Metabolic depression is a ubiquitous characteristic of dormancy, allowing animals to survive off limited endogenous fuels supplies for extended periods of time. Metabolic depression is characterised by a complex set of physiological and biochemical changes (respiratory depression, bradycardia, hypophagia and cessation of renal function), in addition to a remarkable ability to prevent or minimise damage usually associated with such depressive changes (e.g. gut and muscle atrophy, renal failure and ischemic injuries). A coordinated shut-down of systems is essential to surviving long periods of dormancy, however, the mechanisms involved in regulating metabolic depression remain poorly understood. Aestivation, a period of dormancy that occurs in response to desiccating environments, provides an excellent model system for investigating the regulation of metabolic depression, because unlike other forms of dormancy such as hibernation, it is not confounded by changes in temperature or PO2 and thus represents a true intrinsic metabolic depression. The green striped burrowing frog, Cyclorana alboguttata, is an abundant species of burrowing frog inhabiting arid to semi-arid regions of Queensland and Northern New South Wales. During the dry season, C. alboguttata aestivates in an underground clay chamber; the aestivating period lasts on average for nine months a year, but in drought conditions may last for as long as several years. In recent years, C. alboguttata has become a well studied organism for investigating the physiological processes involved in aestivation, especially regarding muscle disuse atrophy and gut maintenance. Despite this, our understanding of this frog’s capacity to metabolically depress remains limited. The first aim of this study was to extend our current knowledge of metabolic depression during aestivation in C. alboguttata. C. alboguttata reduced whole animal metabolism by 82% within five weeks of aestivation. The effects of aestivation on mass specific in vitro tissue metabolic rate (VO2) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO2 was elevated and there was no change in the VO2 of small intestine tissue slices. Organ size was also affected by aestivation, with significant reductions in the mass of all tissues, except the gastrocnemius. These reductions in organ size, combined with changes in mass specific VO2 of tissue slices, resulted in further energy savings to aestivating animals. Mitochondrial VO2 was significantly reduced during aestivation, by an average of 83%; in addition respiratory control ratios (RCRs), a measurement of mitochondrial coupling efficiency, significantly increased during aestivation, suggesting increased mitochondrial coupling efficiency. The second aim of this study was to investigate the potential role of the endogenous opioid system (EOS) and thyroid hormones in the regulation of metabolic depression in C. alboguttata. When incubated in the presence of opioid agonists, liver and muscle tissue slices from Cyclorana alboguttata underwent tissue- and agonist- specific metabolic depression. In most cases, the effect of opioid agonists on metabolism was more pronounced in tissue slices from aestivating animals. The delta (δ) opioid agonist DADLE was the most consistent in producing a metabolic depression, however the effect of DADLE on the metabolism of tissue slices was not reversible by the general opioid antagonist naloxone. Gene transcript levels for the δ-opioid receptor remained constant in the brain and in the muscle of aestivating C. alboguttata compared to controls. Opioid receptor XOR1 transcript levels also remained constant in the brain during aestivation compared to controls. There was a significant reduction in total thyroid hormone concentration in the plasma of C. alboguttata during aestivation, with thyroxine (T4) decreasing by 75% and an 84% reduction in triiodothyronine (T3). We also examined changes in gene expression of deiodinase 2 (D2) and deiodinase 3 (D3), the major activating and deactivating enzymes of thyroid hormones, respectively. There were no significant changes in gene expression of D2 in the brain or muscle, or of D3 in the brain; however there was a significant down-regulation of D3 transcripts in muscle tissue. In the liver, D2 was significantly down-regulated and D3 showed an increase in transcript levels that was approaching significance. During dormancy energy conservation is a key priority and as such dormant animals undergo a major metabolic depression to conserve their limited endogenous fuel supplies. At the same time, dormant animals are faced with the challenge of maintaining functional organs for immediate use upon arousal. In this study C. alboguttata appeared to selectively down- or up-regulating individual tissues, using both changes in metabolic rate and morphology. This strategy would allow maximal energy savings during aestivation without compromising organ functionality and survival at arousal. In addition, the frogs appeared to decrease rates of mitochondrial proton leak to a greater extent than ATP synthesis, consistent with an increase in mitochondrial coupling efficiency. Again, this ability to selectively down-regulate one process to a greater extent than another allows energy savings to be maximised without compromising processes essential for survival. C. alboguttata can survive in a dormant state for several years and it is hypothesised that these frogs become more energy efficient during aestivation. The regulation of metabolic depression still remains poorly understood. In this study the potential role of the EOS and thyroid hormones in regulating metabolic depression was investigated. The results of this study suggest the δ-opioid system may be responsible for the initiation of metabolic depression, while the mu (μ) and kappa (κ) opioid systems may be important during long term maintenance of aestivation. In addition, the EOS may exert its control over metabolism via both receptor and non-receptor mediated pathways. The significant down-regulation of total thyroid hormones, as well as the changes in gene expression of the thyroid deiodinases indicates thyroid hormones may also play a significant role in regulating metabolic depression. This suggests that the regulation of metabolic depression may involve several interacting systems, or perhaps a ‘systems cascade’.

Diss. (sammanfattning) Stockholm : Karol. Inst., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Virginia, 2009. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

In this diploma work influence of needle age at Rubisco activity and content in Norway spruce (Picea abies) was studied. The plants were cultivated in conditions with ambient (A) CO2 concentration (350 µmol CO2/mol) and elevated (E) CO2 concentration (700 µmol CO2/mol). Sampling was done two times during the growing season (in the middle of June and in the end of September) were taken. Initial and total Rubisco activities were measured spectrophotometrically. Rubisco content was determined by SDS–PAGE method. Rubisco activity in 18-months-old needles was in E higher than in A. Rubisco contents in current-year needles and one-year-old needles were in A higher than in E in September. These differences were statistically significant that demonstrates the down-regulation of Rubisco content in conditions of elevated CO2 concentration. It seems the course of activities and content depending on age of the needles are antiparallel, that means that decrease of content is followed by increase of activity.

No description available.

Antisense RNA (asRNA) strategies are identified as an effective and specific method for gene down-regulation at the post-transcriptional level. In this study, the major purpose is to find a correlation between the expression level and minimum free energy to enable the design of specific asRNA fragments. The thermodynamics of asRNA and mRNA hybridization were computed based on the fluorescent protein reporter genes. Three different fluorescent proteins (i) green fluorescent protein (GFP), (ii) cyan fluorescent protein (CFP) and (iii) yellow fluorescent protein (YFP) were used as reporters. Each fluorescent protein was cloned into the common pUC19 vector. The asRNA fragments were randomly amplified and the resulted antisense DNA fragments were inserted into the constructed plasmid under the control of an additional inducible plac promoter and terminator. The expression levels of fluorescent reporter protein were determined in real time by plate reader. Different results have been observed according to the fluorescent protein and the antisense fragment sequence. The CFP expression level was decreased by 50 to 78% compared to the control. However, with the GFP, the down-regulation did not exceed 30% for the different constructs used. For certain constructs, the effect was the opposite of expected and the expression level was increased. In addition, the YFP showed a weak signal compared to growth media, therefore the expression level was hard to be defined. Based on these results, a thermodynamic model to describe the relationship between the particular asRNA used and the observed expression level of the fluorescent reporter was developed. The minimum free energy and binding percentage of asRNA-mRNA complex were computed by NUPACK software. The expression level was drawn as a function of the minimum free energy. The results showed a weak correlation, but linear trends were observed for low energy values and low expression levels the CFP gene. The linear aspect is not verified for higher energy values. These findings suggest that the lower the energy is, the more stable is the complex asRNA-mRNA and therefore more reduction of the expression is obtained. Meanwhile, the non-linearity involves that there are other parameters to be investigated to improve the mathematical correlation. This model is expected to offer the chance to "fine-tune" asRNA effectiveness and subsequently modulate gene expression and redirect metabolic pathways toward the desired component. In addition, the investigation of the localization of antisense binding indicates that there are some regions that favors the hybridization and promote hence the down-regulation mechanisms. / Master of Science

Tetranychus urticae es una especie plaga importante en cítricos que puede también alimentarse de otras especies asociadas a la cubierta vegetal de este cultivo. Para determinar el efecto de la gestión de la cubierta vegetal en el control biológico de este ácaro, hemos estudiado la dinámica tanto de ácaros Tetranychidae como Phytoseiidae en cuatro parcelas comerciales de mandarino clementino en las que se aplicó tres estrategias diferentes de gestión de la cubierta vegetal: (1) suelo desnudo, (2) cubierta espontánea y (3) cubierta sembrada de Festuca arundinacea. Los resultados apuntan a que tanto los enemigos naturales (mecanismos "top-down") como la planta huésped (mecanismos "bottom-up") juegan un papel importante en la regulación de los ácaros Tetranychidae. Por un lado, la selección de dos razas de T. urticae especializadas en F. arundinacea y en Citrus clementina, en la cubierta y en el árbol respectivamente, cuando esta gramínea se utiliza como cubierta podría explicar en parte los resultados obtenidos (regulación "bottom-up") ya que esto impediría a los especímenes de una planta huésped colonizar con éxito la otra. Los ensayos de trasplante recíproco realizados muestran que las dos demos de T. urticae recogidas de clementina y F. arundinacea difieren considerablemente en su éxito en el desarrollo en el huésped alternativo y esto indica la existencia de fenómenos de adaptación local. Esta adaptación se traduciría en mecanismos "bottom-up" que evitarían que los ácaros que habitan en la cubierta colonicen con éxito la copa de los árboles. Por otro lado, la composición cualitativa de las comunidades de Phytoseiidae asociados a las diferentes cubiertas podría ser clave en la regulación de las poblaciones de T. urticae y Panonychus citri (regulación "top-down"). Los ácaros Phytoseiidae tipo I y II, depredadores especializados en Tetranychidae, se encuentran de manera consistente en la cubierta de F. arundinacea y esto puede explicar la mejor regulación de las poblaciones de ácaros Tetranychidae en los árboles asociados a esta cubierta. Por el contrario, la disposición más regular de fuentes de alimentación alternativas (polen) en la cubierta natural en relación con la cubierta de F. arundinacea, podría explicar la mayor abundancia de Phytoseiidae tipo IV en la primera. Como consecuencia, los Phytoseiidae tipo I y II, más eficaces en el control de Tetranychidae, podrían sufrir las consecuencias de ser competitivamente inferiores que el Phytoseiidae generalista tipo IV que explota el polen en la cubierta espontánea. Este hecho, en combinación con los períodos de escasez de presa, podría dar lugar a su desaparición del agroecosistema y resultar en un deficiente control de los ácaros Tetranychidae en los árboles asociados a una cubierta natural. Haciendo balance de gastos e ingresos, la cubierta más favorable fue la de F. arundinacea (entre 44,4 y 74,5% de reducción de costes en relación con la más cara). Festuca arundinacea como cubierta vegetal es una estrategia de control biológico por conservación muy recomendable para los productores de clementina. Aunque su uso no redujo las poblaciones de ácaros en los árboles por debajo del umbral económico, la disminución en la necesidad de tratamientos, hace que la adopción de esta táctica sea una alternativa beneficiosa tanto ecológica como económicamente. Nuestros resultados apuntan a la cubierta de F. arundinacea, que no permitió el establecimiento de Tetranychus evansi y ofrece una mejor regulación de P. citri y T. urticae que en suelo desnudo o cubierta natural, como la más adecuada para un control más sostenible de los ácaros Tetranychidae en cítricos.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.