<p> The mode of chromosome duplication during meiosis and mitosis in Haplopappus gracilis was investigated. Tritiated thymidine was incorporated into the pollen mother cells during premeiotic interphase, and the cells were allowed to reach the tetrad stage. The autoradiographs prepared from the tetrads showed an unequal distribution of grains over their nuclei, suggesting a conservative mode of chromosome duplication during meiosis. Seedlings were fed with tritiated thymidine for the duration of one cell cycle and also for the duration of several cell cycles. The autoradiographs prepared from the root tip cells, thus treated, showed both labelled and unlabeled chromatids in the anaphases of all the experiments, thus again suggesting a conservative mode of chromosome duplication. A chromosome model to explain the results is discussed. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work attempts to make a study of the chemical composition of the essential oil from leaves of seven Lippia gracilis genotypes. In addition, we investigated the effect of water stress on the chemical composition of the essential oil (EO) of the species. A total of 32 volatile compounds were identified with predominance of phenolic monoterpenes: carvacrol, thymol and p-cymene. The seven genotypes were divided into two chemotypes: thymol chemotype, represented by the 106 genotype, and carvacrol chemotype, formed by the 107, 108, 109, 110, 201 and 202 genotypes. Biotransformation reactions have also been carried out using Botryosphaeria sp and Aspergillus niger as biocatalyst. In addition to the major components of the L. gracilis essential oil (carvacrol and thymol), the monoterpenes (+)-pulegone, geraniol, (-)-citronellol, (+)-limonene, rac-camphor and the two enantiomers of the fenchone were evaluated as substrates for biotransformation reactions. Botryosphaeria sp. catalyzed the hydroxylation of the camphor and fenchone at different positions, the carbon 6-endo of the camphor and the carbon 6-exo of the fenchone was the major site of addition of the OH group. Aspergilus niger catalyzed the hydroxylation of fenchone enantiomers and the isomerization of geraniol, resulting in formation of 6-exo-hidroxifenchone and of rac-linalool, respectively. / No presente trabalho foi realizado o estudo da composição química do óleo essencial das folhas de sete genótipos de Lippia gracilis Schauer, além de verificar o efeito do estresse hídrico na composição química e no teor de óleo essencial (OE) produzido pela espécie vegetal. Foram identificados 32 compostos no OE de L. gracilis com predominância de monoterpenos. Os principais componentes identificados foram os monoterpenos fenólicos carvacrol, timol e p-cimeno. Os sete genótipos foram divididos em dois quimiotipos, o quimiotipo timol, representado pelo genótipo 106, e o quimiotipo carvacrol, formado pelos genótipos 107, 108, 109, 110, 201 e 202. Também foram realizadas reações de biotransformação de terpenos voláteis por duas espécies fúngicas: Botryosphaeria sp. e Aspergillus niger. Além dos principais componentes do óleo essencial de L. gracilis, carvacrol e timol, foram testados como substrato os monoterpenos (+)-pulegona, geraniol, (-)-citronelol, (+)-limoneno, rac-cânfora e o par de enantiômeros da fenchona. Botryosphaeria sp. catalisou a hidroxilação da cânfora e da fenchona em diferentes posições, o carbono 6-endo da cânfora e o carbono 6-exo da fenchona foram os principais sítio de hidroxilação. Aspergillus niger promoveu hidroxilação dos enantiômeros da fenchona e isomerização do geraniol, majoritariamente originando a 6-exo-hidroxifenchona e rac-linalol nas reações, respectivamente.

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.

Die Photoaktivierte Adenylatzyklase PAC ist in E. gracilis an der Phototaxis beteiligt und besteht aus den zwei unterschiedlich großen Proteinen PACalpha und PACbeta. Beide besitzen jeweils zwei FAD bindende (BLUF) Domänen F1 und F2 sowie zwei Zyklasedomänen C1 und C2. An den Zyklasedomänen findet die Umsetzung von ATP in cAMP statt und die BLUF-Domänen werden für die Lichtaktivierung benötigt. Für diese Arbeit wurde PAC und Mutanten davon heterolog in Oocyten von Xenopus laevis exprimiert. PAC besitzt bereits im Dunkeln Adenylatzyklaseaktivität, die durch Belichtung erhöht werden kann. Die Zunahme der Aktivität erfolgt mit einer Zeitkonstante von unter 100 ms, die Abnahme nach der Belichtung hat eine Zeitkonstante im Bereich von 10ms. Das für die katalytische Umsetzung in allen Klasse III Nukleotidzyklasen benötigte Dimer zweier Zyklasedomänen ist in PAC das Dimer aus C1 und C2. Durch Messungen mit PAC-Mutanten, bei denen jeweils eine Zyklasedomäne defekt war, konnte gezeigt werden, dass diese Dimerisierung in PACalpha intermolekular auftritt. Ebenso wurde gezeigt, dass ein solches Dimer aus Zyklasedomänen von PACalpha und PACbeta bestehen kann. Der Austausch der Zyklasedomänen von PACalpha durch Zyklasedomänen der Guanylatzyklasen GCY35 und GCY36 aus C. elegans führte zu einem Verlust der Zyklaseaktivität. Die Proteine wurden aber zumindest teilweise korrekt gefaltet, was durch Dimerbildung mit Knockoutmutanten von PAC in Koexpressionsexperimenten gezeigt werden konnte. Die Fusionsproteine aus PACalpha und den CNG-Kanälen CNGA2 und OLF führten in Oocyten zu einer deutlich geringeren Leitwertänderung als eine Expression der Einzelproteine. Sowohl bei einer N-terminalen Fusion des Kanals an PAC als auch bei der C-terminalen Fusion war es jeweils der Kanal, der im Fusionsprotein stark gehemmt war. Eine Deletion des C-Terminus von PACalpha führte zu einem nicht funktionsfähigen Protein, das auch in Koexpression mit PAC-Knockoutmutanten keine messbare Adenylatzyklaseaktivität zeigte. Wurde die F2-Domäne deletiert, so verlor PAC ebenfalls seine Zyklaseaktivität vollständig. Die C1-Domäne war aber korrekt gefaltet, was durch eine Koexpression mit PAC-Mutanten gezeigt werden konnte, die in einer ihrer Zyklasedomänen defekt waren. Beide Chimären aus PACalpha und PACbeta besaßen Adenylatzyklaseaktivität. Diese war bei der Chimäre mit dem C-terminalen Teil von PACalpha deutlich höher als bei der Chimäre mit dem C-terminalen Teil von PACbeta, was darauf hindeutet, dass im C-terminalen Teil von PAC der Grund für den Aktivitätsunterschied zwischen PACalpha und PACbeta liegt. Für die Veränderung der Substratspezifität von einer Adenylat- zu einer Guanylatzyklase waren Mutationen an mindestens drei Aminosäuren erforderlich. Die ebenfalls hergestellten Einzel- und Doppelmutanten verhielten sich wie der Wildtyp oder hatten eine deutlich eingeschränkte Adenylatzyklaseaktivität. Bei der Tripelmutante PACalpha K250E T319G S329Y war Guanylatzyklaseaktivität nachweisbar, die aber geringer war als die noch vorhandene Adenylatzyklaseaktivität. Die Quadrupelmutante PACalpha K250E D317K T319G S329Y zeigte ebenfalls lichtinduzierbare Adenylatzyklaseaktivität, die ca. 0,3% der Aktivität der Wildtyp-PACalpha entsprach. Die Guanylatzyklaseaktivität dieser Mutante war ca. dreifach höher als deren Adenylatzyklaseaktivität. Somit konnte gezeigt werden, dass sich durch die Mutation weniger einzelner Aminosäuren die Substratspezifität von PAC von ATP nach GTP verschieben lässt. / The photoactivated adenylyl cyclase PAC is involved in phototaxis in E. gracilis. It consists of two subunits of different size which are called PACalpha and PACbeta. Both of them harbour two FAD-binding domains (F1, F2) and two cyclase domains (C1, C2). PAC and mutants of PAC have been heterologously expressed in Oocytes of Xenopus laevis. Already in darkness PAC shows a basal level of adenylyl cyclase activity, which can be increased by illumination. The increase in cyclase activity occurs with a time constant lower than 100 ms, whereas the decrease after illumination has a time constant around 10 ms. The dimer of two cyclase domains, which is necessary for catalytic conversion in all class III cyclases, is formed of C1 and C2 in PAC. In electrophysiological experiments with PAC mutants which were defective in either of the cyclase domains it has been shown, that this dimer in PACalpha occurs intermolecularly. Furthermore it has been shown, that this dimer can occur between PACalpha and PACbeta. Mutants of PACalpha where the cyclase domains have been substituted by the cyclase domains of the guanylyl cyclases GCY35 and GCY36 from C. elegans lost their ability to produce cAMP. However coexpression experiments with PAC knockout mutants indicated correct translation of the substitution mutants. Expression of fusion proteins of PACalpha with the CNG channels CNGA2 and OLF showed less light-inducable conductance changes than the expression of the single protein. In both the N-terminal and C-terminal fusion of the channel to PACalpha it was the channel which was the most affected part of the fusion protein. Deletion of the C-terminus of PACalpha results in a non-functional protein, which in coexpression with PACalpha knockout mutants shows no measurable cyclase activity. When deleting the F2-domain, PACalpha also loses its cyclase activity completely. However, the C1-domain was transcribed correctly, which could be shown by coexpression with a C1-knockout mutant. Both PACalpha-PACbeta chimeras showed adenylyl cyclase activity. Whereas the activity in the chimera with the C-terminal part of PACbeta showed little cyclase activity, the chimera possessing the C-terminal part of PACalpha showed adenylyl cyclase acitvity, which was comparable to the wildtype of PACalpha. This indicates that the part of PAC which is responsible for the difference in cyclase activity between PACalpha and PACbeta must be present in the C-terminal half of PAC. For altering PAC’s substrate specificity it was necessary to mutate at least three amino acids. The single and double mutants of PACalpha which were generated resulted in wildtypelike behaviour or reduced adenylyl cyclase activity. The triple mutant PACalpha K250E T319G S329Y showed guanylyl cyclase activity which was lower than its remaining adenylyl cyclase activity. The quadruple mutant PACalpha K250E D317K T319G S329Y also showed adenylyl cyclase activity, which was about 0.3% of the activity in PACalpha wildtype. The guanylyl cyclase activity of this mutant was about threefold higher than its adenylyl cyclase activity. Thus, it could be shown, that by mutating few single amino acids the substrate specificity of PACalpha was shifted from ATP to GTP.

No description available.

Universiẗat, Diss., 2004--Bielefeld.

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-04-16Bitstream added on 2014-06-13T19:55:57Z : No. of bitstreams: 1 fioresi_tb_me_jabo.pdf: 1222534 bytes, checksum: 8a60817f69089bec8947b5422404e7c7 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Um dos fatores importantes na criacao de larvas de peixes e o uso de alimento natural, tal como o fitoplancton, o qual pode ser cultivado em instalacoes especiais, a baixo custo com alto valor nutricional e elevada taxa de crescimento. Muitos fatores afetam o cultivo de algas como luz, nutrientes, temperatura e pH, sendo esses fatores complexos e dificeis de serem analisados separadamente, principalmente, as interacoes entre incidencia de luz e limitacao de nutrientes. Entretanto, a restricao a luz e mais impactante do que a limitacao de nutrientes, desde que a luz afeta diretamente a utilizacao dos compostos disponiveis no meio de cultura. Estudos tem sido enfatizados focalizando o cultivo de algas para a producao de alimento, utilizando-se meios alternativos como forma de baratear o custo de producao. O objetivo do presente trabalho foi avaliar e comparar o efeito do esterco suino no desenvolvimento de Ankistrodesmus gracilis, cultivada em laboratorio em dois meios: com esterco gin natura e esterco biodigerido. O experimento foi realizado no periodo de 22 dias nos volumes, de 2L e 250L, analisando a biologia, valor nutricional e a qualidade da agua do meio de cultura. Biovolume, teor de cinzas, comprimento total e concentracao de lipideos foram significativos (p<0,05) entre os volumes cultivados. O teor de proteina nao foi significativo (p>0,05) somente no meio com esterco biodigerido. As maiores densidades de A. gracilis foram observadas no volume de 2L, tendo a amostra cultivada em meio com esterco biodigerido ao longo do experimento maior densidade, com o pico ao redor do 5o dia, 6,2 x 107 celulas.ml-1. Ortofosfato, pH, alcalinidade, oxigenio dissolvido e temperatura da agua nao foram significativos (p>0,05) entre volumes e o nitrito nao foi detectado... / One of the most important factors in successfully fish rearing is probably the use of natural feed, such as phytoplankton, which may be grown in specially designed installations. So that high nutritional algae could be produced at low cost it is important to reach light production of algae and high growing rate. Many factors affect algae culture growth such as light, nutrients, temperature and pH since they directly involve algae density interactions between light incidence and limitation of nutrients are very complex and the effect of factor can not be analyzed separately. However, light restriction caused more impact than limiting of nutrients, since energy affected directly the utilization of the compounds available in the medium. Several studies focus on the culture of algae for food production through alternative methods to lower production costs. The effect of swine manure fertilization in the development of Ankistrodesmus gracilis was mensured on two medium: in natura and biodigested, during 22 days, in two volumes, 2L and 250L. The biology, nutritional value and water quality of culture medium were analyzed. Biovolume, ash rate, total length and lipids were higher (p<0.05) between treatments. Protein rate was different (p>0.05) only in the biodigested treatment. Highest A. gracilis densities were reported in 2L throughout the experiment the biodigested treatment had the highest density, 6.2 x 107 cells.ml-1, on the 5th day. Orthophosphate, pH, alkalinity, dissolved oxygen and water temperature were only different (p<0.05) between volumes. Nitrite, however, was not reported in the 2L volume treatment. Significant differences (p<0.05) between media were only reported for DBO5. Light demand was also... (Complete abstract, click electronic access below)

Orientador: Adriana Zerlotti Mercadante / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-28T11:46:40Z (GMT). No. of bitstreams: 1 Squina_FabioMarcio_M.pdf: 21730351 bytes, checksum: a93d531f3b4f081de1a75a819883dd6a (MD5) Previous issue date: 2001 / Resumo: Os carotenóides compõem um grupo de pigmentos naturais amplamente distribuídos na natureza com grande diversidade de estruturas e funções. O interesse pelos carotenóides tem aumentado onsideravelmente nos últimos anos, havendo uma potencial demanda por fontes naturais destes compostos nas indústrias de alimentos, farmacêutica e cosmética. As principais fontes industriais de carotenóides são a síntese química e a extração a partir de plantas, porém são poucos os carotenóides que possuem métodos economicamente eficientes de produção. Atualmente, apesar do pequeno número de carotenóides produzidos por biossíntese microbiana, esforços têm sido direcionados no desenvolvimento de processos biotecnológicos para a sua produção. Os carotenóides em microrganismos estão localizados intracelularmente, portanto além de uma elevada quantidade de carotenóides, os organismos selecionados para o desenvolvimento de processos biotecnológicos devem apresentar outras possibilidades, como o cultivo em resíduos ou substratos de baixo custo, produção de carotenóides com alto valor agregado, ou de outros compostos de interesse comercial, como ácidos graxas insaturados ou polissacarídeos. A primeira etapa deste trabalho foi o desenvolvimento de uma metodologia para extração e separação dos carotenóides de Rhodotorula rubra, R. glutinis, R. araucariae, R. minuta e R. lactosa. Dos métodos de extração testados, a utilização de areia tratada como agente abrasivo para o rompimento da parede celular e liberação dos carotenóides, apresentou grande eficiência ¿Observação: O resumo, na íntegra poderá ser visualizado no texto completo da tese digital. / Abstract: Not informed. / Mestrado / Mestre em Ciência de Alimentos