The physiological function of the plant enzyme alternative oxidase has long been a topic of debate. The cyanide-resistant alternative oxidase (AOX), along with the cytochrome c oxidase (COX), catalyzes the reduction of oxygen to water in the electron transport chain of mitochondrial respiration. Although respiration via the alternative pathway (AP) results in approximately one third of the ATP production as respiration via the cytochrome pathway (CP), the AP is utilized by all plants and some fungi and animals. This “energy wasteful” pathway has been proposed to reduce oxidative stress in plant cells under a variety of stressful conditions. Virtually all previous work on the AP has been performed on laboratory-grown plants in controlled environment conditions; thus, there is little knowledge of how the AP responds to unstable conditions and multiple environmental stresses in the field. This thesis presents new methodology for studying AP respiration and the AOX protein in field-grown plants, and investigates how the AP responds to natural changes in environmental conditions in the field in several plant species grown in diverse ecosystem types. The experimental work presented here also investigates how AP activity is related to changes in total rates of respiration, and questions whether abundance of the AOX protein determines electron partitioning to the AP. AP partitioning (or relative changes in AP partitioning) varied over seasonal timescales in each of the experimental studies. Chapter 3 reports on two species of Chionochloa, a native New Zealand tussock grass growing along an altitudinal gradient. In Chapter 4, seasonal variation was studied in two tree types: Populus x canadensis, a deciduous angiosperm, and Pinus radiata, an evergreen gymnosperm. Quercus rubra trees were studied along an urban-rural gradient originating in New York City in Chapter 5. In a highly exposed and variable environment, relative changes in AP partitioning in two species of Chionochloa were correlated with the previous day’s integrated light. In Quercus rubra, the AP was instead related to temperature changes: relative AP partitioning increased in response to seasonally low temperature in trees grown at colder, more rural field sites, while at the warmer, urban sites, it increased in response to high summer temperatures. Each of these environmental conditions that were related to increases in the AP (high light, low temperatures, and heat) are potentially stressful to plants. Thus, it is possible that the increases in AP respiration observed in these studies served to oxidize excess reducing equivalents generated through stressful conditions. In Chapter 4, although AP partitioning in Populus x canadensis and Pinus radiata varied seasonally, these changes were not directly related to environmental parameters. However, AP partitioning in Populus x canadensis was clearly shown to be dependent on measurement temperature. In each of the studies presented here, changes in the AP were not related to abundance of the AOX protein. AOX protein abundance showed consistent seasonal patterns in the two deciduous angiosperms, Populus sp. and Quercus sp, and was correlated with seasonal changes in temperature in Chionochloa spp. However, the lack of correlation between protein abundance and AP partitioning indicates that the AP is subject to post-translational control and likely varies more rapidly than protein levels. In each of Chapters 3 – 5, there was no clear impact of changes in AP partitioning on rates of total respiration. As the AP produces less ATP than the CP, I hypothesized that increases in AP activity would lead to higher respiration rates in order to meet a plant’s energy demands. However, in Populus x canadensis and Quercus rubra, respiration rates remained stable during sharp increases in AP partitioning, indicating that, at least under certain conditions, increases in AP activity are accompanied by a decrease in the CP. In some of the first research studying AP partitioning in field-grown plants, this thesis illuminates possible mechanisms, functions, and implications of the AP. Over a range of plant taxa and environmental settings, this work shows that the AP does respond to stressful conditions in the wild, but that this does not result in increased respiration. Lastly, the methods presented here to study AP activity and AOX proteins in the field enable future studies to further probe the specific responses of AOX to natural stresses.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 101-112). Available also in a digital version from Dissertation Abstracts.

Considerando-se a semelhança morfológica e ocorrência simpátrica de Anastrepha striata Schiner e Anastrepha bistrigata Bezzi, este trabalho propôs identificar formas para melhor caracterizá-las através de análises morfométricas do acúleo e asas de populações simpátricas e alopátricas dessas espécies, e molecular, por meio da análise de seqüências parciais do gene da citocromo oxidase I (COI). O trabalho também teve por objetivo desenvolver iniciadores espécie-específicos que possam vir a ser utilizados em estudos ecológicos em regiões em que ambas as espécies ocorram simpatricamente. As populações analisadas A. striata (seis populações) e A. bistrigata (duas populações) foram provenientes de diferentes regiões do Brasil, Colômbia e Bolívia. Para os estudos morfométricos, os acúleos e asas foram montados em lâminas para captura e análise de imagens com auxílio de câmera fotográfica acoplada ao microscópio estereoscópico, utilizando-se de software apropriado. Foram traçados e mensurados oito segmentos no acúleo e 16 na asa direita de cada indivíduo, obtendo-se assim as medidas nas quais se basearam as análises. Nos estudos moleculares, os produtos de amplificação obtidos para a COI foram seqüenciados e analisados para o estudo da caracterização molecular das diferentes populações desses insetos e desenvolvimento de iniciadores espécie-específicos. Os resultados indicam a existência clara de distinção morfométrica entre as duas espécies e confirmaram as identificações baseadas na largura e comprimento do acúleo. Os dados morfométricos mostraram também a ausência de padrões geográficos distintos entre as populações de A. striata e A. bistrigata. A. striata apresentou maior variação morfométrica entre suas populações quando comparada à diferenciação encontrada para A.bistrigata. Nas duas espécies, populações encontradas em maiores altitudes apresentaram maiores dimensões. A análise de nucleotídeos da seqüência da COI obtida para as populações de A. striata em questão indicou a ocorrência de alta similaridade entre as mesmas. As análises realizadas também indicaram que a divergência genética encontrada no fragmento da COI analisado para as diferentes populações de A. striata não permitiu o agrupamento de acordo com a distribuição geográfica dessas populações. A análise do fragmento do gene da COI permitiu o desenvolvimento de iniciadores com divergência suficiente entre as espécies em questão para permitir a amplificação de porção específica do gene da COI, que possibilita a diferenciação entre as mesmas. Os iniciadores propostos reúnem características que permitem a sua utilização em reações de PCR multiplex para a diferenciação entre espécimes de ambas as espécies. / Because of the fact that Anastrepha striata Schiner and Anastrepha bistrigata Bezzi are very similar morphologically and may also occur sympatrically, this study aimed to propose different ways to characterize them by using wing and aculeus morphometry, and mitochondrial DNA sequencing. This work also aimed to develop species-specific primers to allow for the differentiation between both species in sympatric areas. Six A. striata and two A. bistrigata populations were obtained from different regions from Brazil, Colombia and Bolivia. Specimens from different populations had their aculeus and wings removed and slide mounted for image acquisition under a stereomicroscope and morphometric analysis. Data collection for morphometry were taken for each specimen on eight segments of the aculeus and on 16 of the right wing. The molecular characterization consisted of the sequencing and analysis of a fragment of the cytochrome oxidase I (COI) gene, and the development of species-specific primers. Morphometry did not indicate any consistent variation to allow for geographical characterization of different populations, but indicated A. striata and A. bistrigata are consistently different morphologically. Populations of either species from higher altitudes were larger than the ones at lower altitudes, and A. striata displayed a much higher intraespecific variation in morphology than A.bistrigata. COI nucleotide sequences for A. striata were very similar among the populations studied, and the molecular analysis did not yield groups that were related geographically. The COI analysis allowed the development of a set of species-specific primers that were divergent enough to allow for the discrimination of A. striata and A. bistrigata in multiplex PCR reactions.

A análise da estruturação populacional de espécies codistribuídas permite a comparação de padrões de estruturação, fornecendo informações acerca dos fatores que influenciam a diferenciação em espécies pertencentes ao mesmo ecossistema. Este projeto teve como objetivo principal analisar a variabilidade intraespecífica em caranguejos habitantes de manguezais, codistribuídos ao longo do Oceano Atlântico Ocidental, por meio de ferramentas moleculares e morfológicas, visando testar a hipótese de elevada estruturação populacional em manguezais. Para este fim, cinco espécies foram utilizadas como modelo (Aratus pisonii, Goniopsis cruentata, Sesarma rectum, Uca thayeri e Ucides cordatus) e avaliadas por meio de marcadores mitocondriais COI e 16S e nuclear H3 e análises morfológicas comparativas e de morfometria. Os dados moleculares revelaram dois padrões, indicando elevada estruturação populacional para as espécies A. pisonii e U. thayeri e ausência de estruturação para G. cruentata, S. rectum e U. cordatus. Os dados morfológicos, no entanto, não acompanham esses padrões, já que não foram encontradas diferenças morfológicas ou morfométricas associadas aos grupos evidenciados pelas análises moleculares. A ausência de fluxo gênico entre regiões para algumas espécies deve-se, muito provavelmente, à existência de fatores que não se limitam ao isolamento por distância, mas também devido a diferenças na duração do estágio larval e a diferenças bruscas em alguns fatores abióticos, como a salinidade, por exemplo, que, associados às diferentes características do desenvolvimento larval de cada espécie, culminam na existência de estruturas populacionais diferentes. Além disso, os padrões de diferenciação genética observados concordam com os cenários biogeográficos propostos para o Atlântico Ocidental, no qual mudanças e flutuações geológicas, climáticas e oceanográficas, resultantes do fechamento do Istmo do Panamá e de ciclos glaciais na América do Norte, promoveram divergência genética. / The analysis of population structure of codistributed species allows the comparison of patterns of population structuring, providing information on the factors that influence the differentiation in species belonging to the same ecosystem. This project aimed to analyze the intraspecific variability of crabs inhabiting mangroves, codistributed along the western Atlantic Ocean, by means of molecular and morphological tools, in order to test the hypothesis of high populational structure in mangroves. The genetic variability of five species used as models (Aratus pisonii, Goniopsis cruentata, Sesarma rectum, Uca thayeri and Ucides cordatus) was assessed using the mitochondrial genes COI and 16S and the nuclear H3 and comparative morphological analysis and morphometry were performed. The molecular data revealed two patterns, indicating high population structure for the species A. pisonii and U. thayeri and absence of structure for G. cruentata, S. rectum and U. cordatus. The morphological data, however, do not follow these patterns, because there were no morphological or morphometric differences associated with the groups evidenced by the molecular analyzes. Possibly the absence of gene flow between regions for some species is due to factors not limited to isolation by distance, but also because of differences in the larval stage duration and in withstanding some abiotic factors, such as salinity. These factors associated with different characteristics of larval development can culminate in differences on population structure. In addition, the genetic differentiation patterns observed agree with biogeographical scenarios proposed for the western Atlantic, where geological, climate and oceanographic fluctuations, resulting from the Isthmus of Panama closure and cyclical glaciation in North America, promoted genetic divergence.

No description available.

Urophycis Gill, 1863 é um gênero de peixes marinhos demersais de médio porte, popularmente conhecidos como abróteas, distribuídos ao longo da costa do Atlântico Ocidental, do Canadá à Argentina. Atualmente são reconhecidas sete espécies válidas dentro deste gênero. Apesar de sua importância comercial, até então poucos estudos taxonômicos e biogeográficos haviam sido realizados com estas espécies. Três espécies foram descritas do Atlântico Sul ocidental: U. brasiliensis (Kaup, 1858), descrita de Motevideo, Uruguai, U. latus Miranda Ribeiro, 1903 e Urophycis mystacea Miranda Ribeiro, 1903, ambas descritas do Rio de Janeiro, Brasil. Urophycis mystacea é morfologicamente semelhante à U. cirrata (Goode & Bean, 1896), descrita do Golfo do México nos Estados Unidos, sendo frequentemente confundidas. Os objetivos desta contribuição foram (1) redescrever U. brasiliensis, e alocar U. latus como um sinônimo júnior ; (2) redescrever U. cirrata e alocar U. mystacea como um sinônimo júnior; (3) estabelecer diagnoses e mapas de distribuição atualizados para ambas espécies. Tecidos e exemplares foram obtidos com pescadores e em coleções científicas. Foram utilizados métodos tradicionais de morfologia para comparação dos exemplares, além de técnicas de identificação molecular utilizando o gene mitocondrial citocromo oxidase I (COI). Testes para a amplificação da região controle do DNA mitocondrial de U. brasiliensis foram realizados e discutidos. / Urophycis Gill, 1863 is a genus of marine, demersal, mid-sized fish, popularly known as codlings or hakes, and distributed along the the Western Atlantic coast, from Canada to Argentina. Seven species are currently recognized as valid within this genus. Despite its economical importance, a few taxomomic an biogeographic studies had been carried out with those species. Three species were described from western South Atlantic: U. brasiliensis (Kaup, 1858), described from Montevideo, Uruguay, U. latus Miranda Ribeiro, 1903 and Urophycis mystacea Miranda Ribeiro, 1903, both described from Rio de Janeiro, Brazil. Urophycis mystacea is morfologically similar to U. cirrata (Goode & Bean, 1896), described from Gulf of Mexico, USA, and these species are repeteadly confused. The objectives of this contributions were (1) redescribe U. brasilienis, placing U. latus as a junior synonym; (2) redescribe U. cirrata, placing U. mystacea as a junior synonym; (3) provide updated diagnosis and maps of distributions for both species. Tissues and specimens were obtained from fisherman and scientific collections. Traditional morphological methods were used to compare the specimens, besides molecular identification techniques using the mithocondrial gene citochrome c oxidadse I (COI). Testst for amplification of the DNA mitochondrial control region in U. brasiliensis were performed and discussed.

Urophycis Gill, 1863 é um gênero de peixes marinhos demersais de médio porte, popularmente conhecidos como abróteas, distribuídos ao longo da costa do Atlântico Ocidental, do Canadá à Argentina. Atualmente são reconhecidas sete espécies válidas dentro deste gênero. Apesar de sua importância comercial, até então poucos estudos taxonômicos e biogeográficos haviam sido realizados com estas espécies. Três espécies foram descritas do Atlântico Sul ocidental: U. brasiliensis (Kaup, 1858), descrita de Motevideo, Uruguai, U. latus Miranda Ribeiro, 1903 e Urophycis mystacea Miranda Ribeiro, 1903, ambas descritas do Rio de Janeiro, Brasil. Urophycis mystacea é morfologicamente semelhante à U. cirrata (Goode & Bean, 1896), descrita do Golfo do México nos Estados Unidos, sendo frequentemente confundidas. Os objetivos desta contribuição foram (1) redescrever U. brasiliensis, e alocar U. latus como um sinônimo júnior ; (2) redescrever U. cirrata e alocar U. mystacea como um sinônimo júnior; (3) estabelecer diagnoses e mapas de distribuição atualizados para ambas espécies. Tecidos e exemplares foram obtidos com pescadores e em coleções científicas. Foram utilizados métodos tradicionais de morfologia para comparação dos exemplares, além de técnicas de identificação molecular utilizando o gene mitocondrial citocromo oxidase I (COI). Testes para a amplificação da região controle do DNA mitocondrial de U. brasiliensis foram realizados e discutidos. / Urophycis Gill, 1863 is a genus of marine, demersal, mid-sized fish, popularly known as codlings or hakes, and distributed along the the Western Atlantic coast, from Canada to Argentina. Seven species are currently recognized as valid within this genus. Despite its economical importance, a few taxomomic an biogeographic studies had been carried out with those species. Three species were described from western South Atlantic: U. brasiliensis (Kaup, 1858), described from Montevideo, Uruguay, U. latus Miranda Ribeiro, 1903 and Urophycis mystacea Miranda Ribeiro, 1903, both described from Rio de Janeiro, Brazil. Urophycis mystacea is morfologically similar to U. cirrata (Goode & Bean, 1896), described from Gulf of Mexico, USA, and these species are repeteadly confused. The objectives of this contributions were (1) redescribe U. brasilienis, placing U. latus as a junior synonym; (2) redescribe U. cirrata, placing U. mystacea as a junior synonym; (3) provide updated diagnosis and maps of distributions for both species. Tissues and specimens were obtained from fisherman and scientific collections. Traditional morphological methods were used to compare the specimens, besides molecular identification techniques using the mithocondrial gene citochrome c oxidadse I (COI). Testst for amplification of the DNA mitochondrial control region in U. brasiliensis were performed and discussed.

Sprague-Dawley rats were administered daily, subcutaneous injections of methylmercuric chloride at a dose of 5 mg/Hg/kg beginning on postnatal day 5. By their fourth postnatal week, animals exhibited a constellation of neurological signs of motor impairment which resembled the cerebral palsy syndrome of humans perinatally exposed to methylmercury. Routine histological examination of the brain revealed no gross differences between methylmercury-treated (MeHg), normal control (NC) or weight-matched littermates. The histochemical localization of the mitochondrial enzyme cytochrome oxidase (CO) was utilized in Experiment I to examine possible alterations in the metabolic activity of motor nuclei which might contribute to the observed movement and postural disorders. A population of intensely-staining cytochrome oxidase neurons (ICONs) in the magnocellular portion of the red nucleus (RMC) and interrubral mesencephalon (IRM) were conspicuously present in all MeHg animals at the onset of motor impairment. These morphologically, histochemically, and anatomically distinct neurons did not exhibit intense CO staining in control animals. Conversely, a significant decrease was demonstrated in the oxidative metabolic activity of many neurons in the substantia nigra, zona reticulata of MeHg animals. In Experiment II, the postnatal appearance of ICONs was morphometrically quantified in MeHg animals sacrificed at PND 14, 16, 18, 20, 22, or 25. The histochemically-defined onset of increased metabolic activity in ICONs was first observed on PND 16, at least one week before the onset of clinical signs of neurological impairment. This was the earliest manifestation of methylmercury neurotoxicity yet described in this animal model. A subsequent four-fold increase in the total number of ICONs at PND 18 was followed by a gradual decrease in number to PND 25. Significantly more of the ICONs were found in the IRM than in the RMC at PND 18 & 20. The possibility that the increased activity of ICONs may result from disinhibition of specific afferents to the red nucleus was addressed by introducing either hemidecortication or hemicerebellectomy on PND 10 and then morphometrically determining the deviation from symmetry in the bilateral distribution of the total number of ICONs in the RMC and IRM at PND 22. The distribution of ICONs was symmetrical and not different in either hemidecorticate or unoperated controls. A significant (36%) decrease in the total number of ICONs was observed in both the RMC and IRM contralateral to hemicerebellectomy. The identical ipsilateral regions did not differ from control or hemidecorticate MeHg animals. In Experiment III, the anatomical distribution of major histocompatability complex antigens (MHC) in the brain of MeHg animals was examined using immunohistochemical methods. MHC immunoreactivity was widely distributed throughout the brain of MeHg animals. Areas with low immunoreactivity, or lack of it, stand out and include all of the hippocampus, thalamus, pyriform and entorhinal cortex, and lateral cerebellar hemispheres. Moderate staining intensity was observed in neocortical areas, basal forebrain, caudate-putamen and cerebellar vermis. Strong immunoreactivity was found in red nucleus, substantia nigra, cingulate cortex, retrosplenial cortex, presubiculum, parasubiculum and vestibular nuclei. It was suggested that the increased activity of ICONs likely contributes to the movement and postural disorders resulting from methylmercury intoxication. The increased activity in ICONs was determined to be, at least partially, dependent upon cerebellar input. The results are discussed with reference to the toxic effects of methylmercury and specifically to the susceptibility of GABAergic interneurons in perinatal trauma. Possible analogies are drawn between the mechanisms of methylmercury-induced cerebral palsy syndrome and those of other developmental movement and postural disorders. / Medicine, Faculty of / Graduate

<p>In the present thesis the density functional B3LYP has been used to study reactions of NO and O₂ in redox active enzymes.</p><p>Reduction of nitric oxide (NO) to nitrous oxide (N₂O) is an important part in the bacterial energy conservation (denitrification). The reduction of NO in three different bimetallic active sites leads to the formation of hyponitrous acid anhydride (N₂O₂^2-). The stability of this intermediate is crucial for the reaction rate. In the two diiron systems, respiratory and scavenging types of NOR, it is possible to cleave the N-O bond, forming N₂O, without any extra protons or electrons. In a heme-copper oxidase, on the other hand, both a proton and an electron are needed to form N₂O.</p><p>In addition to being an intermediate in the denitrification, NO is a toxic agent. Myoglobin in the oxy-form reacts with NO forming nitrate (NO₃ ^-) at a high rate, which should make this enzyme an efficient NO scavenger. Peroxynitrite (ONOO^-) is formed as a short-lived intermediate and isomerizes to nitrate through a radical reaction.</p><p>In the mechanism for pumping protons in cytochrome oxidase, thermodynamics, rather than structural changes, might guide protons to the heme propionate for further translocation.</p><p>The dioxygenation of arachidonic acid in prostaglandin endoperoxide H synthase forms the bicyclic prostaglandin G₂, through a cascade of radical reactions. The mechanism proposed by Hamberg and Samuelsson is energetically feasible.</p>

In the present thesis the density functional B3LYP has been used to study reactions of NO and O2 in redox active enzymes. Reduction of nitric oxide (NO) to nitrous oxide (N2O) is an important part in the bacterial energy conservation (denitrification). The reduction of NO in three different bimetallic active sites leads to the formation of hyponitrous acid anhydride (N2O22-). The stability of this intermediate is crucial for the reaction rate. In the two diiron systems, respiratory and scavenging types of NOR, it is possible to cleave the N-O bond, forming N2O, without any extra protons or electrons. In a heme-copper oxidase, on the other hand, both a proton and an electron are needed to form N2O. In addition to being an intermediate in the denitrification, NO is a toxic agent. Myoglobin in the oxy-form reacts with NO forming nitrate (NO3 -) at a high rate, which should make this enzyme an efficient NO scavenger. Peroxynitrite (ONOO-) is formed as a short-lived intermediate and isomerizes to nitrate through a radical reaction. In the mechanism for pumping protons in cytochrome oxidase, thermodynamics, rather than structural changes, might guide protons to the heme propionate for further translocation. The dioxygenation of arachidonic acid in prostaglandin endoperoxide H synthase forms the bicyclic prostaglandin G2, through a cascade of radical reactions. The mechanism proposed by Hamberg and Samuelsson is energetically feasible.