Interactions of Adeno-associated virus Rep78 protein with the host cell

Abstract : Adeno-associated virus (AAV) is a small DNA virus belonging to the familiy of Parvoviridae. Its genome contains two genes : the rep gene encoding four non structural proteins (Rep78, 68, 52 and 40) implicated in transcription, replication and site-specific integration of the viral DNA and the cap gene encoding three capsid proteins. AAV does not cause any disease, but is studied in view of its potential use to treat several diseases. An interesting property of AAV is its antiproliferative effect. Two elements of AAV can inhibit cell growth. Firstly, the single stranded viral DNA is recognized in cells as damaged DNA leading to either a G2 block or cell death depending on p53 status. Secondly, the two larger Rep proteins (Rep78 and 68) also arrest the cell cycle when they are expressed at high levels. Rep78 in particular induces a complete cell cycle arrest in all the phases, including S phase. Such a strong S phase arrest is rarely seen in other conditions. It was thus interesting to determine how Rep78 could induce it. We found that this strong block is the consequence of Rep78's effects on at least two pathways. Rep78 induces a DNA damage response by producing nicks in the cellular chromatin. Furthermore, Rep78 can bind to the cellular phosphatase Cdc25A and prevent its binding to its substrates CDK2 and CDK1, thus inhibiting its activity. A mutational analysis of Rep78 protein determined that its endonuclease activity is responsible for the DNA damage response and its zinc finger domain for Cdc25A inhibition. The combined expression of two mutants each defective for one of these activities, or these two activities obtained independently of Rep78, could restore the complete cell cycle block, indicating that these two effects of Rep78 are likely to explain completely the cell cycle block it induces. Secondly, the lack of pathogenicity of AAV, its broad range of infection and its ability to integrate site-specifically in human chromosome 19 make it an interesting potential vector for gene therapy. However site-specific integration is only possible in the presence of Rep78/68 whose gene is removed in recombinant AAV vectors. In this part of the study, we tried to introduce Rep protein separately from recombinant AAV vectors to promote their site-specific integration. For that purpose, a fusion protein, TAT-Rep, comprising Rep78/68 joined to the human immunodeficiency virus Tat protein was produced. It had the ability to enter cells and remain active there for a short period. Its activity was sufficient to mediate transcription from the p5 promoter, second-strand synthesis of a recombinant AAV and probably site-specific integration. Résumé : Le virus associé à l'adénovirus (AAV) est un petit virus à ADN qui fait partie de la famille des Parvoviridae. Son génome contient deux gènes : le gène rep code pour quatre protéines (Rep78, 68, 52 et 40) qui participent à la transcription, la réplication et l'intégration du virus et le gène cap code pour les trois protéines de capside. AAV ne produit pas de maladie, mais pourrait au contraire être utilisé pour en soigner. Sa bénignité, sa capacité à infecter différents types de cellules et son intégration spécifique en font un vecteur potentiel pour la thérapie génique. Pour qu'il puisse s'intégrer spécifiquement, il a besoin de la protéine Rep78 ou 68, mais ce gène doit être enlevé des vecteurs pour la thérapie génique. Le but de la première partie de cette étude était d'introduire Rep78 ou 68 dans des cellules en même temps qu'un AAV recombinant, mais indépendamment afin de permettre une intégration spécifique. La stratégie utilisée était de produire une protéine de fusion (TAT-Rep) qui peut entrer dans des cellules si elle est présente dans leur milieu. Cette protéine entrait bien dans les cellules et y était active favorisant ainsi l'intégration spécifique. Une deuxième propriété d'AAV, son effet anti-prolifératif, est intéressante dans le cadre de certaines maladies comme le cancer. Deux éléments d'AAV en sont responsables. D'abord, son ADN simple brin active une réponse cellulaire à l'ADN endommagé et arrête les cellules en G2 ou provoque leur mort. De plus, la protéine Rep78 d'AAV peut fortement bloquer le cycle cellulaire à toutes les phases, même en phase S, ce qui est rare. C'est pourquoi nous avons essayé de comprendre cet effet. Nous avons remarqué que Rep78 doit agir sur deux fronts pour obtenir ce fort bloc. D'un côté, Rep78 introduit des coupures simple brin sur l'ADN de la cellule ce qui active une réponse cellulaire à l'ADN endommagé qui passe par ATM. D'un autre côté, Rep78 lie une phosphatase cellulaire, Cdc25A, et l'empêche ainsi de lier ses substrats CDK2 et CDK1 et donc d'être active. Finalement, à l'aide de mutants de Rep78, nous avons déterminé que l'activité endonuclease de Rep78 était nécessaire pour induire une réponse cellulaire via ATM et que le domaine C-terminal appelé «zinc finger » était responsable de la liaison avec Cdc25A. En co-exprimant deux mutants, qui n'ont chacun qu'un des effets de Rep78, ou en obtenant les deux effets de Rep78 indépendamment d'elle, nous avons obtenu un bloc complet du cycle cellulaire similaire à celui obtenu avec Rep78. Il est donc probable que ces deux effets de Rep78 sont suffisants pour expliquer comment elle arrive à arrêter le cycle cellulaire si efficacement.

A reportagem televisiva como género jornalístico: o caso da TCV

Esta monografia faz uma incursão pelos meandros da reportagem enquanto género jornalístico. Depois de uma breve análise das circunstâncias que proporcionaram o desenvolvimento dos enunciados jornalísticos ao longo de diferentes épocas, este estudo atém-se na reportagem, tida como um dos géneros mais correntes na praxis jornalística, com foco, entretanto, no meio televisivo. Assim, o cerne deste trabalho monográfico passa a ser a reportagem televisiva, onde abordamos as suas especificidades bem como todos os mecanismos que envolvem a sua produção. Esta abordagem é, por fim, ancorada com uma análise das reportagens na estação de televisão pública nacional, a TCV.

Myélite transverse et neuromyélite optique chez l'enfant. A propos de trois observations [Transverse myelitis and optic neuromyelitis in children. Apropos of 3 case reports]

We report on three children with acute transverse myelitis. One of them also had optic neuritis. In connection with these three cases, we discuss etiology, diagnosis and outcome of transverse myelitis in childhood and possible involvement of the optic nerve. Our observations show the variability of spinal cord deficit and the fact that there can be a good prognosis, despite severe initial neurological deficits and massive elevation of basic myelin protein in children with ATM.

Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy.

PURPOSE: Evidence has accumulated in recent years suggestive of a genetic basis for a susceptibility to the development of radiation injury after cancer radiotherapy. The purpose of this study was to assess whether patients with severe radiation-induced sequelae (RIS; i.e., National Cancer Institute/CTCv3.0 grade, > or =3) display both a low capacity of radiation-induced CD8 lymphocyte apoptosis (RILA) in vitro and possess certain single nucleotide polymorphisms (SNP) located in candidate genes associated with the response of cells to radiation. EXPERIMENTAL DESIGN: DNA was isolated from blood samples obtained from patients (n = 399) included in the Swiss prospective study evaluating the predictive effect of in vitro RILA and RIS. SNPs in the ATM, SOD2, XRCC1, XRCC3, TGFB1, and RAD21 genes were screened in patients who experienced severe RIS (group A, n = 16) and control subjects who did not manifest any evidence of RIS (group B, n = 18). RESULTS: Overall, 13 and 21 patients were found to possess a total of <4 and > or =4 SNPs in the candidate genes. The median (range) RILA in group A was 9.4% (5.3-16.5) and 94% (95% confidence interval, 70-100) of the patients (15 of 16) had > or =4 SNPs. In group B, median (range) RILA was 25.7% (20.2-43.2) and 33% (95% confidence interval, 13-59) of patients (6 of 18) had > or =4 SNPs (P < 0.001). CONCLUSIONS: The results of this study suggest that patients with severe RIS possess 4 or more SNPs in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro.

Polymorphisms in DNA-repair genes in a cohort of prostate cancer patients from different areas in Spain: heterogeneity between populations as a confounding factor in association studies

Background: Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. Objective: To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. Design, Setting, and Participants: A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArrayH NT Cycler. Outcome Measurements and Statistical Analysis: Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. Results and Limitations: We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. Conclusion: Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

Boca seca y boca ardiente

La saliva es esencial para mantener la salud oral. El 99% de su composición es agua. El desencadenante más común de boca seca es la toma de medicamentos, generalmente acompañada de escasa ingesta de líquidos. Entre sus complicaciones cabe destacar xerostomía, disgeusia y ardor bucal, entre otras. Estos síntomas son tríada frecuente del síndrome de boca ardiente (SBA). Su etiología es imprecisa y multifactorial, sin datos clínicos o de laboratorio específicos. En su patogenia intervienen factores locales, sistémicos y psicológicos. Es común la asociación de hábitos parafuncionales evidentes, dentarios y linguales, con repercusión en tejidos duros (desgaste, ATM) y blandos (lesiones facticias, irritación de papilas linguales). Afecta más a mujeres en edad perimenopáusica, en proporción hasta 7:1 respecto a los hombres. A menudo se asocian ansiedad o depresión. En este trabajo se propone la parafunción deglutoria como parte integrante del síndrome.

Séquelles radio-induites et tests prédictifs [Radiation-induced sequelae: toward an individual profile]

The impact of curative radiotherapy depends mainly on the total dose delivered homogenously in the targeted volume. Nevertheless, the dose delivery is limited by the tolerated dose of the surrounding healthy tissues. Two different side effects (acute and late) can occur during and after radiotherapy. Of particular interest are the radiation-induced sequelae due to their irreversibility and the potential impact on daily quality of life. In a population treated in one center with the same technique, it appears that individual radiosensitivity clearly exists. In the hypothesis that genetic is involved in this area of research, lymphocytes seem to be the tissue of choice due to easy accessibility. Recently, low percentage of CD4 and CD8 lymphocyte apoptosis were shown to be correlated with high grade of sequelae. In addition, recent data suggest that patients with severe radiation-induced late side effects possess four or more SNP in candidate genes (ATM, SOD2, TGFB1, XRCC1 et XRCC3) and low radiation-induced CD8 lymphocyte apoptosis in vitro.

New telecom services over next generation SDH

Our main aim in this report is to use Next generation SDH to solve the problem associated with the new telecom services. We have tried to analyze the different services and in this way identified some drawbacks which can be seen as hindrances in supporting these services. In this thesis we will first try to have idea of the past SDH technology and how the next generation SDH came into effect overriding the drawbacks of the past SDH technology. Our main concern throughout the report will be the way we can use next generation SDH to provide quality telecommunication services. In the section dealing with the telecommunication services through next generation SDH we will consider how we can transport Ethernet services through the Next generation SDH and what are the benefits to the customer and the service provider in using next generation SDH as a carrier. We will also see to improve to the ATM services through Next generation SDH. And finally towards the end I have identified some possible future work can be done in this area.

UMTS-radioverkon ja GSM-runkoverkon yhdistävän moduulin järjestelmätestaus

Tämän diplomityön aiheena oli tutkia ATM-moduulin järjestelmätestaustarpeita. ATM-moduuli on osa Nokian kolmannen generaation matkapuhelinverkkoarkkitehtuuria. Työ tehtiin Nokia Networks Oyj:lle MSC&HLR-tuotelinjan järjestelmätestausjaokselle. Työssä käsitellään aluksi GSM- ja UMTS-verkkojen arkkitehtuureja, protokollia ja toimintoja, sekä tutustutaan ATM-tekniikkaan, jota käytetään UMTS-liityntäverkon tiedonvälityksessä. Tämän jälkeen perehdytään ATM-moduulin rakenteeseen ja toimintoihin. Työssä perehdytään järjestelmätestauksen eri osa-alueisiin, niiden testilaitteille asettamiin vaatimuksiin sekä hahmotellaan järjestelmätestauksen päälinjoja.

New TTCN interfaces for WCDMA base station testing

WCDMA tukiasema (Node B) on osa UMTS-järjestelmän radioverkkoa. Node B on tärkeä verkkoelementti, jonka tarkoituksena on yhdistää mobiilikäyttäjät verkkoon. Telecom –ohjelmisto (TCOM SW) on vastuussa suuresta osasta Node B:n toiminnallisuutta. TCOM SW:n testaukseen käytetään paljon resursseja, jotta ohjelmiston oikeasta toiminnasta ja laadusta voidaan varmistua. System component testing on testausvaihe, jossa järjestelmän (Node B) osa (system component, tässä diplomityössä TCOM SW) testataan ennen sen integroimista muuhun järjestelmään. Tähän tarvitaan testityökalu ja testitapausten toteutus. Node B TTCN Tester (testeri) on työkalu, jota käytetään Node B:n ohjelmiston testauksessa. Testitapaukset toteutetaan TTCN-testinotaatiota käyttäen ja testataan testerin avulla. TCOM SW:n system component –testausvaihetta varten testeriin lisättiin uudet rajapinnat, joiden avulla voidaan simuloita Node B:n ATM-ohjelmistoa sekä WPA- ja WTR-yksiköitä. Tässä diplomityössä toteuttiin TTCN testitapaukset uusille rajapinnoille. Testitapaukset tekivät TCOM SW system component –testausvaiheen riippumattomaksi Node B:n ATM-ohjelmistosta sekä WPA- ja WTR-yksiköistä. Lisäksi TCOM SW:n toiminnan testaus näissä rajapinnoissa voidaan tästä lähtien tehdä automaattisesti. Testitapauksien toiminta varmistettiin testeriä käyttäen. Tulokset olivat hyviä, uudet testitapaukset ja TTCN rajapinnat toimivat oikein lisäten testauksen tehokkuutta.

Handling of TDM Connections with Cello Media Gateway

UMTS is a 3rd generation telecommunication system, which introduces new network architecture. The change in the network architecture introduces new logical network nodes and changes the role of existing nodes in the network. This architecture changes the current vertically specialized network into a horizontally layered structure. In practice, the layering means that different levels in network hierarchy are separated, and they communicate over well-specified interfaces. The Connectivity Layer, at the bottom of the UMTS network architecture, contains Media Gateways (MGW). The GSM radio access network and UMTS access network are connected to the connectivity network via a MGW. External networks, e.g. ISDN networks, are accessed via other MGWs. The user plane is transported across the connectivity network between/via MGWs. ATM network is used as the backbone in Ericsson’s UMTS core network release 2.0. The main goal of this thesis is to study how the MGW is used to bridge ATM and TDM networks. The Circuit Emulation Service (CES) for ATM is studied, as the conversion from TDM to ATM is made according it. The transportation is made using AAL2 and the issues that it has with voice traffic are studied. The implementation and usage of TDM switching service in MGW are described in detail.

Laajakaistaliitynnät - xDSL-tekniikat ja muut vaihtoehdot

Tietoliikenteen suuria linjoja ovat mm. siirtonopeuksien kasvu, IP-tekniikan yleistyminen ja Internet-liitäntöjen kautta käytettävien palvelujen valtaisa lisääntyminen. Kotien verkkoliitännöissä eräänä haasteena on vielä tarjota kohtuullisilla kustannuksilla suuri liikennöintikapasiteetti, mikä voidaan tehdä usealla eri tavalla. Kotien laajakaistaiseen tulevaisuuteen johtaakin useita polkuja, joita tässä diplomityössä on kartoitettu. Työssä on esitelty erilaisia kotitalouksiin soveltuvia nopeita langallisia ja langattomia liitäntätekniikoita. Painopiste on nykyisessä valtavirrassa, kuparikaapelia käyttävässä xDSL-tekniikkaperheessä ja niihin pohjautuvissa verkoissa mukaan lukien ATM-pohjaiset runkoverkot Käytännön liitäntöjen laatua ja toimivuutta on selvitetty mittaamalla perinteistä ADSL-liitäntää sekä uudempaa tekniikkaa edustavaa SHDSL-liitäntää. Liittymiä mitattiin sekä xDSL-mittarilla että spektrianalysaattorilla. Mittauksissa kiinnitettiin huomiota kupariparien toimintaan, kuormituksen ja häiriöiden vaikutukseen sekä liittymän toimintaan käyttäjän kannalta. Mittauksissa todettiin, että kuparikaapeli sietää hyvin häiriöitä myös uusien tekniikoiden käytössä. Tulevaisuutta kartoitettiin haastattelututkimuksella, josta työhön on koostettu operaattoreiden, konsulttien ja käyttäjien näkemyksiä laajakaistaliitäntöjen ja niiden kautta saatavien palveluiden kehittymisestä. Tehty tutkimus osoittaa, että ADSL-tekniikka hyödyntää hyvin sille varattuja taajuusalueita. SHDSL-tekniikassa voidaan lähettää ja vastaanottaa suuria datamääriä molempiin suuntiin.

Desenvolvimento, caracterização e estabilidade de geleia tradicional de umbu-cajá

Objetivou-se desenvolver geleia tradicional de umbu-cajá, caracterizá-la quanto a parâmetros físicos, químicos, microbiológicos e sensoriais, e avaliar sua estabilidade durante o armazenamento por seis meses em condições ambientais. Para processamento, foram utilizados 44% de polpa diluída de umbu-cajá, 1% de pectina de alto teor de metoxilação (ATM) e 55% de açúcar cristal. A formulação foi submetida à cocção em tacho aberto de aço inoxidável até teor de sólidos solúveis totais de cerca de 68 ªBrix. A geleia foi envasada em recipientes de vidros transparentes, caracterizada e estocada em temperatura e umidade relativa médias de 23,25 ºC e 81%, respectivamente, com acompanhamento por meio de análises físicas e químicas a cada 30 dias de armazenamento. Os resultados da caracterização química evidenciaram produto com elevado teor de carboidratos, baixos conteúdos de cinzas e proteínas e valor calórico de 256 kcal/100g. Não foi verificado contagem dos microorganismos pesquisados (bolores e leveduras, coliformes totais e termotolerantes, Staphylococcus, bactérias mesófilas e Salmonella). Constatou-se alta aceitabilidade, com índices de aceitação superiores a 70% para todos os atributos sensoriais investigados (cor, aparência, aroma, consistência, sabor, doçura e impressão global) e intenção de compra de 67,5%, indicando potencial para industrialização e comercialização. O armazenamento promoveu aumento significativo nos valores de pH, sólidos solúveis totais (SST), relação SST/ATT e firmeza e reduções significativas na acidez total titulável (ATT), atividade de água, luminosidade, intensidades de vermelho e amarelo, croma, ângulo de tonalidade, extrusão e adesividade. Constatou-se tendência à estabilidade dos valores de umidade e de sólidos totais. O processamento do umbu-cajá para elaboração de geleia mostrou-se viável, apresentando-se como mais uma opção de renda para pequenos produtores do semiárido brasileiro.

Polymorphisms in DNA-repair genes in a cohort of prostate cancer patients from different areas in Spain: heterogeneity between populations as a confounding factor in association studies

Background: Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. Objective: To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. Design, Setting, and Participants: A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArrayH NT Cycler. Outcome Measurements and Statistical Analysis: Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. Results and Limitations: We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. Conclusion: Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

Ação promotora do berílio em catalisadores da síntese do estireno

The catalytic dehydrogenation of ethylbenzene in presence of steam is the main commercial route to produce styrene. The industrial catalysts are potassium- and chromia-doped hematite which show low surface areas leading to bad performance and short life. In order to develop catalysts with high areas, the effect of beryllium on the textural properties and on the catalytic performance of this iron oxide was studied. The influence of the amount of the dopant, the starting material and the calcination temperature were also studied. In sample preparations, iron and beryllium salts (nitrate or sulfate) were hydrolyzed with ammonia and then calcinated. The experiments followed a factorial design with two variables in two levels (Fe/Be= 3 and 7; calcination temperature= 500 and 700ºC). Solids without any dopant were also prepared. Samples were characterized by elemental analysis, infrared spectroscopy, surface area and porosity measurements, X-ray diffraction, DSC and TG. The catalysts were tested in a microreactor at 524ºC and 1 atm, by using a mole ratio of steam/ ethylbenzene=10. The selectivity was measured by monitoring styrene, benzene and toluene formation. It was found that the effect of beryllium on the characteristics of hematite and on its catalytic performance depends on the starting material and on the amount of dopant. Surface areas increased due to the dopant as well as the nature of the precursor; samples produced by beryllium sulfate showed higher areas. Beryllium-doped solids showed a higher catalytic activity when compared to pure hematite, but no significant influence of the anion of starting material was noted. It can be concluded that beryllium acts as both textural and structural promoter. Samples with Fe/Be= 3, heated at 500ºC, lead to the highest conversion and were the most selective. However, catalysts prepared from beryllium sulfate are the most promising to ethylbenzene dehydrogenation due to their high surface area which could lead to a longer life.