This is the first research by using suppression subtractive hybridization (SSH) to analysis the gene expression in marine macroalga Ulva fasciata Delile against excess copper toxicity, and it gives us a comprehensive understanding of the tolerant mechanism while macroalgae face to the excess copper. Suppression subtractive hybridization was used to identify genes differentially expressed following exposure to 50 £gM CuSO4 for 6- 12h in a marine macroalga Ulva fasciata Delile. In this work, 69 genes were identified, of which 55 were up-regulated and 14 were down-regulated. According to the database of Gene Ontology (GO), these genes were classified into 10 categories as follows: 1. Transcription; 2. Translation, ribosomal structure and biogenesis; 3. Posttranslational modification, protein turnover, chaperones; 4. Photosynthesis; 5. Cell redox homeostasis; 6. Stress; 7. Metabolism; 8. Energy production and conversion; 9. Transport; 10. Function unknown. According to the results, we suggest that the responses of U. fasciata against excess copper toxicity are mainly through increase of the energy production for providing sufficient energy to many metabolic pathways, and control of the Fe homeostasis and redox form of thiol groups for maintaining the cellular redox homeostasis, moreover, expression of photosynthetic genes for letting the photosynthesis work. In addition, to scavenge the ROS is by expression of stress-related genes, meanwhile, the proteins, DNA and lipids damaged by ROS (reactive oxygen species) and copper are repaired by expression of the other categorical genes. Over and above, the genes expressing in the metabolism category might maintain the amino acids homeostasis and increase the purine content, and subsequently increase the tolerant capacity of U. fasciata against excess copper toxicity. In addition, the concentrations of antioxidants and the activities and gene expression of antioxidant enzymes were determined in Ulva fasciata Delile by a 4-day exposure to 0, 5, 10, 20 and 50 £gM CuSO4. These results demonstrate that the maintenance of antioxidant homeostasis and the induction of activities of antioxidant enzymes via enhanced gene expression are used by U. fasciata to cope with the Cu-induced oxidative stress, but the defense capacity cannot sufficiently alleviate oxidative damage occurring under the condition of higher Cu concentrations. Moreover, according to the results from the expression of genes involved in the control of redox homeostasis and antioxidant defense was studied in macroalga Ulva fasciata Delile in response to CuSO4 (5 and 50 £gM) and ROS (H2O2 and O2£»-), we suggest that ROS involved in up-regulation of antioxidant defense-related genes and the expression of genes of antioxidant defense enzymes and UfMsrA (methionine sulfoxide reductase A) are associated with long-term adaptation of U. fasciata to Cu excess and transcription of redox- related genes and UfGr (glutathione reductase) is up-regulated for short-term acclimation. Promoters play a key role in regulating gene expression. Based on the analysis of cis-acting elements on UfMsr promoters, we suggested that the signal transduction pathway of copper stress in U. fasciata is related to that of other stresses and of defense-related plant hormones, however, Ca2+ and calmodulin might participate in it. To sum up, U. fasciata could resist oxidative damage caused by excessive copper through the regulation on the molecular level.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Differential gene expression plays a key role in developmental pathways within an organism. Examples of such pathways include primary sex determination signaling and the formation of secondary sexual characteristics. This dissertation is focused on the use of suppression subtractive hybridization (SSH) to identify genes that are differentially expressed and involved in some aspect of sexual development in the Mediterranean fruit fly (medfly), Ceratitis capitata. In the course ofthis project, a method for sexing individual specimens from pre-adult stages was developed. This method was used to collect sex-specific RNAs at different developmental stages for use in SSH. A total of25 subtraction products were obtained across all the stages examined. Analysis of these products revealed that approximately half were similar to cytoplasmic ribosomal proteins and mitochondrial ribosomal RNA The remaining products represent putative medfly homologs of other previously identified genes or potentially novel genes One ofthe subtraction products, representing a potentially novel gene, was characterized in detail. This gene, named mapotge', represents a novel medfly gene that appears to encode a polypeptide of 299 amino acids. The N-terminus of this polypeptide contains a BTB-POZ domain. This domain functions as a protein-protein interaction motif found in a wide range of organisms from humans to Drosophila that mediates protein dimerization and oligomerization. The temporal expression pattern of mapotge' was determined using RT-PCR and Northern blot analysis. These revealed that the transcript is expressed throughout embryogenesis in both females and males, and in adult females that are > 0.5 days post-eclosion. Minimal expression is observed in female and male third instar larvae, early pupae, and in adult males. Studies were also initiated to characterize the representation of additioual sequences containing a BTB-POZ domain in the medfly genome. This was performed using Southern blot analysis and degenerate primers for the polymerase chain reaction (PCR). These results indicate the presence of at least three sequences in the medfly, in addition to 'mapotge', that contain a BTB-POZ domain. Potential evolutionary relationships ofthe BTB-POZ domain sequences from the medfly and other insect species were also analyzed.

Orientadores: Gonçalo Amarante Guimarães Pereira, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T10:30:44Z (GMT). No. of bitstreams: 1 Leonardo_FlaviaCosta_D.pdf: 6145744 bytes, checksum: 199799932d8959740a6ec52e8963bcb8 (MD5) Previous issue date: 2010 / Resumo: Os cupins são insetos pertencentes à Ordem Isoptera, com mais de 3.000 espécies descritas. São importantes agentes de degradação de madeira e compostos celulósicos em geral. Entretanto, essas mesmas características tornam algumas das espécies danosas por destruírem estruturas de edificações, sendo responsáveis por prejuízos da ordem de milhões de dólares. Uma destas espécies-praga é o Coptotermes gestroi. Originária do sudeste asiático foi introduzida no Brasil no início do século XX, estabelecendo-se ao longo daregião costeira, mas com uma clara expansão para o interior do país. O ciclo de vida de C .gestroi inicia com a eclosão do ovo, a larva originada pode se transformar tanto em um operário (linhagem áptera) como em uma ninfa (linhagem ninfal), a qual pode originar um rei ou uma rainha. Além disso, o operário pode se transformar em soldado, sob indução do hormônio juvenil. Assim sendo, o objetivo do presente trabalho foi a avaliação da expressão gênica nas diversas castas e estágios de vida de C.gestroi visando a identificação de genes relacionados ao ciclo de vida e com possível aplicação biotecnológica. Para isso,foi deita a análise de uma biblioteca de cDNA de cabeça de operários previamente construída. Para melhor compreender genes diferencialmente expressos entre as castas,outras quatro bibliotecas subtrativas foram confeccionadas. Alguns dos genes identificados foram genes selecionados para a validação pelo método de PCR em tempo Real. O seqüenciamento de segunda geração (454) foi realizado com amostras de soldados e operários e mais de 110.000 transcritos foram identificados. Dentre eles destacam-se as Hexamerinas I e II, a epóxido hidroláse do hormônio juvenil e genes relacionados à musculatura. Quanto aos genes com potencial biotecnológico dezenove enzimas envolvidasna quebram da lignocelulose foram identificadas e serão investigadas para confirmar o papel destas neste processo. / Abstract: Termites are insects belonging to the Order Isoptera, with more than 3,000 describedspecies. These insects are important wood and cellulosic compounds degraders. However, these same characteristics make some of them harmful species to mankind, by destroying buildings, accounting for million dollars losses. One of these pest species is Coptotermesgestroi. Originated from Southeast Asia, it was introduced in Brazil in the early twentieth century, establishing colonies along the coastal region, but with a clear expansion into thecountry. The life cycle of C. gestroi works as follows: after hatching from the egg, thelarvae can become either a worker (apterous lineage) or a nymph (nymphal lineage), whichcan lead to a king or a queen. In addition, the worker can become a soldier, under inductionof juvenile hormone. Therefore, the purpose of this study was the evaluation of gene expression in different castes and developmental stages of C.gestroi in order to identifygenes related to different life cycles and biotechnological potential. To accomplish this, weanalized a workers head cDNA library previously prepared. To better understand genes differentially expressed between castes, other four subtractive libraries were constructed. Some of the genes identified were selected for validation by Real Time PCR. Secondgeneration sequencing (454) was performed with samples of workers and soldiers and morethan 110,000 transcripts were identified. Among them stand out Hexamerins I and II, thejuvenile hormone epoxide hydrolase and genes related to muscle formation. As for geneswith biotechnological potential, nineteen enzymes involved in lignocellulose's break downwere identified and will be investigated to confirm their role in this process. / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra x Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 single-tons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription-polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

Dermatófitos são fungos filamentosos com a habilidade de invadir substratos queratinizados e causar dermatofitoses em humanos e animais, penetrando profundamente apenas em hospedeiros imunocomprometidos. Trichophyton rubrum é um fungo antropofílico e cosmopolita, o mais comum agente de micoses superficiais, que usa componentes celulares como proteínas e lipídeos após uma específica regulação de sua expressão gênica governada pelo pH ambiente e sensoriamento celular. A virulência de T. rubrum é relacionada com a secreção de enzimas proteolíticas, um importante fator determinante na invasão, utilização e subseqüente disseminação através do estrato córneo. O objetivo desse estudo foi identificar através de Hibridização Subtrativa Supressiva (SSH) genes de T. rubrum preferencialmente expressos durante o crescimento na presença de queratina e lipídeos, quando T. rubrum degrada fontes de carbono tipicamente encontradas em células epidérmicas. Inicialmente, nós avaliamos as mudanças no pH extracelular durante seu crescimento em queratina e lipídeo (depois de 6, 12, 24, 48, 72 h e 7 dias) em pH inicial 5,0, onde foi observado um gradual aumento do pH basal sob ambas as condições de teste, comparado com a condição glicose (controle). Também identificamos 576 transcritos de T. rubrum diferencialmente expressos por SSH usando conídios cultivados por 72 h em queratina como teste e em glicose como controle. Os genes de T. rubrum ativados codificam proteínas putativas que foram validadas por cDNA dot-blot e northern blot, mostrando similaridade com proteínas fúngicas envolvidas no metabolismo básico, crescimento e virulência, p. ex., transportadores ABC-MDR, MFS e ATPase de cobre, permease, NIMA interactive protein, poliproteína Gag-Pol, fatores de virulência subtilisinas serino-proteases (Sub 3 e 5) e metaloproteases (Mep 3 e 4) e Hsp30. Adicionalmente, entre os 762 clones obtidos na biblioteca da condição lipídeo (72 h), 80 transcritos superexpressos foram confirmados por cDNA dot-blot, revelando 14 unigenes similares à proteínas de vários organismos patogênicos, como glicoproteína 43 kD, transportador MDR, proteína G, quitina sintase e serino/treonina fosfatase. Transcritos do gene TruMDR2, codificador de um transportador ABC, foi isolado tanto na presença de queratina quanto em lipídeo, e a análise da linhagem mutante TruMDR2 de T. rubrum mostrou uma redução na atividade infectante, caracterizada pelo baixo crescimento em unhas humanas comparada com o tipo selvagem. A alta expressão de transportadores por T. rubrum em condições que mimetizam a infecção e a redução na virulência de TruMDR2 durante o modelo in vitro sugerem que transportadores estão envolvidos na patogenicidade de T. rubrum. Outro linhagem mutante (pacC-1) com um nocaute no gene pacC que codifica um fator de transcrição regulado pelo pH local, mostrou a expressão de proteases (Sub 3 e 5 e Mep 4) diminuída após o crescimento em queratina em comparação com o tipo selvagem em análises de northern blots. Essas proteases tem uma atividade ótima em pH alcalino, e nossos resultados indicam uma regulação defectiva do gene pacC de T. rubrum na ativação de proteases. Em conclusão, através do uso de SSH foi possível identificar genes de T. rubrum ativados após tratamentos específicos, o que sugere a importância dos mesmos na interação dermatófito-hospedeiro, instalação e manutenção da doença. Esses resultados disponibilizam novos dados sobre T. rubrum que levarão a um melhor entendimento dos mecanismos moleculares envolvidos no crescimento, metabolismo geral e patogenicidade, e também auxiliar na identificação de novos e efetivos alvos de drogas para dermatófitos. / Dermatophytes are a group of fungi filamentous that have the ability to invade keratinized substrates, causing dermatophytosis in humans and animals and only penetrate deeper if the host is immunocompromised. Trichophyton rubrum is an anthropophilic and cosmopolitan fungi, the most common agent of superficial mycoses, which uses cell components such as proteins and lipids after a specific regulation of its gene expression governed by pH environment and sensing cell. The virulence T. rubrum is related to secretion of proteolytic enzymes, an important factor determinant in the invasion, utilization and subsequently dissemination through the stratum corneum. The aim of this study was to indentify by Suppression Subtractive Hybridization (SSH) T. rubrum genes preferentially expressed during growth in the presence of keratin and lipids, upregulated when this fungus degrades carbon source typically found at epidemic cells. Initially, this work evaluated the changes in the extracellular pH during its growth in keratin and lipid (after 6, 12, 24, 48, 72 h and 7 days) at initial pH 5.0, where we observed a gradual increase of basal pH under both tests when compared to glucose condition (control). Also, we identified 576 T. rubrum transcripts differentially expressed by SSH using conidia cultivated for 72 h in keratin as tester, and in glucose as driver. The T. rubrum genes upregulated encode putative proteins that were validated by cDNA dot-blot and northern blot, showing similarity to fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, permease, NIMA interactive protein, Gag-Pol polyprotein, virulence factors serine-protease subtilisins (Sub 3 and 5) and metalloproteases (Mep 3 and 4) and Hsp30. Additionally, among the 762 clones obtained in a library of lipid condition (72 h), 80 over-expressed transcripts were confirmed by cDNA dot-blot, revealing 14 unigenes similar to proteins of several pathogenic organisms, like glicoprotein 43 kD, MDR transporters, G protein, chitin synthase and serine/threonine-protein phosphatase. Transcripts of TruMDR2 gene, encoding an ABC transporter in T. rubrum, were isolated in the presence of keratin and lipid, and the examination of TruMDR2 mutant T. rubrum showed a reduction in infecting activity, characterized by low growth in human nails compared to wild-strain. The high expression of transporter by T. rubrum in conditions that mimetize the infection and the virulence reduction of TruMDR2 in an in vitro model suggests that transporters are involved in T. rubrum pathogenicity. Another mutant, pacC-1 with a knockout in PacC gene that encodes a transcription factor regulated by local pH, showed the expression of proteases (Sub 3, Sub 5 and Mep 4) decreased after growth in keratin (72 h) in comparison to wild-strain in northern blot analyzes. These proteases have an optimal activity in alkaline pH, and our results indicate a defective regulation of T. rubrum pacC gene in the activation of proteases. In conclusion, by means of SSH to identify genes upregulated in T. rubrum after specific treatments, their importance in the dermatophyte-host interaction, installation and maintenance in the disease is suggested. These results provide new insights about T. rubrum that will contribute to a better understanding of molecular mechanisms about the growth, metabolism and pathogenicity, and may also aid in the identification of novel effective drug targets for dermathophytes.

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No. of bitstreams: 2 Tese - Saulo José Linhares de Siqueira - 2012.pdf: 3918109 bytes, checksum: cfb7931590a50ed74838d6c8cefab1ee (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-03-30 / Species of Trichoderma are commercially applied as biological control agents and are antagonists of important plant pathogenic fungi (as Rhizoctonia, Sclerotinia and Fusarium species) due to its mycoparasitic characteristics. Research has been performed to have a better comprehension of molecular aspects of the biocontrol mechanisms performed by Trichoderma and to find isolates with high antagonistic potential against plant pathogens. In the present study the expression of mycoparasitism-related genes was performed T. asperellum T00 and T. harzianum ALL42 (Enzimologia group ICB/UFG fungal collection) that have great potential as biocontrol agent. Each chapter of this work refers to one of the species studied. In Chapter 1 T. asperellum isolate T00, known to produce high levels of cell wall degrading enzymes, has its β-1,3-glucanases enzymes and genes (tag83 and tag27) studied. The gene tag27 was cloned and characterized and codes to an 27kDa endo-β-1,3glucanase with and 285 aminoacids and 96% similar to a glucanases from T.atroviride. The enzyme was detected when T. asperellum was grown in Rhizoctonia solani or Sclerotinia sclerotiorum cell wall-containing media but not in Fusarium oxysporum cell wall-containing media. The tag83 and tag27 genes was repressed in media containing glucose as carbon source and upregulated in cell wall containing media and during plate confrontation tests with pathogenic fungi. Chapter 2 shows T. harzianum isolate ALL42 genes involved in mycoparasitism against R. solani or S. sclerotiorum detected using subtractive hybridization approach. T. harzianum was grown with R. solani or S. sclerotiorum cell wall as carbon source and the RNA used both as tester and driver in each of two subtractive library constructed. Sequencing analysis resulted in 47 genes related with growth in R. solani cell wall media and 114 genes related with growth in S. sclerotiorum cell wall media. To confirm the obtained data, 18 genes were tested by quantitative real time RT-PCR and 9 were differentially expressed in the same condition of the library they were detected. Five of these genes were also differentially expressed during plate confrontation assay with the respective pathogen, two of them expressed during contact with R. solani (cutinase and alginate lyase) and 3 during contact with S. sclerotiorum (hsp98, serin endopeptidase and a hypotetic gene). The results presented in this study provides additional information about the role of 1,3glucanase genes in mycoparasitism and of other genes related to antagonism against specific pathogens, providing helpful insights in the mechanism of biocontrol performed by Trichoderma. / Espécies do gênero Trichoderma são eficientes antagonistas de fungos fitopatogênicos, como as espécies Rhizoctonia, Sclerotinia e Fusarium, e são comercializados como agentes de controle biológico principalmente por sua característica de micoparasita. Muitos estudos têm sido feitos para compreender as bases moleculares dos mecanismos de biocontrole de Trichoderma e também para encontrar espécies com alto potencial de antagonismo contra fitopatógenos. O objetivo deste trabalho foi analisar a expressão de genes relacionados ao micoparasitismo de T. asperellum T00 e T. harzianum ALL42 (Enzimologia ICB/UFG) que possuem potencial para uso como agente de biocontrole. Este trabalho foi dividido em dois capítulos. O Capítulo 1 se refere ao fungo T. asperellum T00 e suas β-1,3-glicanases (TAG83 e TAG27) que degradam componentes da parede celular de fungos fitopatógenos. O gene tag27 codifica para uma endo-β-1,3glicanase de 27kDa que possui 285 resíduos de aminoácidos e apresentou 96% de similaridade com uma enzima de T. atroviride. A enzima foi secretada em meio de cultura contendo parede celular de Rhizoctonia solani e Sclerotinia sclerotiorum, mas não em meio com parede de Fusarium oxysporum. A expressão dos genes tag83 e tag27 foi reprimida na presença de glicose e ativada tanto na presença de parede celular dos fitopatógenos quanto durante o contato entre os fungos em placa. O Capítulo 2 trata do fungo T. harzianum isolado ALL42 e de genes identificados pela técnica de hibridização subtrativa relacionados especificamente ao biocontrole contra R. solani e S. sclerotiorum. Foram construídas duas bibliotecas subtrativas sendo que os RNAs utilizados como condição teste e controle da subtração foram obtidos de T. harzianum crescido em meio contendo parede celular de R. solani ou S. sclerotiorum. As bibliotecas foram sequenciadas resultando em 47 genes relacionados ao crescimento em meio com R. solani e 114 genes relacionados ao crescimento em meio com S. sclerotiorum. Dos 18 genes escolhidos para validação da biblioteca por RT-PCR em tempo real, 9 se mostraram diferencialmente expressos na condição correspondente à biblioteca em que foram identificados. Dentre estes, 5 genes se mostraram também diferencialmente expressos em experimento de confronto em placa, 2 deles mais expressos contra R. solani (cutinase e alginato liase) e 3 contra S. sclerotiorum (hsp98, serina endopeptidase e um de função hipotética). Os resultados obtidos com as duas linhagens fornecem informações adicionais sobre genes de β-1,3-glicanases, conhecidamente envolvidos no processo de micoparasitismo, e sobre genes relacionados ao antagonismo de patógenos específicos, além de contribuir para o conhecimento relacionado ao biocontrole realizado por fungos do gênero Trichoderma.

To identify known genes or expressed sequence tags (ESTs) which are expressed specifically or preferentially in the chicken hypothalamus and pituitary gland related to highly reproductive performance, two reciprocal cDNA libraries were constructed using a subtractive hybridization strategy. Two different strains, L2 (dam line; n=12) and B (sire line; n=12) of Taiwan Country Chickens (TCCs), which were originated from one single strain and further subjected to 40-wk egg production and comb size, body weight, respectively since 1982, were used in our study. A total of 324 and 370 clones were identified from L2-subtract-B and B-subtract-L2 hypothalamus/pituitary cDNA libraries. 311 and 360 single inserted sequences from each cDNA library, 53 and 23 non-redundant candidate genes were identified. Quantitative reverse-transcription (RT)-PCR were used to validate the association of mRNA expression profiles of the identified candidate genes and high-egg yield trait in another 118 hypothalamuses and pituitary glands that were dissected from seven different chicken stocks, including B-, L2-, Black-, Red-feather TCCs, commercial Single-Comb White Leghorn (WL) layer at National Chung-Hsing University (NCHU) and Red-feather TCCs grouped into high eggs (Red-high) & low eggs (Red-low) to 40 wks of age at National Chiayi University (NCYU). Among identified genes including known genes and novel genes, involving 33 screened genes, Inhibitor-1 of protein phosphatase type 2A (ANP32A), 3-hydroxybutyrate dehydrogenase (BDH), Contactin (CNTN1), Deiodinase iodothyronine type II (DIO2), Inhibitor of growth family, member 3 (ING3), Lysosomal-associated transmembrane protein 4 beta (LAPTM4B), Neural cell adhesion molecule 1 (NCAM1), DJ-1 protein (PARK7), Prostaglandin D2 synthase (PGDS), Prolactin (PRL), Protocadherin 1 (PCDH1), Pleiomorphic adenoma gene 1 (PLAG1), GTP-binding protein SAR1a (SAR1A), Secretogranin II (SCG2), Stathmin 2 (STMN2), T-box protein 2 (TBX2) were up-regulated in B-subtract-L2 cDNA library. Among above-mentioned 16 identified genes, there were 9 genes related to high-egg yield in chickens., including BDH, NCAM1, PCDH1, PGDS, PLAG1, PRL, SAR1A, SCG2, STMN2.

Dermatófitos são fungos filamentosos com a habilidade de invadir substratos queratinizados e causar dermatofitoses em humanos e animais, penetrando profundamente apenas em hospedeiros imunocomprometidos. Trichophyton rubrum é um fungo antropofílico e cosmopolita, o mais comum agente de micoses superficiais, que usa componentes celulares como proteínas e lipídeos após uma específica regulação de sua expressão gênica governada pelo pH ambiente e sensoriamento celular. A virulência de T. rubrum é relacionada com a secreção de enzimas proteolíticas, um importante fator determinante na invasão, utilização e subseqüente disseminação através do estrato córneo. O objetivo desse estudo foi identificar através de Hibridização Subtrativa Supressiva (SSH) genes de T. rubrum preferencialmente expressos durante o crescimento na presença de queratina e lipídeos, quando T. rubrum degrada fontes de carbono tipicamente encontradas em células epidérmicas. Inicialmente, nós avaliamos as mudanças no pH extracelular durante seu crescimento em queratina e lipídeo (depois de 6, 12, 24, 48, 72 h e 7 dias) em pH inicial 5,0, onde foi observado um gradual aumento do pH basal sob ambas as condições de teste, comparado com a condição glicose (controle). Também identificamos 576 transcritos de T. rubrum diferencialmente expressos por SSH usando conídios cultivados por 72 h em queratina como teste e em glicose como controle. Os genes de T. rubrum ativados codificam proteínas putativas que foram validadas por cDNA dot-blot e northern blot, mostrando similaridade com proteínas fúngicas envolvidas no metabolismo básico, crescimento e virulência, p. ex., transportadores ABC-MDR, MFS e ATPase de cobre, permease, NIMA interactive protein, poliproteína Gag-Pol, fatores de virulência subtilisinas serino-proteases (Sub 3 e 5) e metaloproteases (Mep 3 e 4) e Hsp30. Adicionalmente, entre os 762 clones obtidos na biblioteca da condição lipídeo (72 h), 80 transcritos superexpressos foram confirmados por cDNA dot-blot, revelando 14 unigenes similares à proteínas de vários organismos patogênicos, como glicoproteína 43 kD, transportador MDR, proteína G, quitina sintase e serino/treonina fosfatase. Transcritos do gene TruMDR2, codificador de um transportador ABC, foi isolado tanto na presença de queratina quanto em lipídeo, e a análise da linhagem mutante TruMDR2 de T. rubrum mostrou uma redução na atividade infectante, caracterizada pelo baixo crescimento em unhas humanas comparada com o tipo selvagem. A alta expressão de transportadores por T. rubrum em condições que mimetizam a infecção e a redução na virulência de TruMDR2 durante o modelo in vitro sugerem que transportadores estão envolvidos na patogenicidade de T. rubrum. Outro linhagem mutante (pacC-1) com um nocaute no gene pacC que codifica um fator de transcrição regulado pelo pH local, mostrou a expressão de proteases (Sub 3 e 5 e Mep 4) diminuída após o crescimento em queratina em comparação com o tipo selvagem em análises de northern blots. Essas proteases tem uma atividade ótima em pH alcalino, e nossos resultados indicam uma regulação defectiva do gene pacC de T. rubrum na ativação de proteases. Em conclusão, através do uso de SSH foi possível identificar genes de T. rubrum ativados após tratamentos específicos, o que sugere a importância dos mesmos na interação dermatófito-hospedeiro, instalação e manutenção da doença. Esses resultados disponibilizam novos dados sobre T. rubrum que levarão a um melhor entendimento dos mecanismos moleculares envolvidos no crescimento, metabolismo geral e patogenicidade, e também auxiliar na identificação de novos e efetivos alvos de drogas para dermatófitos. / Dermatophytes are a group of fungi filamentous that have the ability to invade keratinized substrates, causing dermatophytosis in humans and animals and only penetrate deeper if the host is immunocompromised. Trichophyton rubrum is an anthropophilic and cosmopolitan fungi, the most common agent of superficial mycoses, which uses cell components such as proteins and lipids after a specific regulation of its gene expression governed by pH environment and sensing cell. The virulence T. rubrum is related to secretion of proteolytic enzymes, an important factor determinant in the invasion, utilization and subsequently dissemination through the stratum corneum. The aim of this study was to indentify by Suppression Subtractive Hybridization (SSH) T. rubrum genes preferentially expressed during growth in the presence of keratin and lipids, upregulated when this fungus degrades carbon source typically found at epidemic cells. Initially, this work evaluated the changes in the extracellular pH during its growth in keratin and lipid (after 6, 12, 24, 48, 72 h and 7 days) at initial pH 5.0, where we observed a gradual increase of basal pH under both tests when compared to glucose condition (control). Also, we identified 576 T. rubrum transcripts differentially expressed by SSH using conidia cultivated for 72 h in keratin as tester, and in glucose as driver. The T. rubrum genes upregulated encode putative proteins that were validated by cDNA dot-blot and northern blot, showing similarity to fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, permease, NIMA interactive protein, Gag-Pol polyprotein, virulence factors serine-protease subtilisins (Sub 3 and 5) and metalloproteases (Mep 3 and 4) and Hsp30. Additionally, among the 762 clones obtained in a library of lipid condition (72 h), 80 over-expressed transcripts were confirmed by cDNA dot-blot, revealing 14 unigenes similar to proteins of several pathogenic organisms, like glicoprotein 43 kD, MDR transporters, G protein, chitin synthase and serine/threonine-protein phosphatase. Transcripts of TruMDR2 gene, encoding an ABC transporter in T. rubrum, were isolated in the presence of keratin and lipid, and the examination of TruMDR2 mutant T. rubrum showed a reduction in infecting activity, characterized by low growth in human nails compared to wild-strain. The high expression of transporter by T. rubrum in conditions that mimetize the infection and the virulence reduction of TruMDR2 in an in vitro model suggests that transporters are involved in T. rubrum pathogenicity. Another mutant, pacC-1 with a knockout in PacC gene that encodes a transcription factor regulated by local pH, showed the expression of proteases (Sub 3, Sub 5 and Mep 4) decreased after growth in keratin (72 h) in comparison to wild-strain in northern blot analyzes. These proteases have an optimal activity in alkaline pH, and our results indicate a defective regulation of T. rubrum pacC gene in the activation of proteases. In conclusion, by means of SSH to identify genes upregulated in T. rubrum after specific treatments, their importance in the dermatophyte-host interaction, installation and maintenance in the disease is suggested. These results provide new insights about T. rubrum that will contribute to a better understanding of molecular mechanisms about the growth, metabolism and pathogenicity, and may also aid in the identification of novel effective drug targets for dermathophytes.