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.

Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by P. carnosa, its genome was sequenced and transcriptomes were evaluated after growth on wood compared to liquid medium. Results indicate that P. carnosa differs from P. chrysosporium in the number and expression levels of genes that encode lignin peroxidase (LiP) and manganese peroxidase (MnP), two enzymes that modify lignin present in wood. P. carnosa has more genes for MnP with higher expression levels than LiP, while the reverse has been observed for P. chrysosporium. The abundances of transcripts predicted to encode lignocellulose-modifying enzymes were then measured over the course of P. carnosa cultivation on four wood species. Profiles were consistent with decay of lignin before carbohydrates. Transcripts encoding MnP were highly abundant, and those encoding MnP and LiP featured significant substrate-dependent response. Since differences in modes of lignin degradation catalyzed by MnP and LiP could affect the ability of each to degrade lignin from different types of wood, their activity on various hardwoods and softwoods were tested. Results suggest that MnP degrades softwood lignin more effectively than hardwood lignin, consistent with high levels of this enzyme in P. carnosa.

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, β-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two β-1,3-endoglucanses while CA whole leaf protein extracts contain at least three β-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 β-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for β-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of β -1,3-endoglucanses.

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.

This study was carried out for understanding of antioxidant mechanisms of lentil under abiotic stress conditions. For this aim, 14 days old lentil seedlings (Lens culinaris Medik cv. Sultan-1) were subjected to drought (20% PEG 6000), and salt (150 mM NaCl ) stress for 6, 12 and 24 hours, for 3, 5 and 7 days. PCR conditions for Mn SOD, Cu/Zn SOD, chloroplastic/mitochondrial GR, CAT and chloroplast /stromal APX antioxidant enzymes were optimized. Then, total RNA was isolated from stressed and non-stressed plant roots and shoots. The gene expression levels of Mn SOD and Cu/Zn SOD were examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. Arabidopsis 18S rRNA was used as internal control in multiplex PCR technique. Relative expression levels of Mn SOD were lower in shoots and roots under salt stress while no significant change was obtained under drought conditions in both tissues. Relative expression levels of Mn SOD were increased on 5th day of salt and drought applications in both shoots and roots. Relative expression levels of Cu/Zn SOD increased after 5th, and on 1st and 7th days of drough treatment in shoots and roots, respectively. On the other hand, expression levels of Cu/Zn SOD increased on 3rd and 5th days of salt treatment in shoot tissues. Although it is nearly impossible to understand the whole antioxidant mechanism of plants under environmental stresses, this study was the first step to learn about molecular background of antioxidant defence mechanisms in lentil.

名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程） 学位授与年月日:平成20年3月25日

Cette thèse porte sur la vérification formelle de systèmes temps réel et procède par transformation de modèle entre l'algèbre de processus temporisée RT-LOTOS et les réseaux de Petri temporels étendus par des chronomètres et des données. Des schémas de traduction de RT-LOTOS vers ces réseaux de Petri étendus sont proposés et formellement prouvés. L'approche transformationnelle développée pour la partie " contrôle " de RT-LOTOS est étendue à la partie " données ". Le langage RT-LOTOS est lui même enrichi d'un opérateur de suspension reprise qui permet de modéliser et vérifier une classe plus large de systèmes temps réel Plusieurs études de cas attestent de l'efficacité des schémas de traduction proposés par rapport à des outils LOTOS ou RT-LOTOS développés antérieurement. L'approche proposée s'avère transposable à d'autres langages de modélisation en particulier le profil UML temps réel TURTLE (Timed UML and RT-LOTOS Environment).

Le sulfure d'hydrogène (H2S) est un métabolite bactérien produit notamment par les bactéries sulfato-réductrices du côlon à partir des acides aminés soufrés, des sulfates/sulfites alimentaires et des sulfomucines. A fortes concentrations, le H2S est un gaz toxique, de par sa capacité à inhiber la cytochome c oxydase, et par conséquent, la respiration mitochondriale. A l'inverse, notre travail montre qu'à faibles concentrations, le H2S induit une énergisation mitochondriale des cellules épithéliales coliques humaines HT 29 Glc-/+ lui conférant aussi un rôle de substrat minéral énergétique. Dans ce travail, nous avons déterminé les concentrations de H2S permettant l'oxydation/détoxication de H2S par les cellules HT 29 Glc-/+ et celles provoquant une inhibition de la consommation d'oxygène. L'oxydation du H2S nécessite la coopération entre la " sulfide oxidation unit " et la chaîne respiratoire. La capacité des cellules HT29 Glc-/+ à oxyder le H2S est associée à la présence des transcrits codant les enzymes constituant la " sulfide oxidation unit " : la sulfure d'hydrogène quinone reductase (SQR), la dioxygenase ETHE1 et la thiosulfate transferase (TST). Nous avons démontré la priorité de l'oxydation du H2S sur les substrats carbonés . En effet, nos résultats suggèrent que les électrons venant de la SQR sont transférés au pool d'ubiquinone aux dépens de ceux venant du complexe I. Nos résultats démontrent que la SQR joue un rôle déterminant pour l'oxydation de H2S. De plus, la détoxication du H2S par les cellules HT29 Glc-/+ augmente au cours de la différenciation spontanée ou induite par un traitement au butyrate. L'augmentation de la détoxication de H2S au cours de la différenciation est associée à une augmentation de la réserve respiratoire soulignant l'importance de la chaîne respiratoire comme composante de la fonction de détoxication du H2S. En situation d'inhibition de la cytochrome c oxydase, la grande capacité des cellules coliques humaines à détoxiquer le H2S pourrait être en partie due à la présence d'un transfert réverse des électrons issus de l'oxydation de H2S de la SQR vers le complexe I. Outre le butyrate, le zinc un autre composé de la lumière colique, exerce un effet protecteur contre la toxicité cellulaire du H2S. Enfin, notre travail a mis en évidence une diminution de l'expression d'un gène codant pour une enzyme de la " sulfide oxidation unit " (la TST) dans le rectum comparée à différents segments du côlon, ce qui pourrait correspondre à des capacités de détoxication du H2S différente en fonctions des segments du gros intestin humain.

Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by P. carnosa, its genome was sequenced and transcriptomes were evaluated after growth on wood compared to liquid medium. Results indicate that P. carnosa differs from P. chrysosporium in the number and expression levels of genes that encode lignin peroxidase (LiP) and manganese peroxidase (MnP), two enzymes that modify lignin present in wood. P. carnosa has more genes for MnP with higher expression levels than LiP, while the reverse has been observed for P. chrysosporium. The abundances of transcripts predicted to encode lignocellulose-modifying enzymes were then measured over the course of P. carnosa cultivation on four wood species. Profiles were consistent with decay of lignin before carbohydrates. Transcripts encoding MnP were highly abundant, and those encoding MnP and LiP featured significant substrate-dependent response. Since differences in modes of lignin degradation catalyzed by MnP and LiP could affect the ability of each to degrade lignin from different types of wood, their activity on various hardwoods and softwoods were tested. Results suggest that MnP degrades softwood lignin more effectively than hardwood lignin, consistent with high levels of this enzyme in P. carnosa.

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No. of bitstreams: 1 rossi_gam_dr_jabo.pdf: 3246904 bytes, checksum: 2ed62292bd3bfa8f9ec88fe41c5bbaa2 (MD5) Previous issue date: 2017-12-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As bactérias pertencentes ao grupo do Bacillus cereus são importantes para indústrias processadoras de leite e produtos lácteos devido à capacidade de sobrevivência aos tratamentos térmicos utilizados durante os processamentos, e consequente deterioração dos produtos e risco à saúde pública. Assim, inicialmente, objetivou-se compreender a estrutura populacional de isolados pertencentes ao grupo do B. cereus na cadeia produtiva do leite e produtos lácteos. Por meio da utilização de amostragem estruturada e técnicas genômicas comparativas, investigou-se quais linhagens específicas e genes estão significativamente associados a estágios de produção específicos e produtos. Os genomas de 69 isolados obtidos de equipamentos, leite cru e produtos lácteos foram comparados com outros 193 disponíveis de diversas origens. A estrutura populacional incluiu os conhecidos grupos filogenéticos II, III, IV, V e VI, e quase todos os isolados dos produtos lácteos pertenceram ao grupo III. A investigação de genes específicos revelou um grande número de isolados carreando aqueles relacionados à produção de toxinas, como o cytK (53,62%), hblA (59,42%), hblC (44,93%), hblD (53,62%), nheA (84,06%), nheB (89,86%), nheC (84,06%), cesA (2,90%) e cesB (2,90%). Os isolados pertencentes aos grupos IV e V possuíram uma prevalência significativamente maior dos genes hblACD e o grupo IV do gene cytK. Os isolados obtidos de produtos lácteos tiveram uma prevalência significativamente menor dos genes cytK e hblACD comparados aos de equipamentos e leite cru/tanques de refrigeração. A análise genômica populacional demonstrou a diversidade dos isolados e a variedade de funções dentro do grupo do B. cereus da cadeia produtiva leiteira, com grande número de isolados potencialmente capazes de causar doenças alimentares. Posteriormente, objetivou-se avaliar a viabilidade do processo de tindalização de leite cru refrigerado e determinar se o processo de pasteurização do leite influencia na expressão dos genes relacionados à produção das toxinas diarreicas pelo B. cereus s.s. em leite experimentalmente contaminado. Para isso, realizou-se um processo de tindalização de leite cru refrigerado e então foi realizada a contaminação com um isolado potencialmente toxigênico, e, posteriormente, o mesmo foi pasteurizado, envasado e mantido em refrigeração durante 10 dias. Em momentos determinados, foram coletadas amostras do produto afim de avaliar a expressão de genes codificadores de toxinas (hblACD, nheABC e cytK). O protocolo de tindalização utilizado permitiu redução logarítmica da população de bactérias do grupo do B. cereus e foi possível detectar a expressão do gene hblA em amostras de leite 5 e 10 dias após a pasteurização. Conclui-se que o modelo proposto foi adequado e que a expressão do gene hblA não foi inibida após a realização da pasteurização do leite, demonstrando o potencial risco aos consumidores decorrentes do consumo de leite pasteurizado contaminado. / The bacteria belonging to Bacillus cereus group are important to dairy industries due their ability to survive to thermal treatments used during processing, and consequently cause food spoilage and risk to public health. Thus, firstly, this study aimed to better understand the population structure of B. cereus group isolates in dairy production chain. Using structured sampling of B. cereus in dairy production chain and comparative genomics techniques, we investigated if specific lineages and genes are significantly overrepresented in particular production stages. The genomes of 69 isolates obtained from equipments, raw milk and dairy products were compared with others 193 avaiable from several origins. The populational structure included the known phylogenetic groups II, III, IV, V and V, and almost all isolates from dairy products belonged to group III. The genes investigation showed a high number of isolates carrying genes related to toxins production, such as cytK (53.62%), hblA (59.42%), hblC (44.93%), hblD (53.62%), nheA (84.06%), nheB (89.86%), nheC (84.06%), cesA (2.90%) and cesB (2.90%). The isolates belonging to groups IV and V had a significant higher prevalence of genes hblACD an group VI of cytK. The isolates obtained from dairy products had a significant lower prevalence of genes cytK and hblACD compared to those from equipments and raw milk/bulk tanks. The populational genomic analyses showed the diversity of isolates and variability of functions in B. cereus group in dairy production chain, with a high number of isolates potentially able to cause foodborne disease. Posteriorly, this study aimed to evaluate the viability of tyndallizating raw milk and to establish if milk’s pasteurization influences on the expression of genes related to the production of diarrheal toxins by B. cereus s.s. using a milk contamined experimentally. For this purpose, the tyndallization of raw milk was performed and later it was contaminated with a potentially toxigenic isolate, and, posteriorly, it was pasteurized, packaged and kept under refrigeration during 10 days. In established periods, samples of this product were collected in order to evaluate the expression of genes related to toxins production (hblACD, nheABC and cytK). The protocol used for tyndallization allowed a logarithmic reduction of population of B. cereus group and the expression of hblA gene was detected in samples from milk after 5 and 10 days after pasteurization. It was concluded that the proposed model was appropriate and the expression of hblA gene was not inihibited after milk pasteurization, highlighting the potential risk for consumers through consumption of contamined pasteurized milk. / 2016/19214-9 / 2014/13104-1