Estudos sobre a diversidade de Bacteria em solos de mangue (Brasil) e marisma (Espanha) são escassos. A vegetação de mangue, composta por espécies como Spartina alterniflora, Rhizophora mangle, Avicennia schaueriana e Laguncularia racemosa, pode ser um dos fatores que determinam a estruturação das comunidades de procariotos. Determinações das estruturas das comunidades e de diversidade de Bacteria podem ocorrer em função das diferentes condições físico-químicas dos solos, refletindo na configuração dos processos biogeoquímicos. O objetivo deste trabalho foi avaliar a variação das estruturas das comunidades de Bacteria e Archaea, bem como a diversidade, em solos de mangue e marisma utilizando DGGE e sequenciamento parcial do rDNA 16S. As estruturas das comunidades de procariotos apresentaram variações em função de condições de vegetação. Proteobacteria e Bacteroidetes estão presentes em todos os solos estudados. A comunidade de Bacteria destes ambientes é dominada por Proteobacteria. Vários dos táxons detectados estão relacionados com ciclos biogeoquímicos importantes para os ambientes estudados. As estimativas não-paramétricas de riqueza de espécies (ACE e Chao1) mostram que solos de mangue e marisma podem conter milhares de espécies de bactérias. As comunidades de Bacteria dos solos de mangue e marisma são significativamene diferentes. Na camada mais superficial do sedimento de mangue predomina Euryarchaeota metanogênicas enquanto que na camada mais profunda predomina Crenarchaeota. Bactérias das ordens Desulfobacterales, Desulfovibrionales e Desulfuromonales podem estar relacionadas com a atividade de sulfato-redução e formação de pirita na camada anaeróbia do perfil de solo de marisma. De uma maneira geral, pode-se concluir que a diversidade e estrutura das comunidades de procariotos de ambientes estuarinos pode variar em função da vegetação estabelecida e do tipo de ambiente. Adicionalmente, solos de mangue e marisma possuem grande diversidade de procariotos, grande parte da qual é desconhecida, podendo representar elevado potencial genético para utilização biotecnológica. / The bacterial diversity in mangrove (Brazil) and marisma (Espanha) soils are largely unknown. Bacterial communities participate in biogeochemicals processes that occurs in soils of estuarine ecosystems. Determinations of the bacterial communities structures and diversity can occur in function of different physico-chemical conditions, reflecting in the biogeochemical processes. The aim of this work was to evaluate the variation of bacterial an archaeal communities structures utilizing DGGE and partial sequencing of 16S rDNA. Bacterial community structures showed more similarity between repetitions samples than the areas under different vegetation. Phylogenetic afiliation shows that several sequences were not clamped into known phyla. Proteobacteria prevails in bacterial communities of mangrove and marisma soils. Several taxa detected are associated to important biogeochemical cycles that occur in estuarine ecosystems. Analysis of species richness showed that mangrove and marisma soils can contain 200 to 6000 species of bacteria. Methanogenic Euryarchaeota was found specially in the upper sample of mangrove sediment analysed whereas the Crenarchaeota was found specially in the lower. Based on the data obtained, it can be concluded that the vegetation is one of the factors affecting the structure of bacterial and archaeal communities in mangrove soils. Additionaly, the effects of edafic factors and seasonal variations have to be considered as determining the prokaryotic community sctuctures, and bacterial and archaeal communities can respond independently to the factors that determine their community structures. Bacterial diversity can vary with the studied estuarine ecosystem. Studies are necessary concerning to diversity of Bacteria, it variation and correlation with biogeochemical process in the mangrove and marisma soils. These soils show a great diversity of bacteria, much of than unknown, which represent a great genetic potential to the biotechnology.

O Brasil é o maior produtor mundial de cana-de-açúcar, sendo o Estado de São Paulo responsável por mais de 50% do total desta produção. Esta cultura tem um enorme impacto sobre a agricultura brasileira e devido a isto, uma melhor compreensão da composição da comunidade microbiana relacionada a ciclagem do nitrogênio (N) nestes solos é de grande importância para o aprimoramento no cultivo da cana-de-açúcar. No entanto, pouco se sabe sobre a relação entre grupos microbianos e essa cultura. Este trabalho visou determinar variações nas comunidades microbianas que participam das transformações do N nos solos de três áreas utilizadas para a produção de cana-de-açúcar (A, F e J), as quais apresentam uma gama de diferentes condições de manejo e características do solo. Cada área foi analisada em triplicatas, e o DNA extraído do solo foi utilizado para metodologias de sequenciamento de segunda geração (metagenômica e sequenciamento do gene 16S rDNA), PCR quantitativo (qPCR) e polimorfismo dos fragmentos terminais de restrição (T-RFLP). A maioria das sequências derivaram de bactérias (98%; base de dados M5NR); seis etapas do ciclo do nitrogênio foram anotadas pela plataforma MG-RAST (base de dados SEED): amonificação do nitrato e nitrito (ANN); fixação de nitrogênio; desnitrificação; óxido nítrico sintase; redução dissimilatória do nitrito e assimilação de amônia. Este último passo mencionado foi o mais abundante (28%) (genes gltb e gltD) e está diretamente relacionado a multiplicação microbiana nos solos.. O segundo processo mais abundante foi ANN (genes nir e nar), responsável por consumir este substrato durante o ciclo. Proteobacteria, Chloroflexi, Verrucomicrobia e Cyanobacteria estão presentes em todas as etapas do ciclo, representando microrganismos que podem participar das vias de transformação do N e, ademais, foi possível descrever um core microbiano (nível de ordem) representado por Sphingomonadales. Algumas variáveis ambientais, como a aplicação de torta de filtro e a produtividade mostraram uma correlação significativa com a estrutura da comunidade microbiana. Isto também foi encontrado para algumas características do solo como fósforo, magnésio e matéria orgânica. Além disso, identificou-se uma alta abundância de microrganismos carregando genes que codificam enzimas da via de redução do nitrato e nitrito. Portanto, apesar de sua complexidade, o estudo das funções microbianas de nitrogênio em solos cultivados com cana-de-açúcar é inovador por acessar de forma conjunta todas as comunidades envolvidas neste ciclo, caracterizadas por sequenciamento, o que evita os problemas inerentes ao cultivo microbiano, além de permitir inferir sobre a hierarquia das variáveis ambientais que influem sobre esse grupos microbianos. / Brazil is the largest world\'s producer of sugarcane and State of São Paulo accounts for over 50% of this production. This crop has a huge impact on Brazilian agriculture and due to this great importance, a better understanding of the composition of the microbial community related to cycling of nitrogen (N) in these soils is of utmost importance for the improvement in the cultivation of sugarcane. However, little is known about the relationship between microbial groups and this crop. This study aimed to determine changes in microbial communities that participate in the transformation of N in soils derived from three areas used for sugarcane production (named A, F and J), which have a range of different management conditions and soil characteristics. Each area was analyzed in triplicate, and the extracted DNA was used to develop next generation sequencing (shotgun metagenomics and 16S rDNA), quantitative PCR (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Most of the sequences derived from Bacteria (98%; M5NR database); six stages of nitrogen cycle were annotated by MG-RAST plataform (SEED database): nitrate and nitrite ammonification (NNA); nitrogen fixation; denitrification; nitric oxide synthase; dissimilatory nitrite reductase and ammonia assimilation. This last mentioned step was the most abundant (28%) (gltB and gltD genes) and is directly linked to microbial growth in soil, since the final product of the reaction is glutamate. The second most abundant process was NNA (nir and nar genes), responsible for consuming this substrate during the cycle. Proteobacteria and Chroloflexi are present at all stages of the cycle, representing microorganisms that can participate in the N transformation and, moreover, it was possible to describe a microbial core (at an order level) represented by Sphingomonadales. Some environmental variables, such as the application of filter cake and productivity showed a significant correlation with the structure of the microbial community. This was also found for certain soil characteristics such as phosphorus, magnesium and organic matter. In addition, it was identified a high abundance of microorganisms carrying genes encoding the enzymes for nitrate and nitrite reduction pathway. Therefore, despite its complexity, the study of microbial functions of nitrogen in soils cultivated with sugarcane is innovative by accessing jointly all communities involved in this cycle, characterized by sequencing, which avoids the problems inherent in microbial cultivation and allows to infer about the hierarchy of environmental variables that influence this microbial groups.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-06-01T21:04:14Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Organização genômica de sequências repetitivas em Pica-paus (aves piciformes).pdf: 2128434 bytes, checksum: 8afa26cc3c248da9e51696791e1dadc1 (MD5) / Made available in DSpace on 2017-06-01T21:04:15Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Organização genômica de sequências repetitivas em Pica-paus (aves piciformes).pdf: 2128434 bytes, checksum: 8afa26cc3c248da9e51696791e1dadc1 (MD5) Previous issue date: 2017-04-05 / A caracterização da quantidade e distribuição da fração de DNA repetitivo em genomas auxilia no entendimento de sua organização cromossômica. As Aves são conhecidas por apresentar uma baixa proporção de DNA repetitivo quando comparada a outras classes de Vertebrados. Entretanto, a ordem Piciformes se destaca por apresentar uma quantidade percentual superior dessas sequências comparado com as outras aves. Com isso, o objetivo deste estudo foi determinar a distribuição de diferentes tipos de sequências repetitivas no genoma de três espécies da família Picidae, Colaptes melanochloros (2n=84), Colaptes campestris (2n=84) e Melanerpes candidus (2n=64), por meio de hibridização in situ fluorescente (FISH) com sondas de rDNA 18S, teloméricas (TTAGGG)n e microssatélites. Os resultados mostraram, nessas três espécies, o cromossomo sexual Z como o maior do complemento, esse fato deve-se ao acúmulo de diferentes sequências de microssatélites. Entretanto o cromossomo W de C. Melanochloros, que é totalmente heterocromático, não apresentou acúmulo destas sequências. Os sítios ribossomais estão organizados em um par de cromossomos com uma constrição secundária e este teve o acúmulo da sequência (CGG)10 nas três espécies. As sondas teloméricas apresentaram marcações nas regiões terminais dos cromossomos e marcações intersticiais em alguns macrocromossomos. As marcações intersticiais indicam fusões entre cromossomos ou acúmulo de sequências repetitivas similares as teloméricas. Com as sondas de microssatélites identificou-se o mesmo padrão de hibridização nas espécies de Colaptes e padrão distinto entre Colaptes e M. candidus. As nossas análises de FISH mostraram várias sequências de microssatélites amplificadas no cromossomo Z nas três espécies analisadas, o que pode explicar o fato deste ser o maior elemento do cariótipo e desta família conter maior quantidade de sequências repetitivas comparadas com outros grupos de aves. Curiosamente, nenhuma das sequências foi encontrada acumulada no cromoss omo W, apesar de desempenharem um papel importante na diferenciação de cromossomos sexuais. Estes resultados evidenciam que, apesar da origem comum proposta para o sistema sexual ZW em aves, esses cromossomos seguiram diferentes trajetórias evolutivas em cada espécie, indicando uma alta plasticidade para a diferenciação cromossômica sexual neste grupo. Este trabalho é o primeiro passo para esclarecer o papel das sequências satélites e microssatélites na diferenciação de cromossomos sexuais. / The characterization of the amount and distribution of the repetitive DNA fractions in genomes assists in the understanding of their chromosomal organization. The Birds are characterized by presenting a low proportion of repetitive DNA when compared to other classes of Vertebrates. However, the order Piciformes stands out for having a higher percentage of these sequences compared to other birds. The objective of this study was to determine the distribution of different types of repetitive sequences in the genome of three species of the family Picidae, Colaptes melanochloros (2n = 84), Colaptes campestris (2n = 84) and Melanerpes candidus (2n = 64) by fluorescence in situ hybridization (FISH) with 18S, telomeric (TTAGGG) and microsatellite rDNA probes. The results showed, in these three species, the sexual chromosome Z as the largest of the complement, this fact is due to the accumulation of different sequences of microsatellites. However, the W chromosome of C. melanochloros, which is totally heterochromatic, did not show accumulation of these sequences. The ribosomal sites are organized on a pair of chromosomes with a secondary constriction and this had the accumulation of the sequence (CGG)10 in the three species. The telomeric probes showed markings in the terminal regions of the chromosomes and interstitial markings on some macrochromosomes. Interstitial markings indicate fusions between chromosomes or the accumulation of repetitive sequences similar to the telomeric ones. With the microsatellite probes the same pattern of hybridization was identified in the Colaptes species, distinct pattern between Colaptes and M. candidus. Our FISH analyzes showed several amplified microsatellite sequences on the Z chromosome in the three species analyzed, which may explain the fact that this is the largest element of the karyotype and that its genome contains the largest number of repetitive sequences compared to other groups of Birds. Interestingly, none of the sequences were found to be accumulated on the W chromosome, although they play an important role in the differentiation of sex chromosomes. These results show that, despite the common origin proposed for the ZW sexual system in birds, these chromosomes followed different evolutionary trajectories in each species, indicating a high plasticity for the sexual chromosome differentiation in this group. This work is the first step to clarify the role of satellites and microsatellite sequences in the differentiation of sex chromosomes.

Made available in DSpace on 2017-07-21T19:59:50Z (GMT). No. of bitstreams: 1 Helena Pistune.pdf: 2191433 bytes, checksum: d64e158aacb1b4070276bdafd47d03ce (MD5) Previous issue date: 2010-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The origin, maintenance and frequency of B chromosomes present in some population of Astyanax have been arousing interest of cytogeneticists groups about a possible beneficial or deleterious effect for the species. These chromosomes generally shows intra and interindividual numerical variation. The aim of this work was to perform a cytogenetical analysis on the origin and molecular differentiation of the B chromosomes of Astyanax scabripinnis from “Das Pedras” stream, Campos do Jordão, SP. The analysis showed a diploid number of 50 chromosomes, karyotypic formula 6m, 22sm, 10st and 12a, besides the intra and interindividual variation of a B macrochromosome. The heterochromatic B macrochromosome shows shape and size similar to the major pair of the A complement (1st metacentric pair). In spermatogonial metaphase was possible to observe three great metacentric chromosomes in cells with 2n=51 chromosomes. However, was observed in pachitene cells with 26 chromosomical elements only one great bivalent, showing that the B chromosome performs arm-to-arm pairing. In situ localization with a B chromosome probe showed this element entirely marked, in addittion to small pericentromerics and terminal sites in the A chromosomes complement, among them, the 24th pair. Besides, the As51 probe showed localization in the major part of the B chromosome, and also 14 autossomical sites. Between the As51 sites there is an interstitial marking in the long arm of the 24th acrocentric pair. On the other hand, the localization of the repetitive sequences Cot-1 DNA, obtained from individuals without B chromosome, showed signals on the pericentromeric and terminal regions of almost all chromosomes, including the B chromosome and the 24th pair. No rDNA site was observed on B chromosome. This data corroborate the hypothesis of the origin of B chromosome through the formation of an isochromosome of the 24th acrocentric pair. Even, the cytogenetical analysis in meiotic cells shows that the B chromosome performs arm-to-arm pairing, a mechanism that can contribute to the transposition of sequences, rising thus the molecular differentiation of the B chromosome in relation to the A complement. Therefore, this work contributes to a better comprehension of the molecular nature of the B chromosome in A. scabripinnis, as well as its origin and evolutionary trends. / A origem, manutenção e frequência dos cromossomos B presentes em algumas populações de Astyanax têm despertado interesse de grupos de citogeneticistas sobre um possível efeito benéfico ou deletério para a espécie. Estes cromossomos geralmente apresentam variações numéricas intra e interindividuais. O objetivo deste trabalho foi realizar uma análise citogenética da origem e diversificação molecular do cromossomo B de Astyanax scabripinnis, proveniente do córrego das Pedras, Campos do Jordão-SP. As análises mostraram um número diplóide de 50 cromossomos, fórmula cariotípica: 6m, 22sm, 10st e 12a, além da variação intra e interindividual de um macrocromossomo B. O macrocromosomo B heterocromático tem forma e tamanho similar ao maior par do complemento A (1o par metacêntrico). Em metáfase espermatogonial foi possível observar três metacêntricos grandes em células com 2n=51 cromossomos. Já em paquíteno com 26 elementos cromossômicos foi observado apenas um bivalente grande, evidenciando que o cromossomo B realiza pareamento braço a braço. A localização in situ da sonda cromossomo B evidenciou este elemento totalmente marcado, além de pequenos sítios pericentroméricos e terminais nos cromossomos do complemento A, entre eles o par 24. Já a sonda As51 mostrou localização na maior parte do cromossomo B, além de 14 sítios autossômicos. Entre os sítios As51 está uma marcação intersticial do braço longo do par acrocêntrico 24. Por sua vez, a localização das sequências repetitivas Cot-1 DNA, obtidas de exemplares sem cromossomo B, mostraram sinais nas regiões pericentroméricas e terminais de quase todos os cromossomos, incluindo o cromossomo B e o par 24. Nenhum sítio de rDNA foi observado no cromossomo B. Esses dados corroboram a hipótese da origem do cromossomo B a partir da formação de isocromossomo do par acrocêntrico 24. Ainda, a análise citogenética em meiose evidencia que o cromossomo B realiza pareamento braço a braço, mecanismo que pode contribuir para a transposição de sequências, auxiliando assim a diversificação molecular do cromossomo B em relação ao complemento A. Dessa forma, este trabalho contribui para uma melhor compreensão da natureza molecular do cromossomo B em A. scabripinnis, bem como de sua origem e suas tendências evolutivas.

At the commencement of this study, some sensitive overseas markets were rejecting chilled vacuum-packed New Zealand lamb due to higher than expected total viable counts, and counts of Enterobacteriaceae, a family of bacteria used to indicate sanitary condition. Of the many factors that influence the bacterial composition of chilled lamb in the overseas marketplace, the meat producer can only exert significant control over: Hygiene, ensuring the bacterial viable count on the meat prior to packaging is as low as possible, and comprised of as few species as possible that are capable of anaerobic growth at chilled meat temperatures. Maintaining the pH of the meat within acceptable limits, by careful animal selection and minimal pre-slaughter stress. Refrigeration temperatures, through rigorous maintenance of the cold-chain. The type of preservative packaging used, which is often limited by regulation in the marketplace. Initial work established that the bacterial microbiota present on the meat contact surfaces in the butchering facilities at some premises, in particular conveyor belting, was excessive and comprised of species that contributed to the high counts on the meat reported above. As a means of improving the hygiene of this process, this study investigated the hypothesis that some species of bacteria were able to form biofilms on the conveyor belt contact surfaces, becoming reservoirs for cross-contamination. This hypothesis was not been proven by this work; the results showing that biofilms were not present and that adequate hygiene of these surfaces instead depends on the ability to remove all meat-based residues from them at the completion of each day's processing. For premises operating interlocking belts from one manufacturer (Intraloxreg), a clean-in-place system is now available that is able to achieve this. Premises operating conventional disinfectant and water sanitisation of either continuous or interlocking belts must ensure that meat residue is completely removed before disinfection. The majority of New Zealand meat industry premises can now demonstrate that their hygienic processes in this area are under control. The microbiota of conveyor belting in this study was found to consist of bacteria from five taxonomic groups; the Flavobacteriaceae, the Actinomycetales, the Bacillus/Clostridium group, and the alpha and gamma branches of the Proteobacteria. The genera present on belts from premises whose hygiene was found to be in control did not contain species known to cause food-borne disease or spoilage of vacuum packaged meats. The bacterial viable count remains the most effective method available at this time for monitoring conveyor belt hygiene. Attempts to develop a monitoring system based on microscopy of an in-situ sampling device were unsuccessful due to an inability to penetrate the meat residue matrix. Denaturing Gradient Gel Electrophoresis (DGGE) may offer an alternative for rapid investigation of diversity, but further work is required before this can be validated for routine use.

Bacteria play important roles in the transformation of nutrients in wetlands, but few studies have examined parameters affecting variation in bacterial community composition between wetlands. We compared the composition of eubacterial and denitrifying bacterial biofilm communities in 32 agricultural constructed wetlands in southern Sweden, and the extent to which wetland environmental parameters could explain the observed variation. Structure and richness of the eubacterial 16S rRNA gene and three denitrifying bacterial enzyme genes (nirK, nirS and nosZ), analysed by molecular fingerprinting methods, varied among the constructed wetlands, which could be partly explained by different environmental parameters. Results from the enzyme gene analyses were also compared to determine whether the practice of using a single denitrifying bacterial gene could characterize the overall community composition of denitrifying bacteria. We found that nirK was more diverse than both nirS and the nosZ, and the band structure and richness of the three genes were not related to the sam environmental parameters. This suggests that using a single enzyme gene may not suffice to characterize the community composition of denitrifying bacteria in constructed agricultural wetlands. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>

Mathematical modeling has been important in the study of biology. Two main challenges with modeling biological problems are the lack of quantitative data and the complexity of biological problems. With the invention of new techniques, like single molecule transcript counting, very quantitative gene expression measurements at the level of single transcript in individual cells can now be obtained. Biological systems are very complex, involving many reactions and players with unknown reaction rates. To reduce the complexity, scientists have often proposed simplified phenomenological models that are tractable and capture the main essence of the biological systems. These simplified models allow scientists to describe the behavior of biological systems with a few meaningful parameters. In this thesis, by integrating quantitative single-cell measurements with phenomenological modeling, we study the (1) roles of Wnt ligands and receptors in sensing and amplification in Caenorhabditis elegans’ P cells and (2) regulation of rDNA transcription in Saccharomyces cerevisiae. The initiation of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified by intracellular signaling. It is challenging to determine the role of proteins towards sensing and amplification as these two processes are intertwined. We integrated quantitative single-cell measurements with phenomenological modeling to determine the roles of Wnt ligands and receptors in sensing and amplification in the P cells of Caenorhabditis elegans. By systematically exploring how P cell polarity is altered in Wnt ligand and receptor mutants, we inferred that ligands predominantly affect sensing, whereas receptors are needed for both sensing and amplification. Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus nontranscribing repeats (OFF) but do not provide any dynamical information. By using the single molecule transcript counting technique complemented with theoretical modeling, we determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions.

Η πραγματική ταξινομική κατάσταση των πληθυσμών του γένους Ligidium, κυρίως αυτών που κατανέμονται στη νότια Ελλάδα, δεν είναι ξεκάθαρη. Αυτό συμβαίνει γιατί η απόληξη του δεύτερου ενδοποδίτη του πλεοποδίου των αρσενικών, που αποτελεί σημαντικό διαγνωστικό χαρακτήρα, ποικίλλει και η εύθραυστη δομή της μπορεί εύκολα να καταστραφεί και κατ’ επέκταση να οδηγήσει σε μια μη ακριβή περιγραφή της κατάστασης αυτού του χαρακτήρα. Οι παλαιότερες μελέτες των φυλογενετικών σχέσεων των ισοπόδων του γένους Ligidium, με χρήση μοριακών δεικτών είναι ολιγάριθμες. Πιο συγκεκριμένα, για τη μελέτη πληθυσμών του γένους Ligidium από τον ελλαδικό χώρο έχει γίνει μόνο μια μελέτη στην οποία χρησιμοποιήθηκε η τεχνική της αλληλούχισης του DNA. Η παρούσα μεταπτυχιακή εργασία αποτελεί την πιο εκτεταμένη φυλογενετική ανάλυση μέσα στο γένος Ligidium. H έρευνα επιλέχθηκε να γίνει σε επίπεδο ατόμων συλλέγοντας πολλούς πληθυσμούς από διαφορετικές περιοχές, οι οποίοι ανήκουν είτε στο ίδιο είτε σε διαφορετικά ποτάμια συστήματα. Συγκεκριμένα χρησιμοποιήθηκαν μοριακοί δείκτες από 26 πληθυσμούς ισοπόδων, οι οποίοι καλύπτουν σχεδόν όλο το φάσμα των ειδών, που ανήκουν στo γένος Ligidium και διαβιούν στον ελλαδικό χώρο. Η πειραματική προσέγγιση περιελάμβανε τον πολλαπλασιασμό αλληλουχιών με τη μέθοδο της αλυσιδωτής αντίδρασης πολυμεράσης (PCR), τον προσδιορισμό της αλληλουχίας τους, και τη στατιστική και φυλογενετική ανάλυσή τους. Οι αλληλουχίες που χρησιμοποιήθηκαν προέρχονται από τους πυρηνικούς γονιδιακούς τόπους ITS1, 5.8s, ITS2 οι οποίοι είναι από τους πιο ευρέως διαδεδομένους μοριακούς δείκτες για μελέτες σε επίπεδο πληθυσμών και ειδών και δεν έχουν προηγουμένως μελετηθεί για άλλα χερσαία ισόποδα. Οι αλληλουχίες επεξεργάστηκαν με τρεις μεθόδους φυλογενετικής ανάλυσης οι οποίες είναι η μέθοδος σύνδεσης γειτόνων (Neighbor-Joining), η Μέθοδος Μέγιστης Φειδωλότητας (Maximum Parsimony) και η Μπεϊεσιανή Συμπερασματολογία (Bayesian Inference). To τελικό σύνολο των στοιχισμένων αλληλουχιών, μετά την αφαίρεση τμημάτων από την αρχή και το τέλος τους, είχε μήκος 614 βάσεων. Σε γενικές γραμμές τα αποτελέσματα που προέκυψαν από τη χρήση των εν λόγω πυρηνικών δεικτών και των τριών φυλογενετικών μεθόδων (BI, MP, NJ), τόσο ως προς τα δέντρα όσο και ως προς τις γενετικές αποστάσεις, δεν θεωρούνται ασφαλή για την εξαγωγή ικανοποιητικών συμπερασμάτων. Επίσης πραγματοποιήθηκε σύγκριση αποτελεσμάτων προηγούμενων μελετών με αυτά που προκύπτουν από τη δική μας έρευνα, καθώς και αξιολόγηση της αξιοπιστίας των μοριακών δεικτών που χρησιμοποιήθηκαν. / Terrestrial isopods (suborder Oniscidea) form a monophyletic group within the crustacean order Isopoda that has conquered almost all terrestrial environments. The family Ligiidae, forming the sister group to all the other Oniscidea (with the possible exception of Tylidae) consists of five genera. The genus Ligidium is the most species-rich genus within Ligiidae and consists of 47 species distributed throughout the northern hemisphere, from western Europe to Japan and North America. The taxonomy of the genus has hitherto been based on a few morphological characters most of which exhibit high intrapopulation variation. As a consequence, the current taxonomy of Ligidium is problematic and the validity of described species cannot be taken for granted. Molecular data for only a few species have been used in broader phylogenetic analyses within terrestrial isopods In the present study we perform the most extensive phylogenetic analysis within the genus Ligidium. We attempt to clarify the taxonomic status of the various populations and try to identify geographical patterns in genealogical affinities that might help us reconstruct the evolutionary history of the respective species. We used three nuclear ribosomal gene segments (ITS1, 5.8s, ITS2) for the phylogenetic analysis of Ligidium populations, which were collected from several locations in Greece, representing almost all species reported from this region. For comparative reasons, we have also included one population from each of the two widespread European species, L. hypnorum, Cuvier, 1792 and L. germanicum, Verhoeff, 1901. Since the taxonomy within the genus is still ambiguous, and because most morphological characters are not dependable for species identification, we chose to perform the analysis at the individual level, sampling several populations from different locations that belong either to the same or to different river systems. Fragments of the three rDNA genes (ITS1, 5.8s, ITS2) were amplified using the polymerase chain reaction (PCR) technique and then individual sequences were determined via automated sequencing. We compare the results which occurred from our study with those of previous ones and we evaluate the plylogenetic utility of the molecular markers that we used. In general the results that came out by the analyses of three following phylogenetic methods Neighbor joining (NJ), Maximum parsimony (MP) and Bayesian Inference (BI), are not considered to be safe for making generalized conclusions.

The 35S rDNA gene clusters on the X and Y chromosomes of Drosophila melanogaster are repeats of approximately 150 to 225 copies. Each are transcribed as a single unit by RNA Polymerase I and modified into the 18S, 5.8S, 2S and 28S ribosomal rRNAs. Reduction in the array copy number results in a bobbed phenotype, characterized by truncated bristles and herniations of abdominal cuticle, due to a decrease in protein production. In some copies within the arrays, R1 and R2 retrotransposable elements are inserted in a conserved region of the 28S gene which represses the transcription of a functional rRNA. Inserted arrays are transcribed at very low levels, but it is not clear how they are identified for repression. Similarly, a subset of uninserted arrays are silenced, and the epigenetic mechanism controlling how this decision is made it is also unknown. The CCCTC binding factor (CTCF) is a boundary element binding protein and a transcriptional regulator found in the nucleolus of differentiated mammalian cells, whose localization requires poly (ADP-ribosyl)ation. We investigated whether CTCF might be involved in the regulation of rDNA expression in Drosophila. Our data show that CTCF is found at the nucleolus of both polytene and diploid nuclei, and we have identified binding sites in the 28S gene, R1 and R2 elements by a bioinformatic approach. ChIP data indicate that CTCF binds only to the site in the R1 retrotransposon. Reduction of CTCF or members of the poly(ADP-ribosyl)ation pathway by RNAi in S2 cells causes an increase in the amount of 35S rDNA gene, R1, and R2 transcripts. In flies, CTCF and PARG mutant alleles show disrupted nucleoli and increased rRNA transcripts. Mutant alleles of CTCF suppress variegation of a P-element inserted in a 35S rDNA array, but not of elements inserted elsewhere in the genome. Consistent with a role for CTCF in rRNA regulation, we found that during oogenesis CTCF is recruited to the nucleolus of nurse cells at early stages when the demand of ribosomes is low and it leaves this compartment in later stages when the cell increases rRNA production. We conclude from these studies that CTCF acts as a regulation of rDNA transcription by RNA polymerase I.

Phytopathogenic bacterium, Xylella fastidiosa, causes a number of economically important diseases, including Pierces disease (PD) of grape and bacterial leaf scorch (BLS) of a number of landscape trees. In Kentucky (KY), BLS affects a number of shade trees including many oak and maple species. In 2001, PD was diagnosed in grapevines in western KY. Xylella fastidiosa is also detected in many asymptomatic landscape plants and grasses. It was the goal of this research to identify hosts of X. fastidiosa around KY and use phylogenetic analysis to compare sequences of the 16S rDNA and gyrase B (gyrB) genes between samples. This research tests the hypothesis that sequence comparison can identify asymptomatic hosts and vectors that serve as a source of inoculum for pathogenic strains of X. fastidiosa. Plant collections were done in urban areas of KY between 2002 and 2004 and samples were tested for the presence of X. fastidiosa by ELISA and PCR. A number of symptomatic and asymptomatic plants were found to be hosts. Primer sets specifically developed for X. fastidiosa were used to amplify part of the 16S rDNA and the gyrB gene from DNA extracted directly from plant tissue. Sequence data from these specifically amplified products were assembled using Phrap, aligned with ClustalW, then phylogenetic analysis was done with Paup 4.0b10 beta. Comparisons with strains outside of Kentucky were also done using X. fastidiosa sequence obtained from NCBI. Maximum parsimony (MP) trees from the 16S rDNA showed a clade of sequence from oak and grass samples that is an outgroup to sequence from NCBI and other samples in this study. According to BLAST, sequences in this outgroup clade seem to be more closely related to the genera Xanthomonas or Stenotrophomonas than Xylella. However, the gyrB gene MP tree showed sequence from three of the samples that were part of this outgroup clade as being closely related to those X. fastidiosa sequences that are part of the ingroup of both 16S rDNA and gyrB trees. The topology difference between the 16S rDNA and gyrB trees suggest there may have been recombination in the genomic region containing one of these genes.