Orientador: Lara Durães Sette / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T06:41:16Z (GMT). No. of bitstreams: 1 Passarini_MichelRodrigoZambrano_D.pdf: 6997453 bytes, checksum: 0a905c32bd8d912eb875c88cff54de8e (MD5) Previous issue date: 2012 / Resumo: O oceano representa um habitat promissor na busca por novos micro-organismos, os quais podem apresentar capacidade de produzir enzimas de interesse industrial diferentes das produzidas por seus parceiros terrestres. Neste contexto, duas amostras da esponja marinha Dragmacidon reticulatum foram coletadas no litoral Norte do Estado de São Paulo, objetivando a caracterização da diversidade fúngica por métodos dependentes e independentes de cultivo, bem como a avaliação da produção, expressão da enzima e caracterização do gene da lacase. Com relação à parcela cultivada das amostras, 108 fungos filamentosos foram isolados. Destes, 64 ribotipos distintos foram submetidos aos experimentos de taxonomia polifásica e aos relacionados com a lacase. Análises macro- e microscópicas, moleculares (genes ribossomais ITS/28S) e pela técnica de MALDI TOF ICMS, resultaram na caracterização de 38 isolados distribuídos em 23 gêneros pertencentes ao Filo Ascomycota e um ao Filo Zygomycota. Este foram posteriormente depositados na Coleção Brasileira de Micro-organismos de Ambiente e Indústria (CBMAI). Dentre os isolados obtidos, uma potencial espécie nova de Penicillium foi identificada. Os resultados da triagem enzimática permitiram a seleção de dois isolados identificados como Nigrospora sp. CBMAI 1328 (0,30 U L-1) e Arthorpyrenia sp. CBMAI 1330 (0,40 U L-1), os quais foram submetidos à uma nova avaliação da atividade e expressão (RT-PCR) com indução de íons cobre e caracterização do genes da lacase. A adição de íons cobre na concentração de 5 mM, proporcionou aumento das atividades enzimáticas dos isolados CBMAI 1328 e CBMAI 1330 em 3,9X (25,2 U L-1) e 1,2X (9,0 U L-1) após 120 h, respectivamente. Os resultados da expressão dos genes da lacase para o isolado CBMAI 1328 foram os mesmos encontrados pela indução com cobre (maior expressão em 5 mM após 120 h), entretanto para o isolado CBMAI 1330, a maior expressão foi após 96 h sem adição de cobre. Os resultados da caracterização dos genes da lacase revelaram a possivel existência de 3 novos putativos genes de lacases marinhas. Com relação à parcela não cultivada, foi possível a identificação de 7 gêneros e de um fungo não cultivado (uncultured fungi) do Filo Ascomycota bem como 3 gêneros e um fungo não cultivado (uncultured fungi) do Filo Basidiomycota a partir da técnica de DGGE e do sequenciamento direto do gene RNAr ITS e do gene 18S. Em ambas as abordagens utilizadas, a maior diversidade foi encontrada na amostra DR9. Os dados derivados do presente trabalho, ressaltam a importância em se utilizar a taxonomia polifásica, bem como empregar metodologias dependente e independente de cultivo (em paralelo) para um melhor e maior conhecimento da real diversidade em amostras ambientais. Estes resultados ampliam o conhecimento dos fungos filamentosos recuperados de esponjas marinhas e demonstram o potencial biotecnológico destes micro-organismos / Abstract: The ocean represents a promissing habitat in the search for new microorganisms, which may have the ability to produce enzymes of industrial interests different from that produced by their terrestrial counterparts. In this context, two samples of the marine sponge Dragmacidon reticulatum were collected on northern coast of São Paulo State, aiming at the characterization of fungal diversity by cultureddependent and -independent approaches as well as the evaluation of the production, characterization and expression of laccase enzyme gene. Regarding the cultivated portion of the samples, around 108 filamentous fungi were isolated, belonging to 64 different taxonomic groups (ribotypes), which were subjected to enzymatic activity assays. Polyphasic taxonomy approaches (macro- and microscopic, molecular - ITS/28S ribosomal genes - and MALDI TOF ICMS analyses) resulted in the characterization of 38 isolates distributed in 23 genera belonging to the phylum Ascomycota and one to the phylum Zygomycota, which were deposited in the Brazilian Collection of Micro-organisms from Environment and Industry (CBMAI). Among the isolates recovered one possible new species of Penicillium was identified. Enzymatic screening allowed the selection of two isolates identified as Nigrospora sp. CBMAI 1328 (0,30 U L-1) and Arthorpyrenia sp. CBMAI 1330 (0,40 U L-1), which were subjected to a new activity evaluation and expression (RT-PCR) with the induction of copper ions and the laccase genes characterization. The addition of copper ions in a concentration of 5 mM resulted in an increase in the enzymatic activities of CBMAI 1328 and CBMAI 1330 strains in 3,9X (25,2 U L-1) and 1,2X (9,0 U L-1) after 120h, respectively. The results of the expression of the laccase genes for CBMAI 1328 strain were the same found by induction with copper (expression increased in 5 mM after 120 h), however for CBMAI 1330 the higher expression was after 96 h without addition of copper. Results of laccase genes characterization revealed the existence of three possible putative new marine laccase genes. Regarding to the uncultured portion of the samples, from the DGGE and direct sequencing of the ITS rRNA gene and 18S gene approaches, was possible to identify seven species of fungi and one uncultured fungus from phylum Ascomycota and three species and one uncultured fungus from phylum Basidiomycota. For both approaches used the greatest diversity was achieved in the DR9 sample. Data derived from the present work highlight the importance of using polyphasic taxonomy as well as applying culturedependent and culture-independent methodologies (in parallel) in order to have a better and greater knowledge of the real diversity in environmental sample. These results expand the knowledge of fungi recovery from marine sponges and demonstrate the biotechnological potential of these micro-organisms / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fungi represent an extremely important group of organisms in terrestrial ecosystems. Among their several important ecological roles, is the mutualistic association with plant roots, forming different types of mycorrhiza. Most studies carried out so far focused on epigeous ectomycorrhizal fungi, which occur above ground. On the other hand, below ground, hypogeous ectomycorrhizal fungi, are still poorly studied. This research aimed to study the diversity of hypogeous ectomycorrhizal fungi (Basidiomycetes) collected in Eucalyptus spp. plantations in the Central Region of Rio Grande do Sul, Brazil, based on morphological and molecular characters. Samples were taken between May 2009 and July 2010. A total of five species belonging to three families were identified. The material was analyzed to determine the morphological and molecular characters. Among the species identified are: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway and Setchelliogaster tenuipes (Setch.) Pouzar. Additionally a second species of Chondrogaster sp. was collected. However, no identity was determined for this species. Furthermore, it is believed it represents a new undescribed species to science. Among the species studied, Descomyces albus is reported for the first time in the State of Rio Grande do Sul. Hysterangium affine and H. inflatum are newly recorded species from Brazil while Chondrogaster pachysporus is recorded for the first in South America. / Os fungos representam um grupo de organismos extremamente importante nos ecossistemas terrestres. Entre as muitas funções por eles desempenhadas está a associação mutualística com as raízes dos vegetais, formando diferentes tipos de micorrizas. A maioria dos estudos realizados até o momento está principalmente focada nos fungos ectomicorrízicos epígeos, os quais ocorrem acima do solo. Por outro lado, os fungos ectomicorrízicos hipógeos, que vivem abaixo da superfície do solo, são ainda pouco estudados. A presente pesquisa tem como objetivo estudar a diversidade de fungos ectomicorrízicos hipógeos (Basidiomycetes) coletados em plantações de Eucalyptus spp. na Região Central do Rio Grande do Sul, Brasil, com base em características morfológicas e moleculares. O período de coleta dos fungos foi de maio de 2009 a julho de 2010. Um total de cinco espécies pertencentes a três famílias foram identificadas. Este material foi analisado para verificar suas características morfológicas e moleculares. Dentre as espécies identificadas estão: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway e Setchelliogaster tenuipes (Setch.) Pouzar. Adicionalmente identificou-se uma segunda espécie de Chondrogaster sp. porém não se chegou a uma espécie conhecida, acreditando tratar-se de uma espécie ainda desconhecida para a ciência. Dentre as espécies estudadas, destacam-se a Descomyces albus que é citada pela primeira vez para o Estado do Rio Grande do Sul, Hysterangium affine e H. inflatum que têm sua ocorrência registrada pela primeira vez no Brasil, além de Chondrogaster packysporus citado pela primeira vez para a América do Sul.

Les systèmes de transport intelligents coopératifs permettent la communication des véhicules entre eux ainsi qu'avec l'infrastructure, afin d'assurer la disponibilité des informations d'une manière plus fiable sur les véhicules, leurs positions et les conditions de la route. Cet échange d'informations pertinentes permet d'améliorer la sécurité routière, réduire les incidents du trafic et d'assurer l'efficacité de la mobilité des véhicules. IEEE 802.11p est standardisé comme la technologie par défaut pour les communications des véhicules. Dans ce contexte, le standard européen ETSI s'attaque en particulier aux applications de la sécurité routière. Pour ce faire, il standardise plusieurs types de messages comme CAM (Cooperative Awareness Message) et DENM (Decentralised Event Notification Message). Les CAMs sont des messages de diffusion à un seul-saut, envoyés par chaque véhicule contenant des informations sur sa position, sa vitesse, sa direction, etc., afin d'assurer une coopération lucide entre les autres usagers de la route (y compris les véhicules). Les DENMs sont envoyés à la détection d'un événement sur la route, comme le cas d'un accident, embouteillages, etc. Si nécessaire, une communication multi-saut, exploitant des algorithmes de routage standardisés, est mise en {oe}uvre pour disséminer ces messages au-delà de la portée du transmetteur. La faiblesse de 802.11p réside dans la congestion du canal radio due à la bande passante limitée (5.9 GHz). Afin de pallier à cela, ETSI a proposé un cadre pour le contrôle de la congestion appelé DCC (Distributed Congestion Control). Celui-ci permet l'échange d'informations, en particulier l'état du canal radio, entre les couches de la pile protocolaire. Ainsi, chaque protocole de communication contrôle ses propres paramètres pour éviter la congestion du canal. Par ailleurs beaucoup d'approches de contrôle de la congestion DCC existent pour les messages CAM tel que le contrôle de la période de génération des CAMs sur la couche Facilities. La puissance de transmission ou le débit sur la couche Accès, etc. En revanche, peu de travaux ont été faits sur DENMs. A cet égard, nous avons proposé une approche DCC sur la couche GeoNetworking qui contrôle les paramètres de routage en se basant sur l'état du canal radio. Une évaluation du dual-DCC, à savoir CAM sur Facilities et DENM sur GeoNet, a démontré l'efficacité de l'approche proposée. En outre, certaines applications tel que la gestion d'une flotte de véhicules, ont besoin d'un centre de contrôle localisé sur Internet qui communique avec la flotte. Pour ce type d'échange, une communication hybride (IP et Géo) est nécessaire. De plus pour assurer la fluidité de la communication, la gestion de la mobilité est primordiale. Tout en restant dans le cadre de l'architecture Mobile IP, nous proposons notre approche d'adressage qui constitue une adresse IP routable avec une adresse GeoNetworking, ce qui permet de traiter le problème d'accessibilité des véhicules en mouvement sur la route à partir d'une entité située sur Internet. Contrairement à Mobile IP, notre approche permet de réduire la surcharge de la signalisation. Et cela grâce au partitionnement de la route en zones de routage (RA) de telle sorte que l'accès à Internet se fait via une passerelle RSU-FA qui contrôle la RA. Chaque RA regroupe un certain nombre de RSUs. / Cooperative intelligent transport systems allow vehicles to communicate with each other as well as with the infrastructure in order to ensure the availability of information more reliably on vehicles, their positions and road conditions. This exchange of relevant information improves road safety, reduces traffic incidents and ensures efficient mobility of vehicles. IEEE 802.11p is standardized as the default technology for vehicle communications. In this context, the European ETSI standard addresses in particular road safety applications. To do this, it standardizes several types of messages such as CAM (Cooperative Awareness Message) and DENM (Decentralized Event Notification Message). CAMs are single-hop broadcast messages, sent by each vehicle containing information on its position, speed, direction, etc., in order to ensure lucid cooperation between other road users (including vehicles). The DENMs are sent when there is a detection of an event on the road, as in the case of an accident, traffic jams, etc. If necessary, multi-hop communication, using standardized routing algorithms, is implemented to disseminate these messages beyond the scope of the transmitter. The weakness of 802.11p lies in congestion of the radio channel due to the limited bandwidth (5.9 GHz). In order to compensate for this, ETSI proposed a framework for congestion control called DCC (Distributed Congestion Control). This allows the exchange of information, in particular the state of the radio channel, between the layers of the protocol stack. Thus, each communication protocol controls its own parameters to avoid congestion of the channel. In addition, many DCC congestion control approaches exist for CAM messages such as the control of the CAM generation period on the Facilities layer. Transmission power or data rate on the Access layer, etc. On the other hand, little works have been done on DENMs. In this regard, we proposed a DCC approach on the GeoNetworking layer which controls the routing parameters based on the state of the radio channel. An evaluation of the dual-DCC, namely CAM on Facilities and DENM on GeoNet, demonstrated the effectiveness of the proposed approach. In addition, some applications such as managing a fleet of vehicles require a localized control center that communicates with the fleet. For this type of exchange, a hybrid communication (IP and Geo) is necessary. Moreover, to ensure the fluidity of communication, the management of mobility is paramount. While remaining within the framework of the Mobile IP architecture, we propose our approach of addressing which constitutes a routable IP address with a geonetworking address, which makes it possible to deal with the problem of accessibility of vehicles moving on the road from of an entity on the Internet. Unlike Mobile IP, our approach reduces the overhead of signaling. This is done by partitioning the road into routing area (RA) in such a way that the access to the Internet is via a RSU-FA gateway that controls the RA. Each RA regroups a number of RSUs.

The aim of my study was to sum up the data about genetic variability of freshwater gastropods living in temporary ponds. My model species were moss bladder snail (Aplexa hypnorum) and the critically endangered ramshorn snail (Anisus vorticulus). Genetic variability was investigated by two mtDNA and two nuclear markers. The differences in genetic variation at the COI gene follow the pattern of different catchments. However, these findings do not agree with the patterns derived from genetic markers ITS1 and 16S. The reasons for the differences between the different markers are discussed in several ways involving few possible historical scenarios, but also possible errors of laboratory methods. In this study, there was only little success in amplification and the most likely reason is the inhibitory effect of snail mucus on DNA amplification. Another problem that occurred when this work was to obtain the sequence of the parasite (fluke) instead Anisus gene using gastropod specific primers. In addition to genetic and related methodological part of this work is the third part concerning the evaluation of environmental factors periodic pools in the presence and absence of the species studied. The fourth part sis focused on the phylogenetical analyses of the european and american Aplexa linneages. Powered by TCPDF...

L’oïdium est une des maladies les plus communes sur chêne en Europe. Cette maladie a été signalée à partir du début du 20ème siècle. Toutefois, peu de données sont disponibles sur l’identité de l’agent causal et de son interaction avec l’hôte. L’objectif de cette étude est d’améliorer les connaissances sur ce pathosystème : (1) caractériser son agent causal avec un marqueur moléculaire (ADN ribosomal) et étudier la distribution spatiale des différentes lignées ; (2) étudier l’adaptation locale du parasite à son hôte ; (3) explorer le déterminisme génétique de la résistance de l’hôte. L’étude de l’ADN ribosomal (ITS=Internal transcribed region et IGS= Intergenic spacer) a mis en évidence l’existence de quatre haplotypes, dont trois seulement avaient été associés à des agents d’oïdium sur chêne en Europe. Erysiphe alphitoïdes, classiquement considéré comme responsable de l’épidémie du XXème siècle est bien l’espèce prépondérante (environ 80% des détections), E. hypophylla et Phyllactinia sp étant détectés à fréquence beaucoup plus faible. Un résultat inattendu est la détection d’une quatrième séquence, présentant 100% d’homologie avec celle de plusieurs agents d’oïdium d’arbres tropicaux et avec Erysiphe quercicola, très récemment décrit sur chêne en Asie et Australie mais jusqu’alors inconnu en Europe. L’ITS de E. quercicola est détecté dans toutes les régions de France, à une fréquence non négligeable (de l’ordre de 15% en moyenne). On le trouve dans les mêmes parcelles et parfois sur les mêmes arbres, voire dans les mêmes lésions que E. alphitoides. La mise au point d’une méthode d’inoculation artificielle a permis d’étudier de façon quantitative l’interaction chêne-oïdium. Une expérimentation d’inoculations réciproques montre que les souches d’oïdium sont plus performantes sur les descendants des arbres dont elles ont été isolées que sur ceux d’autres arbres, suggèrant une adaptation trans- générationnelle. Des inoculations artificielles, complétées par des observations en conditions naturelles, ont également permis de démontrer un déterminisme génétique de la résistance des chênes, avec l’identification de plusieurs QTL. Certains de ces QTL co-localisent avec des QTL de phénologie, en accord avec l’importance de la résistance ontogénique des chênes à l’oïdium. L’ensemble de l’étude amène à reconsidérer l’oïdium du chêne comme un probable complexe d’espèces cryptiques, dont l’histoire de l’invasion et la co-existence en Europe restent à préciser. Les résultats acquis sur l’héritabilité de la résistance et l’adaptation locale du parasite constituent une première étape dans la compréhension des interactions démo-génétiques entre hôte et parasite dans ce pathosystème. / Powdery mildew is the most common disease on oaks in Europe where it was first recorded at the beginning of the 20th century. Yet, little is known about the identity of the causal agent and his interaction with its host. The objective of this study was: (1) to characterize the species responsible of oak powdery mildew with a molecular marker (ribosomal DNA); to study the spatial distribution of these different lineages and (2) to study local adaptation of the parasite to its host (3) to explore the genetic determinism of host resistance. The study of the ITS (internal transcribed region) and IGS (intergenic spacer) diversity revealed the existence of four haplotypes, only three had been already associated with oak powdery mildew in Europe. Erysiphe alphitoïdes, for a long time considered as sole responsible for the epidemic of the XXth century, was the predominant species (~ 80% of detections), E. hypophylla and Phyllactinia sp. were detected at lower frequencies. An unexpected result is the detection of a fourth sequence which show 100% homology with ITS sequences of several powdery mildew agents of tropical trees and Erysiphe quercicola, recently described on oak in Asia and Australia but previously unknown in Europe. E. quercicola ITS was detected in all French regions, at a significant frequency (~ 15%). The study showed that E. alphitoides was often found in association with different ITS types in the same region, the same tree, and even in the same lesion. The development of an artificial inoculation method allowed the quantitative evaluation of the oak-powdery mildew interaction. A reciprocal inoculation experiment showed that powdery mildew strains were more efficient on their mother tress and their descendants than on the other trees, suggesting a trans-generational adaptation. Artificial inoculations, supplemented by observations in natural conditions, have also demonstrated a genetic determination of resistance of oak trees, with the identification of several QTL. Some of these QTL co-localize with QTL controlling phenology, in agreement with the importance of oaks ontogenic resistance to powdery mildew. The entire study leads to reconsider oak powdery mildew as a probable complex of cryptic species; the invasion history and the co-existence in Europe are still to be determined. The results achieved on the heritability of resistance and localization of the parasite are a first step in understanding the demo-genetic interactions between host and parasite in this pathosystem.

Für die Identifizierung bzw. Differenzierung von Candida- Spezies und -Stämmen sowie für die Bestimmung der genetischen und epidemiologischen Verwandtschaft von Stämmen der gleichen Spezies wurde eine PCR-Fingerprint-Technik und eine RFLP-Analyse der amplifizierten ITS-Region angewandt. Das PCR-Fingerprinting amplifiziert anonyme Sequenzen in der chromosomalen DNA, die über das gesamte Genom verteilt sind. Die ITS-Region ist Bestandteil des ribosomalen Operons, welches in ca. 50-100 Kopien/Zelle vorhanden ist. 1.a. Beide molekularbiologischen Verfahren wurden zur Unterscheidung von routinemäßig schwer differenzierbaren klinischen Candida famata und Candida guilliermondii-Isolaten genutzt. Von insgesamt 37 fraglichen Stämmen konnten 31 als C. guilliermondii und 3 als C. famata identifiziert werden, die drei verbliebenen Stämme waren mit diesen Techniken nicht identifizierbar. Mit der Biochemotypie gelang nur die Zuordnung eines der 3 C. famata-Isolate sowie von 23 der 31 C. guilliermondii-Isolate. 14 Isolate wurden mit den konventionellen Methoden gar nicht oder falsch identifiziert. 1b. Mit dem PCR-Fingerprinting wurde auch die Spezieszugehörigkeit phänotypisch veränderter Candida albicans-Isolate überprüft. Alle atypischen Stämme, bei denen solche für C.albicans charakteristischen Merkmale wie die Bildung von Chlamydosporen, die Verwertung der Aminozucker Glukosamin und N-Acetylglukosamin sowie die Assimilation von 2-Ketogluconat und Xylose nicht ausgeprägt waren, wiesen die für C. albicans typischen Fingerprintmuster auf. Unsere Studie zeigte, daß die biochemische Typisierung an Grenzen stößt, wenn typische Stoffwechselreaktionen nicht nachgewiesen werden können. 2. Bei 6 verschiedenen C. albicans-Populationen aus Angola, Madagaskar, Deutschland und Portugal wurde die Variabilität phänotypischer und genotypischer Merkmale untersucht, wobei in diese Analyse auch atypische Stämme miteinbezogen wurden. Während die phänotypischen Eigenschaften, bis auf die der atypischen Stämme, kaum variierten, wurden für die insgesamt 212 C. albicans-Isolate 87 unterschiedliche PCR-Fingerprint-Genotypen nachgewiesen. Eine Analyse der Beziehungen zwischen den Fingerprint-Genotypen wurde mit der UPGMA-Distanz-Methode durchgeführt. 3. Weiterhin wurden Candida-Vaginalisolate von Patientinnen mit rezidivierenden Episoden von Candida-Vaginitis mit Stämmen verglichen, die aus anderen Körperregionen stammten bzw. bei ihren Partnern isoliert wurden. Es konnte gezeigt werden, daß Stammaustausche zwischen den Partnern vorkommen, ein Stamm ohne oder mit geringfügigen genotypischen Veränderungen trotz Therapie persistieren kann und daß eine Reinfektion auch durch einen neuen Stamm möglich ist. Das Problem der Erregeridentifizierung in der mykologischen Labordiagnostik ist sowohl klinisch als auch epidemiologisch relevant. Molekularbiologische Methoden sollen gut funktionierende konventionelle Methoden zur Erregeridentifizierung nicht ersetzen, können aber bei Problemfällen eine wertvolle Ergänzung für die mykologische Diagnostik vorzugsweise in fachständigen Referenzlaboratorien darstellen. / A PCR fingerprinting approach and a RFLP analysis of the amplified ITS region were used to differentiate Candida species and strains as well as to assess genetic and epidemiological relationships of strains belonging to the same species. The PCR fingerprinting amplifies anonymous DNA sequences sampled throughout the whole genome. The ITS region is part of the ribosomal operon which occurs in tandem arrays of nearly 50-100 copies in one cell. 1.a. Both methods were used to identify clinical isolates of C. famata and C. guilliermondii which were difficult to differentiate with routine methods. Out of 37 ambiguous isolates 31 could be identified as C. guilliermondii, 3 as C. famata and the other 3 were not identifiable. Biochemical typing (Api 32 C V.1) identified only 23 out of 31 C. guilliermondii and 1 out of 3 C. famata whereas 14 isolates were misidentified or not identified at all. 1.b. By using the PCR fingerprinting technique strains of C. albicans with altered phenotypes could be identified at species level. Atypical isolates which did not express those characteristics which are thought to be typical for C.albicans like the formation of clamydospores, the ability to metabolise the amino sugars glucosamine and N-acetylglucosamine as the sole carbon source or to assimilate 2-ketogluconate and xylose showed the PCR patterns typical for C.albicans. Our study revealed that biochemical and morphological methods of species identification are limited if some of the key reactions fail. 2. We investigated the variability of phenotypic and genotypic properties of 6 different C. albicans populations from different countries (Angola, Madagascar, Portugal and Germany) including atypical strains. Except for the atypical strains only very little phenotypical variation was observed. However, 87 different genotypes were found among the 212 strains. The relatedness of the fingerprint-genotypes were analysed by measuring genetic distances with the UPGMA method. 3. Vaginal isolates of Candida spp. obtained from patients with recurrent episodes of vaginitis were compared with isolates from different body locations of the women and from their male partners. It has been shown that strain exchanges between the partners occur, that the original strain with or without minor genotypic changes can persist despite of the therapy, but also that reinfection by a new strain is possible. The identification of an ethiological agent by the mycological diagnostic laboratory is of clinical and epidemiological importance. Molecular biological methods should not replace well established conventional methods but they can supplement the identification of fungal pathogens in specialised reference laboratories if diagnosis cannot be achieved easily by conventional diagnostic procedures.

Estudos sobre a diversidade de macroalgas de manguezais no Brasil tem-se baseado apenas em abordagens morfológicas, nas quais os caracteres empregados são instáveis e pouco informativos para a identificação e delimitação de espécies. Neste contexto, macroalgas de manguezais foram investigadas pela primeira vez no litoral brasileiro usando uma abordagem molecular, tendo como alvo de estudo o manguezal da Ilha Barnabé, Baixada Santista, São Paulo. Foram utilizados marcadores moleculares do tipo Barcode, UPA e COI-5P, além do rbcL, amplamente empregado para inferências filogenéticas, e o SSU rDNA. Além desses, os marcadores ITS e tufA foram empregados exclusivamente para as algas verdes, este último sem sucesso. Quinze espécies foram registradas para a área estudada, sendo dez Rhodophyta e cinco Chlorophyta. Destas, duas não novas ocorrências para o Estado de São Paulo, Caloglossa apomeiotica e Boodleopsis vaucherioidea. Das quatro espécies do gênero Bostrychia identificadas neste estudo: \"Bostrychia calliptera\", B. montagnei, B. moritziana e B. radicans, apenas B. montagnei revelou-se uma espécie molecular e morfologicamente bem definida. As demais formaram complexos de espécies com linhagens moleculares distintas. Para B. radicans e B. moritziana as análises relevaram três e duas linhagens moleculares, respectivamente, das sete identificadas para o complexo B. radicans/B. moritziana na literatura. O táxon identificado como \"B. calliptera\" mostrou alta divergência molecular com sequências de B. calliptera do Brasil, apresentando morfologia \"B. pinnata\", um táxon atualmente reduzido a sinônimo de B. calliptera. Espécies do gênero Caloglossa, excetuando C. ogasawaraensis, são de difícil identificação morfológica devido aos tênues caracteres considerados de valor diagnóstico e a sua considerável plasticidade fenotípica. Caloglossa apomeiotica, C. confusa e C. leprieurii foram identificadas essencialmente com o emprego de marcadores moleculares. Caloglossa apomeiotica pode ser segregada das demais pela presença de biesporângios, o que impossibilita uma identificação morfológica segura quando coletados talos inférteis, enquanto C. confusa possui nós fortemente contritos. Os dados moleculares obtidos para Catenella caespitosa a partir de sequências de SSU sugerem que as citações dessa espécie para o litoral brasileiro podem estar equivocadas já que apresentam alta divergência intraespecífica com C. caespitosa do banco de dados. A falta de sequências da localidade tipo, de sequências com marcadores do tipo Barcode e de uma maior amostragem molecular das espécies de Catenella nos bancos de dados, nos impossibilitaram chegar a um resultado conclusivo. A obtenção de sequências para as algas verdes foi extremamente problemática, inviabilizando uma comparação mais ampla entres as espécies coletadas. Das duas espécies coletadas de Boodleopsis foram obtidas apenas duas sequências parciais de rbcL para B. vaucherioidea e nenhuma para B. pusilla. A comparação com a única sequência de Boodleopsis depositada nos bancos de dados, uma sequência parcial de rbcL de B. pusilla, revelou baixa divergência molecular com as nossas sequências de B. vaucherioidea. Além da necessidade de obtenção de sequências completas de rbcL de ambas espécies da área estudada para comparação, uma maior amostragem e o emprego de outros marcadores moleculares são necessários para esclarecer o posicionamento taxonômicos desses dois táxons, cuja coespecificidade não pode ser descartada. Morfologicamente, \"Cladophoropsis membranacea\" é uma espécie facilmente identificada, entretanto sequências de ITS obtidas neste estudo são não comparáveis a nenhuma sequência dessa espécie depositada nos bancos de dados, incluindo sequências da localidade tipo. Reconhecidamente Chadophoropsis é um gênero polifilético e integra o complexo Boodlea que inclui diferentes gêneros de Boodleaceae. A obtenção de sequências de outros marcadores como o SSU rDNA e LSU rDNA assim como uma maior amostragem podem ser informativas para esclarecer a posição das \"C. membranacea\" brasileiras dentro das Cladophorales. Mesmo após inúmeras tentativas não foi possível obter sequências para as duas espécies de Rhizoclonium encontradas, R. africanum e R. riparium, cuja identificação foi feita com base em caracteres morfológicos tradicionais / Studies on the diversity of macroalgae from mangroves in Brazil have been based only on morphological approaches, in which characters used are unstable and uninformative for the species identification and delimitation. In this context, macroalgae of mangroves were investigated for the first time in the Brazilian coast using a molecular approach, having as target of our study the mangrove of the Barnabé Island, Santos, São Paulo. DNA Barcode markers UPA and COI-5P were used, besides the rbcL, largely used for phylogenetic inferences, and also SSU rDNA. In addition to these, the ITS and tufA markers were used exclusively for the green algae, the latter unsuccessfully. Fifteen species were recorded for the studied area, ten Rhodophyta and five Chlorophyta. Of these, two are new records for the State of São Paulo, Caloglossa apomeiotica e Boodleopsis vaucherioidea. Of the four species of the genus Bostrychia identified in this study: \"Bostrychia calliptera\", B. montagnei, B. moritziana and B. radicans, only B. montagnei proved to be a molecular and morphologically well-defined species. The other species formed complexes with different molecular lineages. For B. radicans and B. moritziana, the analyses showed three and two molecular lineages, respectively, of the seven identified for the B. radicans/B. moritziana complex in the literature. The taxon identified as \"B. calliptera\" showed high molecular divergence with sequences of B. calliptera from Brazil, presenting morphology \"B. pinnata\", a taxon currently reduced to a synonym of B. calliptera. Caloglossa species, except C. ogasawaraensis, are difficult to identify due to the subtle morphological characters considered of diagnostic value, and their considerable phenotypic plasticity. Caloglossa apomeiotica, C. confusa and C. leprieurii were identified primarily by the use of molecular markers. Caloglossa apomeiotica can be segregated from the others by the presence of bisporangia, which makes unreliable morphological identification when collected infertile thalli, while C. confusa has strongly constricted thallus nodes. The molecular data obtained for Catenella caespitosa from SSU sequences suggest that the citations of this species for the Brazilian coast may be misleading since they have high intraspecific divergence with C. caespitosa from database. Due to the lack of sequences from the type locality, sequences of DNA barcode markers, and a major molecular sampling of Catenella species in databases, became impossible to reach a conclusive result. The obtaining of sequences for green algae was extremely problematic, making impracticable a broader comparison between the collected species. Of the two collected species of Boodleopsis, only two partial sequences of rbcL were obtained for B. vaucherioidea and none for B. pusilla. The comparison with the unique sequence of Boodleopsis deposited in databases, a partial rbcL sequence of B. pusilla, revealed low molecular divergence with our sequences of B. vaucherioidea. Besides the need to obtain complete sequences of rbcL from both species from the studied area for comparison, an increased sampling and the use of other molecular markers are needed to clarify the taxonomic position of these two taxa, whose conspecificity cannot be disregarded. Morphologically, \"Cladophoropsis membranacea\" is an easily identified species, however, ITS sequences obtained in this study are not comparable to any sequence of this species deposited in databases, including sequences from the type locality. Admittedly. Chadophoropsis is a polyphyletic genus and integrates the Boodlea complex that includes different genera of Boodleaceae. The obtaining of sequences from other markers, such as SSU rDNA and LSU rDNA, well as a larger sampling, may be informative to clarify the taxonomic position of \"C. membranacea\" within the Brazilian Cladophorales. Even after numerous attempts we could not get sequences for the two Rhizoclonium species found in the studied area: R. africanum and R. riparium, whose identification was made based on traditional morphological characters

La presente tesis propone un modelo para la prestación de servicios de valor agregado en el ámbito de los Sistemas Inteligentes de Transporte (Intelligent Transportation Systems -ITS) dando solución a los problemas de integración, interoperabilidad, compatibilidad y escalabilidad provocados por la gran cantidad de tecnologías que componen el escenario de los ITS. / Con el fin de integrar todos los elementos que componen los sistemas ITS, el modelo propuesto se basa en las Arquitecturas Orientadas a Servicios (Services Oriented Architecture -SOA) ya que es una solución altamente exitosa en el contexto de las tecnologías de la información y que en otros ámbitos ha contribuido a la prestación de servicios. / Las principales aportaciones del trabajo de investigación son: la creación de un modelo para la prestación de servicios ITS de valor agregado; la creación de un nuevo concepto para la unidad de carretera (Roadside Unit -RSU) la cual integra el paradigma SOA para ofrecer sus funcionalidades en un esquema basado en servicios (Roadside Unit as a Service -RSUAAS); la creación de un catálogo que relaciona las tecnologías y los servicios ITS y que sirve de elemento fundamental para lograr la especialización de los fabricantes; La creación de una arquitectura que aprovecha las ventajas del esquema orientado a servicios para ofrecer servicios de valor agregado al usuario ITS a partir de las tecnologías.

La carrera por dotar a los vehículos de mayor seguridad y de comodidades hasta hace poco inimaginables, ha espoleado los avances en novedosos sistemas inteligentes de transporte, o intelligent transportation systems (ITS). De hecho, tal es la cantidad de líneas de trabajo y ámbitos de la ciencia que conforman los ITS, que la investigación se ha visto diversificada en gran medida en el último lustro. Uno de los campos que más interés procesa (si no el que más) es el de la telemática vehicular. Los nuevos servicios de a bordo englobados por la informática y las telecomunicaciones no paran de crecer en el ámbito científico, e incluso los modelos comerciales empiezan a incorporarlos en forma de sistemas de navegación integrados o mecanismos de tele-asistencia, por ejemplo.La tesis doctoral se encuadra dentro de este ámbito, mediante la definición de una plataforma integral para la provisión de servicios telemáticos tradicionales y de carácter ubicuo. La investigación puntual en los principales subsistemas de a bordo y del lado de la infraestructura, completa las piezas del puzzle que comprende un sistema de carácter genérico de despliegue de servicios ITS. La adecuación de un vehículo prototipo de referencia se ha enriquecido con una unidad de a bordo basada en un computador de propósito general, que incluye una propuesta de plataforma software modular basada en pasarela de servicios. El sistema de navegación toma como referencia a GPS, y explota las capacidades de los sistemas de aumento de señal, o satellite based augmentation systems (SBAS). De esta manera, se mejora la precisión y la disponibilidad, pero, sobre todo, se añade la funcionalidad necesaria para la monitorización de la integridad del sistema. La red vehicular cubre el hueco existente entre las soluciones poco flexibles basadas en redes de infraestructura, y las demasiado localizadas y distribuidas (VANET), mediante una arquitectura de comunicación overlay que funciona sobre unas resurgidas redes celulares. Finalmente, la plataforma se completa con un soporte remoto adicional de la infraestructura, para la definición de servicios pervasivos con capacidades de monitorización de la red viaria, procesamiento global, e inferencia descentralizada de información contextual adaptada a las preferencias de los usuarios. / Due to the growing interest that current society has in new technologies, new products in the fields of information and communication technologies are emerging in new environments still unexploited. In this way, vehicles are a perfect frame for installing a lot of useful functionalities traditionally available at work or home environments. However, this expansion needs a suitable hardware and software support adapted for the market and user demands. The on-board software architecture developed in the frame of the thesis covers some of these issues and acts as the basis for the rest of proposals presented in the work. Integration of telematics and vehicular communications in next-generation cars will turn the provision of contextual information into the cornerstone of vehicular services. In the world of intelligent transportation systems (ITS) such capability has a key role in the transmission of traffic incidences. Although there are multitude of solutions for the problem of sharing information between vehicles and between the vehicle and the infrastructure, none presents a valid general communication architecture for the notification, storage, management and provision of context-aware information on traffic and current location. The proposal given in the thesis offers a solution using an integrated vehicle to vehicle (V2V) and vehicle to infrastructure (V2I) communication paradigm enriched with an information management system. The infrastructure manages all the collected safety hazards heard from vehicles and the interesting information to be provided to the user, and adapts these to the car's context and driver's preferences. Moreover, positioning requirements of this new generation of ITS services are continuously growing. New applications in the vehicular domain require positioning sensors with a high level of accuracy, availability, continuity and integrity. The proposed navigation and networking architectures are of special relevance in the thesis and, as it is explained, both of them imply novel designs not only in the vehicle, but also in the remote and road side support. In addition to the system design, a real vehicle prototype and the whole implementation of the system are presented. This test-bed has been also used to perform multitude of experimental evaluations, which demonstrate the suitability of the whole proposed platform.

I storstadsregionerna blir trafikläget allt mer ansträngt för vart år som går. Inflyttningen fortsätter i oförändrad takt och fler människor måste försöka samsas om samma utrymme. Situationen på vägarna börjar bli ohållbar och det måste till att dessa problem löses snart för att utvecklingen i regionerna inte ska stagnera. Möjligheter för ytterligare utbyignationer finns dock i en begränsad grad och man måste börja se till andra lösningar. Inom området för intelligenta transportsystem(ITS) erbjuds många nya tillämpningar där man med ny teknik försöker hitta lösningar till dagens trafikproblem. Ett led i detta är att samla in och distribuera information om restider på vägarna, för att försöka fördela trafiken mer jämt över hela vägnätet. Det finns olika metoder för att hämta in den här typen av information, men den här rapporten fokuserar sig vid att beskriva system baserat på Floating Car Data(FCD). Arbetet som beskrivs i rapporten har i huvudsak analyserat fyra olika restidsuppskattnings-modeller och jämfört dessa med varandra. Modellerna baserar sina beräkningar på observationer från oidentifierade fordon, dvs att observationerna inte har någon identitetsstämpel som kan kopplas till ett specifikt fordon. Två av modellerna betraktar länkarna som en helhet och utför beräkningarna med detta som grund, medan de två andra delar upp varje länk i mindre segment vilket skapar möjlighet för en större noggrannhet. Modellerna testades inledningsvis på simulerad data baserat på trafikmätningar i Göteborgstrakten. Alla beräkningar begränsades ner till länknivå och inte hela vägnät. Detta p.g.a. att det initialt var för komplicerat att skapa en map- matchingmetod som skulle krävas för genomföra beräkningar på olika länkar samtidigt. Efter genomförda tester på simulerad data prövades modellerna även på en reella datamängd hämtad från projektet Probe i Stockholmsområdet. Resultaten från de utförda testerna visar på att det inte skiljer sig nämnvärt i restidsuppskattningarna mellan de olika modellerna. Sträckan som valdes att analyseras i de simulerade fallen, påverkades inte av några större störningar eller flödesvariationer. Det resulterade i att alla modellerna genererade likvärdiga restider. Även i fallet med den reella datamängden, som innehöll större flödesvariationer över tiden, kunde de olika modellernas uppskattningar inte skiljas åt nämnvärt. Slutsatsen är att trafiken i allmänhet inte har så kraftiga förändringar i flödet över tiden, att det krävs särskilt avancerade modeller för att beräkna restider på länknivå. I alla fall inte om man bortser från incidenter. De framräknade restiderna och den information som dessa ger, bör främst användas för direkt trafikstyrning för att uppnå önskat resultat. Människor förlitar sig mer till sina egna erfarenheter i kända områden, så information av den här typen lämpar sig mer som hjälpmedel för den enskilde individen vid resor i okänd trafik.