an abnormal increasednumber of lentigines in sun-exposed areas with the typical appearance of hypo- andhyperpigmentation spots. Moreover, pigmentary phenotype (i.e. distribution of hypoandhyper-pigmented spots) of XP-C patients examined over a long period of time revealeda relatively spatially stable profile. Analysis of the hypo- and hyper-pigmentedareas of the skin isolated from XP-C patients showed that the number of melanocytesand melanin quantity are significantly decreased in the hypo-pigmented area. Proteomicanalysis on hypo- and hyper-pigmented spots identified some molecular targets thatwere expressed differently in these spots. To investigate in detail the molecular mechanismunderlying the effect of XPC deficiency on pigmentation, human reconstructedskin, which mimics many morphological and molecular characteristics of normal humanskin was used for modeling XPC. Our results showed that silencing of XPC inmelanocyte using CRISPR/ Cas9 technology could be used as a useful model to counteractthe scarcity of patient samples.

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Les mutations dans les gènes codant pour les facteurs NER donnent lieu à des maladies autosomiques récessives telles que Xeroderma pigmentosum (XP), le syndrome de Cockayne (CS) et la trichothiodystrophie (TTD). Les phénotypes associés à ces syndromes génétiques se caractérisent par une sensibilité extrême à la lumière UV, avec prédisposition accrue à certains cancers (pour XP et XP / CS combiné, principalement), ainsi qu’un retard mental et des signes de progeria (pour CS et XP / CS combiné). Si on peut admettre une corrélation entre réparation de l'ADN endommagé et sensibilité aux UV / cancer, celle avec les symptômes neurologiques/progéroïdes est encore sujet à débat. Une explication pourrait provenir du rôle des facteurs NER dans la régulation de la transcription. Nous proposons une vue d’ensemble des roles de XPG et XPC dans la régulation de la transcription en absence des stress exogènes et comment CSA et CSB orchestrent l’arret de la transcription après une attaque génotoxique. XPC était capable d’interagir stablement avec la methyltransferase NSD3. Des mutations dans XPC altèrent le transcriptôme et la distribution des H3K36me3. Les mutations dans XPG dérégulent l’expression génique et XPG est capable d’etre recruté sur l’ensemble du genôme avec TFIIH. CSA et CSB faisant partie de la machinerie ubiquitin/proteasôme, régulent le recrutement de facteurs fixant l’ADN et contrôlant le programme transcriptionnel après irradiation aux UV. Nos donnés mettent en évidence le rôle des facteurs NER dans la transcription et leur défaut d’action provoque les maladies XP et XP/CS. En plus, nos données fournissent des explications sur le méchanisme d’arrêt de la transcription après un stress genotoxique et pose la question de l’origine du phenotype CS. / Mutations in genes coding for NER factors give rise to autosomal recessive diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). The phenotypes associated with these genetic syndromes spans from extreme sensitivity to UV light, with increased predisposition to cancer (for XP and combined XP/CS, mostly), mental retardation and progeria (for CS and combined XP/CS). Whether the correlation between defective DNA repair reactions and UV-sensitivity/cancer may be more intuitive, a link with neurological/progeroid symptoms is still a matter of debate. As a possible explanation, it has been proposed a connection between NER and transcription regulation. We propose additional insights on XPG and XPC roles in transcription regulation in absence of exogenous stress and how CSA and CSB orchestrate transcription arrest due to genotoxic attack. XPC was able to stably interact with NSD3 methyltransferase. Mutations in XPC also disturbed the transcriptome and the H3K36me3 distribution. Mutations in XPG deregulate gene expression and XPG is able to be recruited genome wide together with TFIIH. CSA and CSB can, as part of the ubiquitin/proteasome machinery, regulate the recruitment timing of DNA binding factors and control transcriptional program after UV irradiation. Hence, our data shed more light in NER factors role in transcription and their defective action as a cause of XP and XP/CS disorders. Additionally, our data provide explanations on the mechanism of transcription arrest following genotoxic stress and pose questions about the origins of CS phenotype.

Host cell reactivation (HCR) of UV-irradiated (UV'd) herpes simplex virus type 2 (HSV-2), capacity of UV'd cells to support HSV-2 plaque formation and UV enhanced reactivation (UVER) of UV'd HSV-2 were examined in human fibroblasts. The cells were derived from four Cockayne's Syndrome (CS) patients, 5 xeroderma pigmentosum (XP) patients and 5 normal patients. Survival curves for HCR of HSV-2 plaque formation showed 2- components. HCR was not significantly different in the CS strains and an XP variant strain compared to normal, whereas all excision deficient strains showed a significant reduction in HCR. The o37 values for the delayed capacity curves were in the range 8.6-12.4 J/m2 for the normal strains, 3.1-5.1 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.40-1.98 J/m2 for the XP excision deficient strains examined. UVER was also examined for HSV-2 UV-irradiated to survival levels of 10-2 and 10-3 in unirradiated cells. Maximum delayed UVER was observed in normal strains at a UV dose of 15 J/m2 to the virus. Maximum UVER in CS cells was detected at a UV dose of 5 J/m2 to the cells, in XP excision deficient cells maximum UVER occurred at doses ranging from 0.5-2.5 J/m2 to the cells, and in XP variant maximum UVER occurred at 10 J/m2 to the cells. In all cell strains the level of UVER increased with increasing UV dose to the virus. Results are discussed in terms of the repair defects of CS and XP cells and their relationship to possible viral repair functions. In addition, the possible existence of an inducible DNA repair response is discussed in terms of the results of this study. / Thesis / Master of Science (MSc)

Le xeroderma pigmentosum (XP) est une maladie génétique rare caractérisée par une hypersensibilité aux ultraviolets (UV) et une forte incidence des tumeurs cutanées. Les cellules des patients XP sont incapables d’éliminer les lésions induites dans l’ADN par les UV en raison d’un dysfonctionnement du mécanisme de réparation par excision de nucléotides (NER). Plusieurs groupes de complémentation ont été identifiés dans le syndrome XP, parmi lesquels le groupe XP-C représente la majorité des patients à travers le monde.Au cours de mon travail de thèse, j’ai développé un modèle de correction ciblée par recombinaison homologue (RH) d’une délétion de deux nucléotides au niveau de l’exon 9 du gène XPC aboutissant à l’apparition prématurée d’un codon stop. Afin de stimuler la RH, deux types de nucléases ingéniérées sont utilisées : les méganucléases et les TALENs. J’ai observé que la méthylation de la séquence ciblée pouvait affecter l’activité de celles-ci et donc l’efficacité du ciblage de gène. Cependant, deux approches ont été développées pour résoudre ce problème : l’utilisation d’un agent déméthylant (5-aza-2’-désoxycytidine (5azadC)) ou la création d’une endonucléase insensible à la méthylation. L’utilisation des méganucléases en combinaison avec la 5azadC a permis de stimuler la fréquence de coupure de presque 20 fois dans des fibroblastes XPC et la TALEN modifiée permet une augmentation de 40 fois. Avec ces deux stratégies j’ai obtenu des événements de correction génétique par introduction d’une matrice de réparation dans le locus ciblé avec une fréquence proche de 3%. La caractérisation des clones corrigés avec la TALEN XPC montre la correction génomique des deux nucléotides dans l’exon 9, une restauration de l’expression de la protéine XPC et une résistance cellulaire après irradiation UV traduisant le rétablissement des fonctions de la NER. Cette étude représente la première preuve de correction génétique de cellules déficientes en protéine XPC en utilisant une approche ciblée. / Xeroderma pigmentosum (XP) is a rare inherited genetic disorder characterized by an UV hypersensitivity and a severe predisposition to skin cancers. Cells from XP patients are deficient in nucleotide excision repair (NER) of UV‐induced DNA lesions. Several complementation groups have been identified in the XP syndrome and the XP-C group represents the majority of XP patients around the world. During my PhD work, I developed a model of targeted correction by homologous recombination (HR) in order to correct a deletion of two nucleotides in the ninth exon in XPC gene leading to a premature stop codon. To stimulate HR, I used two types of engineered endonucleases : meganucleases and TALEN. I observed that the target methylation status could affect the endonuclease activities and therefore XPC gene correction. Nervertheless, I developed two approaches to overcome this methylation sensitivity : use of a demethylating agent (5-aza-2-deoxycytidine (5azadC)) or a specific engineering of TALEN. Using 5azadC with meganuclease allowed to stimulate the cutting frequency by nearly 20 fold in XPC fibroblasts and the engineered TALEN allowed a 40 fold-increase in frequency. With both strategies I obtained genetic correction events by repair matrix introduction in the targeted locus with a near 3% frequency. The characterization of corrected clones with the XPC TALEN shows genomic correction in the ninth exon, a restoration of the XPC protein expression and cell survival following UV exposure, thus demonstrating fully recovered normal repair activity by NER. This study represents the first evidence of genetic correction of XPC-deficient cells by a targeted approach.

Mais de 95% de luz ultravioleta (UV) que atinge a superfície da Terra corresponde ao comprimento de onda da luz UVA (315-400 nm). A luz UVA induz no DNA danos pela absorção direta e indireta dos fótons, bem como por intermédio de cromóforos. Pacientes com Xeroderma Pigmentosum Variante (XP-V) possuem mutações na DNA polimerase η (pol eta), que promove a síntese translesão dos danos induzidos pela luz solar. Na ausência dessa polimerase há aumento da mutagênese, provável causa de câncer de pele em pacientes XP-V. O objetivo deste trabalho foi caracterizar os mecanismos de indução de danos no genoma e mutagênese por luz UVA, em células derivadas desses pacientes. Os resultados indicam que a exposição à luz UVA resultou em maior sensibilidade de células XP-V comparadas à linhagem controle. Os níveis de fosforilação da histona H2AX (gerando γH2AX - indicador de indução de danos no genoma) e detecção de danos diretos e indiretos no DNA apresentou um aumento significativo em células XP-V irradiadas com luz UVA. Curiosamente, na ausência de pol eta houve uma redução na capacidade de remoção das lesões formadas. Além disso, a irradiação com luz UVA causou forte bloqueio de replicação do DNA e parada do ciclo celular em fase S em células XP-V, desencadeando importantes respostas mediadas por ATR/Chk1. Surpreendentemente, o antioxidante N-acetilcisteína (NAC) resultou em diminuição da sensibilidade celular, dos níveis de γH2AX, da parada de forquilha de replicação e do ciclo celular, reduziu os efeitos citotóxicos da inibição de ATR, melhorou o reparo de lesões no DNA e preveniu a carbonilação de proteínas em células XP-V irradiadas com luz UVA. Investigamos também a indução de mutagênese nas células irradiadas com luz UVA, através de sequenciamento de exoma de clones celulares. Os dados indicaram um aumento significativo da mutagênese em células XP-V irradiadas comparadas a células controle, e pela avaliação dos tipos de mutações encontradas verificamos uma frequência bastante alta de transições C>T, provavelmente como consequência de replicação errônea de dímeros de pirimidina. Entretanto, também identificamos indução significativa de transversões C>A, provavelmente devido a lesões oxidadas no genoma. Curiosamente, ao compararmos células XP-V com células controle detectamos um aumento desse último tipo de mutação na ausência de pol eta, provavelmente devido a lesões endógenas, produzidas pela oxidação do DNA. Além disso, análises in silico mostraram que células XP-V irradiadas com UVA apresentaram assinatura mutacional similar ao observado para tumores de pele. Os dados claramente indicam que células XP-V são sensíveis a irradiação com UVA e que os danos promovidos no DNA, incluindo aqueles causados por estresse oxidativo, desencadeiam respostas celulares e mutagênese nesses pacientes. Assim, além de apontar que UVA pode gerar efeitos deletérios na pele de pacientes XP-V, nossos dados também contribuem para a compreensão de como esses comprimentos de onda podem atuar em células humanas em geral / More than 95% of ultraviolet-light (UV) that reaches the Earth surface corresponds to UVA wavelengths (315-400 nm). UVA-light induces DNA damage through direct and indirect absorption of photons, as well as, intermediated by chromophores and by oxidation mechanisms. Xeroderma Pigmentosum Variant (XP-V) patients are defective in DNA polymerase η (pol eta) that performs translesion synthesis of sunlight induced DNA damage. Absence of pol eta results in increased mutagenesis, which is probably responsible for high frequency of skin cancer in XP-V patients. The goal of this work was to characterize the mechanisms of UVA-induced genome DNA damage and mutagenesis in cells derived from these patients. The results indicate that UVA irradiation increased cell death of XP-V compared to control cell line. The phosphorylation of the histone H2AX (generating γH2AX, an indicator of genotoxic stress) and DNA damage was highly increased in UVA irradiated XP-V cells. Curiously, however, in the absence of pol eta, there was a reduction in the capacity of cells to remove DNA damage from genome. Moreover, UVA irradiation triggered strong DNA synthesis blockage and cell cycle arrest in S phase, resulting in important responses mediated by the ATR/Chk1 pathway in XP-V cells. Interestingly, the antioxidant N-acetyl cysteine (NAC) resulted in decreased cell sensitivity, γH2AX levels, fork stalling and cell cycle arrest, reduced the cytotoxic effect of ATR inhibition, improved DNA repair and prevented the protein carbonylation in XP-V cells irradiated with UVA. The mutagenesis by UVA-light was also investigated by exome DNA sequencing of cellular clones. The data indicated a significative increase of mutagenesis in XP-V irradiated cells compared to control cells, and the identification of mutation types indicated a high increase of C>T transitions, probably as result of error-prone replication of pyrimidine dimers. Nevertheless, the induction of C>A transversions were also detected, probably due to oxidized DNA damage. Curiously, when XP-V and control cells were compared, in the absence of irradiation, these transversions were also detected, maybe due to endogenous oxidation of DNA. In addition, in silico analyses showed that UVA-irradiated XP-V cells had a mutation signature similar to the observed for skin cancer. The data demonstrate XP-V cells are sensitive to UVA-light and DNA damage, including by oxidative stress, trigger cell responses and induce mutagenesis in these patients. Therefore, besides showing that UVA-irradiation may generate deleterious effects in the skin of XP-V patients, the data also contribute to understand how these light wavelengths may damage human cells in general

La réparation par excision de nucléotides (NER) permet l'élimination des lésions provoquant une distorsion de la double hélice de l’ADN. Ces lésions sont induites par plusieurs agents environnementaux comme les rayons UV, ainsi que par certaines drogues chimio- thérapeutiques tel que le cisplatine. Des défauts dans la NER conduisent à de rares maladies autosomiques héréditaires : La xérodermie pigmentaire (XP), le syndrome de Cockayne (CS), le syndrome de sensibilité aux UVSS et la trichothiodystrophie (TTD). Ces maladies sont associées soit à une prédisposition élevée au cancer de la peau et / ou à de graves anomalies du développement neurologique. Le groupe de patients XP-A représente le deuxième groupe (XP) le plus fréquent, et possède la forme la plus sévère combinant cancer de la peau avec un haut risque de dégénérescence neurologique. À date, aucune explication n`a été proposée pour les symptômes neurologiques observés chez ces patients. Nous avions suggéré ainsi que la protéine XPA possède d`autres fonctions dans d`autres processus cellulaires, ceci en interagissant avec des partenaires protéiques différents de ceux déjà connus. Afin de confirmer cette hypothèse nous avions réalisé une étude protéomique à grande échelle en combinant la spectrométrie de masse à une immunoprécipitation en Tandem d`affinité (TAP), afin d`identifier de nouvelles protéines interagissant directement avec XPA. Nous avions montré que XPA peut interagir avec MRE11, la protéine clé de la réparation par recombinaison homologue. Des études additionnelles sont requises pour confirmer cette interaction et comprendre sa fonction / To maintain genome integrity and ensure the continuation of transcription, helix distorting DNA lesions induced by UV and other environmental mutagens are eliminated through a highly-versatile DNA repair pathway: nucleotide excision repair (NER). Mutations in 11 genes (XPC, XPE, XPB, XPD, XPG, XPA, XPG, TTD-A, CSA, CSB and UVSSA), among the 30 genes directly involved in NER, have been associated with the human genetic disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS), trichothiodystrophy (TTD), and UV-sensitive syndrome (UVSS). Patients of these syndromes display a wide variety of clinical features that range from normal development with extreme predisposition to cancer, to neurodevelopmental defects associated with premature aging abnormalities. The connection between DNA damage and neurodegeneration remains unclear, i.e. cannot be explained by a DNA-repair deficiency alone, implying that various repair factors perform other functions beyond the repair process. XP-A is the second most common form of XP. XP-A cells have very low levels of NER activity and are sensitive to killing by UV light. It is one of the most severely affected XP groups, with the onset of cutaneous features, skin cancer, ocular features, and severe early onset neurological disease. Therefore we hypothesize that XPA interacts with cellular proteins that regulate its functions either in UV damage repair or in neurological development. To test this, our major aim was to carry out a large-scale proteomics investigation to identify novel interacting partners for XPA in the absence or presence of genotoxic stress, thus providing clues on the origins of neurodegeneration observed in many XP-A patients. We provide evidence that XPA can interact with MRE11, the key factor in repair of double strand breraks by homologous Recombination. Future experiments will be aimed at determining the impact of the XPA/MRE11 interaction functions in cells.

Mutações em genes da via de reparo de DNA por excisão de nucleotídeo estão associados a doenças genéticas raras, como Xeroderma pigmentosum, Síndrome de Cockayne e Tricotiodistrofia. Este trabalho teve como objetivo identificar mutações responsáveis por essas Síndromes em pacientes brasileiros. Inicialmente o projeto teve como foco o estudo da comunidade de Araras, no interior de Goiás, onde existe uma das maiores incidências de pacientes XP no mundo. Duas mutações foram identificadas no gene POLH, associadas a dois efeitos fundadores independentes na região. Os dezessete pacientes, entre 10 e 80 anos, caracterizados geneticamente apresentam uma grande heterogeneidade das manifestações clínicas, com fenótipos brandos à extremamente agressivos. Esse estudo se estendeu a mais dezessete pacientes de todo o Brasil, incluindo outros grupos de complementação de XP, CS e TTD. Na segunda parte deste trabalho avaliamos a resposta das linhagens XP-V ao tratamento com cisplatina. Foi possível observar que essas linhagens são mais sensíveis aos danos induzidos pela cisplatina, encontram problemas na fase G1/S de replicação e possuem um aumento da marcação high-level para histona gH2AX. / Mutations at DNA repair genes are associated with rare genetic diseases such as Xeroderma pigmentosum, Cockayne syndrome and Trichothiodystrophy. This study aimed to identify mutations responsible for these syndromes in Brazilian patients. Initially the project focused on the study of Araras community in the state of Goiás, where there is one of the densest known areas of XP patients. Two mutations were identified at POLH gene, associated with two independent founders effects in the region. The seventeen patients between 10 and 80, characterized genetically a wide range of heterogeneity of clinical symptoms with mild to extremely aggressive phenotypes. Brazilian patients from other places were studied, including seventeen other patients with XP, CS and TTD phenotypes. In the second part of this work, we evaluated the response of XP-V cell lines treated with cisplatin. It was observed that Pol eta deficient cells are more sensitive to damage induced by cisplatin, as well as encounter problems in the S phase replication after 24 hours of treatment, and have increased high-level fluorescence for gH2AX.

O NER é uma via de reparo de DNA capaz de lidar com uma ampla variedade de lesões. Participam do NER diversas proteínas, entre elas a endonuclease XPG. Pacientes que possuem mutações no gene XPG apresentam a síndrome XP, e em alguns casos XP/CS. Investigamos a natureza genética de dois pacientes XP-G, que são irmãos e apresentam fenótipo moderado. As células destes pacientes demonstraram alta sensibilidade à luz UVC. Quando expostas a um agente oxidativo, apenas células XP-G/CS exibiram sensibilidade. Identificamos duas mutações missense no gene XPG desses pacientes, e comparamos com outras mutações existentes. Observamos que as mutações possuem um impacto negativo na funcionalidade de XPG. A proteína com a mutação p.Ala28Asp exibiu uma atividade residual em testes de complementação. Os resultados indicam que o fenótipo XP-G desses pacientes é causado por duas mutações missense em heterozigose composta, e que células portadoras dessas alterações exibem respostas diferenciadas frente aos estresses genotóxicos causados pela luz UV e pelo agente oxidativo utilizado. / NER is the most flexible of all known DNA repair mechanisms. XPG is an endonuclease that participates in the final steps of NER. Mutations in this gene may result in the human syndrome XP and, in some cases, in the XP/CS. We investigated the genetic nature in two XP-G patients, siblings and mildly affected. The cells from these patients demonstrated the high UV sensitivity typical of this syndrome. When exposed to an oxidative agent, only XP-G/CS cells exhibited sensitivity. We identified two missense mutations in the XPG gene of these patients, and a comparison with other known mutations is presented. These mutations have a negative impact in the function of XPG. The protein harboring the mutation p.Ala28Asp exhibited residual activity in complementation tests. These results indicate that the phenotype of XP-G patients is caused by two missense mutations in a compound heterozygous manner, and that the cells carrying these alterations exhibit different responses against genotoxic stress caused by the UV light and by the oxidative agent used.

Um dos mais versáteis mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (nucleotide excision repair- NER). Defeitos genéticos associados a esta via podem gerar diferentes síndromes com deficiência de reparo. Dentre essas, Xeroderma pigmentosum (XP) é a que apresenta maior sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e, em alguns casos, degeneração neurológica progressiva e envelhecimento prematuro. Na primeira parte deste projeto é apresentado o uso de adenovírus recombinantes portando genes da via de NER para identificar a deficiência gênica de três pacientes portadores de XP. Na segunda parte do trabalho os estudos de reparo de DNA são estendidos a modelos animais, com deficiências nos mesmos genes carregados pelos vetores adenovirais. A expressão gênica do vetor foi avaliada pela detecção de proteína e por visualização da fluorescência de EGFP na pele dos animais infectados. Em resumo, este trabalho apresenta o uso eficiente de vetores adenovirais portando genes de reparo em ensaios in vitro e in vivo, e descreve duas mutações deletérias no gene XPC de pacientes XP brasileiros, incluindo uma mutação nova. / One of the most versatile mechanisms of DNA repair is the nucleotide excision repair (NER). Genetic defects in NER can generate different syndromes. Among these, Xeroderma pigmentosum) presents the highest sensitivity to sunlight, resulting in a large increase in the incidence of skin cancer, especially in areas exposed to the sunlight, and in some cases, progressive neurological degeneration and premature aging. In the first part of this project, adenoviral vectors carrying NER genes were used to identify genetic deficiency of three XP patients. The second part of work was extended to animal models, deficient for the same XP genes carried by adenoviral vectors. The genetic expression of vector was evaluated by detection of protein and EGFP fluorescence visualization in the skin of animals transduced. In summary, this work presents the use of adenovirus, carrying DNA repair genes for in vitro in vivo studies reports two deleterious mutations in Brazilian XP patients, including a new mutation.