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Although the eDNA sequences for the 10 k:Da (hsp 10, hsp 1 0) and the 60 k:Da (hsp60, cpn60) heat shock proteins have been obtained for a number of mammalian species, until very recently information was not available on the functional genes encoding these proteins. The primary objective of this work was to clone and sequence the functional genes for these proteins from CHO, Chinese hamster ovary cells. Screening of a lambda EMBL3 CHO genomic library with the CHO hsp 10 eDNA identified a clone containing the putative hsp 10 functional gene. A -5.5 kb fragment was isolated from one of these clones by enzymatic digestion and -3.3 kb was sequenced. The clone was found to contain consensus regulatory sequences upstream of the putative transcription initiation site, + 1, including two Sp 1 binding sites, a CAAT box, and a single heat shock element, HSE, but lacked a TATA box. The coding region consists of four exons, identical to the hsp10 CHO eDNA sequence, separated by three introns, of 200 bp, 600 bp and 1600 bp in size, containing conserved splice sites. Screening of the same EMBL3 CHO genomic library with the CHO hsp 10 eDNA also resulted in isolation of a full length processed pseudogene with -90 % identity to the eDNA. This pseudogene lacked introns, contained a poly(A) tract, as well as various single bp changes, additions and deletions. The upstream region of this pseudo gene was found to contain similarity to the human LINE sequence, a DNA repetitive element. PCR amplification ofCHO-WT genomic DNA resulted in isolation offive additional processed pseudogenes, corresponding to the central -270 bp of the CHO hsplO eDNA. All the pseudogenes displayed a high degree of similarity to the CHO hsp 10 eDNA sequence despite the presence of numerous mutations. Prior to this report, pseudogenes had not been found associated with hsp 10. The identification of these pseudogenes suggests the presence of a multi gene family for this heat shock protein in the CHO genome. Previously, a semi-processed pseudogene, Gel, was identified for hsp60 from CHO cells which contained a single -87 bp intron near its 3' end (Venner eta/., 1990). From this pseudo gene, a fragment containing the -87 bp intron was isolated for use as a probe to screen a lambda EMBL3 CHO genomic library. This resulted in isolation of several positive clones, two of which were purified, a -1.0 kb fragment amplified by PCR and then sequenced revealing two additional semi-processed pseudogenes, designated .A4 and .AS. These pseudo genes were found to be homologous to the GC 1 clone, containing many similar mutations as well as the -87 bp intron. Utilizing CHO-WT genomic DNA, a separate PCR amplification resulted in isolation of a -2.5 kb fragment which was partially sequenced and found to correspond to the putative hsp60 functional gene. The fragment contained one exon, which was identical to the CHO hsp60 eDNA in the region sequenced, and two introns of800 bp and 1500 bp. This fragment can now provide an ideal probe for isolation ofthe CHO hsp60 functional gene. / Thesis / Master of Science (MSc)

En esta Tesis Doctoral se ha abordado el estudio de la respuesta al frío en el flavedo de frutos cítricos, simulando tratamientos cuarentenarios requeridos para evitar la propagación de plagas con la exportación. Desafortunadamente, hay variedades sensibles que desarrollan síntomas de daño por frío durante este tratamiento. En este trabajo se utilizaron frutos de Clementina de Nules (CN), variedad tolerante, y de Clementina Fortune (F), sensible, para analizar a nivel transcriptómico las respuestas de adaptación y de desarrollo de daños durante la exposición al frío. Para ello (1) se generaron dos genotecas de cDNA (una de longitud completa) representativas de flavedo de frutos CN sometidos a estrés por frío; (2) los cDNAs novedosos obtenidos se incluyeron (10,3%) junto con otros (12672 ESTs totales) para generar la primera micromatriz del Proyecto de Genómica de Cítricos; (3) mediante ésta micromatriz se abordó el análisis transcriptómico de CN y F. Los resultados manifestaron que en ambas variedades se produjo una represión de procesos biosintéticos, principalmente de proteínas y también de lípidos, y se activó su catabolismo. Algunas diferencias transcriptómicas podrían ser causantes, al menos en parte, de la tolerancia de CN, al igual que el retraso de la respuesta al frío en F. Por último, también se detectó un programa de expresión tardío exclusivo de F, que podría relacionarse con el desarrollo de daños. / Royo Brun, C. (2010). Respuesta de los frutos cítricos a las bajas temperaturas: estudio mediante micromatrices [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8642

A critical event during the life cycle of Dictyostelium discoideum is glycogen turnover. This process is catalyzed by glycogen phosphorylase-2 (gp-2). Since gp-2 expression is first induced during the transition from growth to differentiation, understanding how this gene is controlled may provide some insight into the process of differentiation. In order to identify the trans-acting factors responsible for activating gp-2 expression, cDNA plasmid libraries of amoebae, and cells at 8 h and 12 h of development were generated. The long-term goals of this project involve screening expression libraries with identified cis-acting elements from the gp-2 promoter to yield the DNA binding proteins responsible for gene regulation. For this approach to succeed, a high-quality cDNA library is essential. The library must contain full-length cDNA that represents the complexity of mRNA present during the developmental stage of interest. Hence, all three libraries were subjected to extensive testing prior to and following cloning. RNA quality and the fidelity of the time points were determined by Northern blot analysis and by RTPCR for several marker genes. Following cDNA synthesis, the cDNA was assessed for complexity and full-length synthesis by PCR and radioactive primer extension, respectively. Ligation of the cDNA into a vector was performed using several ratios of vector:insert in order to ensure that long cDNA species were included in the plasmid library. Finally, the presence of the marker genes was confirmed by PCR amplification of plasmid extracted from bacteria transformed with the plasmid library. / Master of Science

Neste trabalho visamos otimizar o isolamento da BnP1, uma SVMP presente no veneno de B. neuwiedi, e isolar disintegrinas desse veneno, ambas originadas de precursores tipo P-II, analisando as seqüências de cDNA e a ação sobre plaquetas. A purificação da BnP1 foi por exclusão molecular e troca iônica; duas novas disintegrinas, D2 e D4, foram isoladas por cromatografia em fase reversa; e outra disintegrina, MS, foi caracterizada a partir do SDS-PAGE. As proteínas foram parcialmente seqüenciadas por espectrometria de massas. Diferentes cDNAs foram clonados a partir da glândula de veneno de B. neuwiedi e os precursores da BnP1 e MS se localizaram em um cluster que inclui SVMPs classe P-I e P-II. O precursor de D4 correspondeu a uma SVMP tipicamente P-II. A BnP1, ao contrário das disintegrinas, não inibiu a agregação plaquetária induzida por diferentes agonistas. Este trabalho indica ainda que a biossíntese de SVMPs da classe P-II pode envolver processamento de mRNA, um novo mecanismo de geração de diversidade das SVMPs. / The aim of this study was to optimize the isolation of BnP1, a SVMP from B. neuwiedi venom, and isolate new disintegrins from this venom, both originated from P-II class precursors, and analyze the sequence of their precursors and their action on platelets. BnP1 was isolated by size exclusion and anion-exchange chromatography; the disintegrins D2 and D4 by reverse-phase chromatography and MS was isolated from SDS-PAGE. These proteins were partially sequenced by mass spectrometry. Several SVMPs cDNAs were cloned from B. neuwiedi venom gland, and BnP1 and MS precursors localized in a cluster enclosing P-I and P-II SVMPs. MS cDNA was tipical of P-II class of SVMPs. BnP1, unlike the disintegrins, was not able to inhibit platelet aggregation induced by different agonists. This work suggested that the biosynthesis of class P-II SVMPs include mRNA processing as a further step to generate venom diversity.

Made available in DSpace on 2016-06-02T20:20:34Z (GMT). No. of bitstreams: 1 4606.pdf: 10556895 bytes, checksum: debf5633a053f88ca7dd4618edef9175 (MD5) Previous issue date: 2012-07-06 / Universidade Federal de Minas Gerais / The plant biomass is a large-scale available resource and one of its more important applications is the second-generation ethanol production. However, the enzyme cost is one of the biggest barriers for economically viable ethanol from biomass. Therefore, it is important to identify fungal strains that can produce high concentrations of plant biomass-degrading enzymes. The aim of this work was to clone, study the gene expression and characterize the plant biomass-degrading transcript set of the filamentous fungus Trichoderma harzianum IOC-3844. A total of 1,543 highquality reads from the Trichoderma harzianum IOC-3844 cellulose induced cDNA library were organized into 1,002 transcripts representing 167 contigs and 835 singlets. Of these 1,002 transcripts 646 had unknown functions and 356 showed associated functions. Among the transcripts with associated functions, we found 20 transcripts related to plant biomass deconstruction. The real time PCR analysis of Trichoderma harzianum IOC-3844 mycelia grown for 36 and 60 hours in cellulose, revealed that the levels of the following mRNAs were induced by at least 2,000-fold when compared to uninduced mycelia: cbh1, cbh2, egl1, egl2, egl3, egl7 and swo1. In some cases, the values were higher than 100,000-fold. Among the transcripts analyzed by real time PCR, cbh1, cbh2 and egl7 exhibited the highest expression levels. The Trichoderma harzianum IOC-3844 exhibited a repertoire with high expression of plant biomassdegrading transcripts. The enzymes EGIII and Xyn2 were recombinantly expressed in Pichia pastoris, showing good quality purification and good enzymatic activity. The heterologous expression assays made possible future studies aiming at the industrial application of the enzymes. Therefore, this strain showed potencial to produce biomassdegrading enzymes for second-generation ethanol production and to be a source of enzymes for the paper industry / A biomassa vegetal é um recurso disponível em larga escala e uma das suas mais imporantes aplicações é a produção de etanol de segunda geração. No entanto, o custo das enzimas é um dos maiores entraves para a produção economicamente viável deste etanol. Neste contexto, é importante encontrar organismos produtores de grandes quantidades de enzimas que degradam a biomassa. Os objetivos deste estudo foram clonar, estudar a expressão gênica e caracterizar o conjunto de enzimas que degradam a biomassa vegetal, do fungo filamentoso Trichoderma harzianum IOC-3844. Um total de 1.543 seqüências de boa qualidade, geradas a partir de uma biblioteca de cDNA do Trichoderma harzianum IOC-3844, induzido por celulose, foi organizado em 1.002 transcritos, sendo 167 representados por mais de uma seqüência e 835 representados por apenas uma seqüência. Destes transcritos, 356 tiveram função associada e 646 não tiveram. Com isso, entre os transcritos com função associada, foram listados 20 transcritos envolvidos com degradação de biomassa vegetal. Análises de PCR em tempo real do micélio de Trichoderma harzianum IOC-3844, crescido por 36 e 60 horas em celulose, mostraram níveis de mRNA mais de 2.000 vezes mais representados para os transcritos cbh1, cbh2, egl1, egl2, egl3, egl7 e swo1, quando comparados com o micélio não induzido. Em alguns casos as maiores representatividades alcançaram valores superiores a 100.000 vezes. Entre os transcritos analisados o cbh1, o cbh2 e o egl7, mostraram os mais altos níveis de expressão. O Trichoderma harzianum IOC-3844 exibiu um repertório com alta expressão de transcritos envolvidas na degradação de biomassa vegetal. As enzimas EGIII e Xyn2 foram expressas em sistema recombinante com uso da levedura Pichia pastoris, apresentando facilidade de purificação e boa atividade enzimática. Os ensaios de expressão heteróloga viabilizaram estudos posteriores que visam a aplicação industrial das enzimas. Assim, esta cepa mostrou potencial para produzir enzimas que degradam a biomassa para a produção de etanol de segunda geração, e para ser fonte de enzimas para a indústria de papel.

Neste trabalho visamos otimizar o isolamento da BnP1, uma SVMP presente no veneno de B. neuwiedi, e isolar disintegrinas desse veneno, ambas originadas de precursores tipo P-II, analisando as seqüências de cDNA e a ação sobre plaquetas. A purificação da BnP1 foi por exclusão molecular e troca iônica; duas novas disintegrinas, D2 e D4, foram isoladas por cromatografia em fase reversa; e outra disintegrina, MS, foi caracterizada a partir do SDS-PAGE. As proteínas foram parcialmente seqüenciadas por espectrometria de massas. Diferentes cDNAs foram clonados a partir da glândula de veneno de B. neuwiedi e os precursores da BnP1 e MS se localizaram em um cluster que inclui SVMPs classe P-I e P-II. O precursor de D4 correspondeu a uma SVMP tipicamente P-II. A BnP1, ao contrário das disintegrinas, não inibiu a agregação plaquetária induzida por diferentes agonistas. Este trabalho indica ainda que a biossíntese de SVMPs da classe P-II pode envolver processamento de mRNA, um novo mecanismo de geração de diversidade das SVMPs. / The aim of this study was to optimize the isolation of BnP1, a SVMP from B. neuwiedi venom, and isolate new disintegrins from this venom, both originated from P-II class precursors, and analyze the sequence of their precursors and their action on platelets. BnP1 was isolated by size exclusion and anion-exchange chromatography; the disintegrins D2 and D4 by reverse-phase chromatography and MS was isolated from SDS-PAGE. These proteins were partially sequenced by mass spectrometry. Several SVMPs cDNAs were cloned from B. neuwiedi venom gland, and BnP1 and MS precursors localized in a cluster enclosing P-I and P-II SVMPs. MS cDNA was tipical of P-II class of SVMPs. BnP1, unlike the disintegrins, was not able to inhibit platelet aggregation induced by different agonists. This work suggested that the biosynthesis of class P-II SVMPs include mRNA processing as a further step to generate venom diversity.

<p>Worldwide obesity is an increasing problem. Apart from the fact that obesity greatly  impairs the health, quality and length of life for the affected individuals, it is also has the  potential to become a major socioeconomic problem in a near future. However preventive  actions require an understanding of the cause. Before the psychological influence on  eating can be evaluated a profound understanding of the biological regulatory system and  how this interacts with the food consumed is required. On the assumption that food  consumption is regulated by interplay between food and genes, the food itself may  influence the genes that regulate consumption, hence change the expression levels of the  genes regulating food intake.     To evaluate the interplay between food and gene expression, the project contained several  parts, reflecting different aspects of the area of research. The feeding studies had in  common that they were initial trials in a larger project. The results of these will be  evaluated and used in combination with further studies.     The mice typed for food preference illustrate the complexity of the feeding regulatory  system by pointing out the differences between individuals even in a relatively small  group of animals. Mice in general like food high in fat and here the animals that showed a  preference for sugar also showed a significant increase in their intake of chow. Since  chow consists mainly of carbohydrates the results might indicate a preference not for  sucrose in particular but for carbohydrates in general. The effect this may have on other  studies is still unclear as further studies are needed to determine whether the difference  may be the result of an innate genetic difference.      Leucine has been previously shown to reduce the total caloric intake. When given in  combination with palatable food the addition of Leucine primarily reduced the intake of  chow. From a dietary perspective this would translate to a preference to sweets and fast  food at the expense of food with more nutritious content.     The RT-PCR analysis’s gives clues to how the energy regulatory circuitry responds to the  intake of selected macronutrients. When it comes to gene expression there is a significant  effect of macronutrients on the gene expression levels. The common theme for many of  the genes tested seems to be down regulation of satiety signals, as if to support over  feeding on palatable diets and in many cases sucrose in particular.     The intake of macronutrients such as sugar or fat has been showed to have an effect on  the feeding regulatory circuitry, demonstrated by the change in gene expression levels.   The response to said macronutrients is site specific which is clearly shown both by RTPCR analysis of samples from different parts of the brain, such as the brainstem or  hypothalamus, and by immunohistochemistry of selected areas. The  immunohistochemistry also confirms that the novel Oxytocin receptor-antagonist, who is  injected IP, actually passes over the blood-brain barrier and has an actual affect on the  regions of interest. The areas affected by the antagonist can be visualized and identified  through the staining of active sites.</p>

Real-time PCR method is a type of PCR which allows continual monitoring of DNA amplification during every cycle of its process. It is mostly used for gene expression analysis. Based on the results of previous experiments, we decided to test out the effect of anticoagulants EDTA, heparin, sodium citrate and CPDA on the expression of selected genes of the immunological spectrum and further, to test how the time period between drawing the blood and processing of blood sample influences mRNA levels of selected genes that are determined by changes in gene expression and/or mRNA degradation. To quantify mRNA of the studied genes, we isolated total RNA from the peripheral blood leucocytes and transcribed it into cDNA by using the reverse transcription PCR. This cDNA served as a template for the real-time PCR. To examine the changes of the expression caused by the effect of each particular anticoagulants, peripheral blood derived from 10 volunteers was used (each donor's blood was taken into 3 vacuum tubes with EDTA, heparin and sodium citrate anticoagulant agents). Next to that, we obtained 10 buffy coat samples in transfusion blood bags with CPDA anticoagulant agent. Compared to blood cells influenced by one of the three anticoagulant agents present in vacuum tubes, cells from transfusion bags affected...

A produção brasileira de laranjas destina-se à indústria de suco concentrado, principal produto citrícola, sendo o estado de São Paulo o maior produtor e exportador do país. Devido à diminuição da variabilidade genética causada pela multiplicação de plantas cítricas por enxertia, esta cultura se tornou alvo constante de inúmeras pragas e doenças. Dentre estas doenças que vem causando crescentes prejuízos para a citricultura brasileira destaca-se a pinta preta causada pelo fungo Guignardia citricarpa Kiely. O combate desta doença é feito por inúmeras pulverizações de fungicidas que aumenta significativamente o custo para os produtores. Uma possível alternativa para o combate a este patógeno seria a produção de plantas cítricas resistentes a doença, através de transformação gênica. Mas para isto, é necessário conhecer os mecanismos de respostas de defesa da planta e identificar quais genes podem estar relacionados com a defesa. Na tentativa de elucidar as respostas de defesa deste hospedeiro foram feitas análises anatômicas de frutos de laranja doce (Citrus sinensis Osbeck cv. Pêra) inoculados com o patógeno e análise transcricional de duas bibliotecas de cDNA, uma de albedo de frutos sadios(BAFS) e outra de albedo de frutos inoculados com o patógeno (BAFC). As análises anatômicas mostraram que os danos causados pelo patógeno na planta iniciam-se 24 horas após a inoculação com as lesões das células do epicarpo, e continuam gradativamente até 72 horas apresentando a diminuição na quantidade de amido e acúmulo de compostos fenólicos. Foi obtido 184 ESTs válidas da BAFS e 370 da BAF. A anotação e categorização destas sequencias apresentaram que o perfil transcricional encontrado nas duas bibliotecas foi semelhante, porém, na BAFC, foram encontrados transcritos pertencentes à defesa da planta como a catalase, a glutationa e monooxygenases. Está analise mostrou que a resposta de defesa da planta inicia-se com lesões nas células, que a interação é custo-intensiva (redução da reserva de amido) para o hospedeiro, e que a defesa da planta está relacionada a muitos genes de defesa. / The Brazilian production of oranges is mainly for the industry of concentrated juice, it is the most important citrus product, and the state of São Paulo is the largest producer and exporter of the country. Due to the low genetic variability caused by the multiplication of citrus plants by grafting, this culture became constant target of many pests and diseases. Among all the diseases that lead to elevated damage to the Brazilian citrus plantation, one is very relevant, the Black Spot disease caused by the fungus Guignardia citricarpa Kiely. The control of this disease is done by many sprays of fungicides that significantly increase the production cost to the producers. A possible alternative to control this pathogen is the development of resistant citrus plants to disease through genetic transformation. To that, it is necessary to understand the mechanisms of plant defense response and identify the genes related to the defense. In an attempt to elucidate the responses of this host both,anatomical analysis of sweet orange fruits (Citrus sinensis Osbeck cv. Pêra) inoculated with the pathogen and transcriptional analysis of two cDNA libraries of albedo section were done. One was from health fruits (BAFS) and another from albedo of fruits inoculated with the pathogen (BAFC). The analysis showed that the anatomical damage caused by the pathogen in the plant shall begin 24 hours after inoculation showing lesions on epicarp cells, and continue gradually until 72 hours also showing a decrease in the amount of starch and accumulation of phenolic compounds. It was obtained 184 valid ESTs of the BAFS and 370 from BAFC. The annotation and categorization of these sequences showed that the transcriptional profile in the two libraries were similar, however, in BAFC, were found transcripts belonging to plant defense such as catalase, the glutathione and monooxygenases. These analyses showed that the plant defense response begins with lesions in the cells, being it cost-intensive interaction for the host, (with reduction of starch reserves) and the plant defense to resistance is related to many genes.