At the cellular level, the stress response involves the synthesis of a highly conserved family of heat shock proteins (Hsps). These proteins are essential for maintenance of cellular homeostasis, both in times of stress and in normal cell functioning. Some of the most abundant forms of Hsps in the cell are members of the 70 kDa family. Intracellular heat shock protein 70 (Hsp70) expression in response to proteotoxicity is a highly conserved cellular stress response, but little is known about the role of extracellular Hsp70 (eHsp70) in fish. In order to begin characterizing eHsp70 in fish, the hypothesis that an acute stressor will elevate plasma Hsp70 levels in rainbow trout (Oncorhynchus mykiss) was tested. Subsequent in vitro studies examined whether eHsp70 level was modulated by cortisol and if this involved the action of the glucocorticoid receptor (GR), a ligand-activated transcription factor. The effect of cortisol on the eHsp70 response is important to consider because this steroid is elevated as a result of stressor exposure to allow for short-term allocation of energy stores to cope with stress. Cortisol is the primary corticosteroid in fish and exerts its main effects by binding to either GR or mineralocorticoid receptors (MR). Furthermore, eHsp70 has been previously implicated as having important immunoregulatory roles in mammalian models, but nothing has yet been reported in fish. To this end, a hypothesis tested here was that eHsp70 levels will increase after exposure to the bacterial endotoxin lipopolysaccharide (LPS), and that this response is modulated by cortisol. Finally, research on the effects of exogenous Hsp70 has not been reported in lower vertebrates; however, the relevance of this protein in intercellular signaling, especially in regards to immune regulation, is gaining increasing importance in mammalian models. Therefore, an experiment to determine whether Hsp70 would elicit upregulation of key immunoregulatory cytokines was also conducted. To accurately measure the low levels of Hsp70 in the plasma, a competitive antibody-capture enzyme-linked immunosorbent assay (ELISA) was developed. In the in vivo study, fish exposed to an acute heat shock (1h at 10°C above ambient temperature) exhibited a significant elevation in red blood cell Hsp70 levels over a 24 h period. There was also a significant increase in plasma Hsp70 levels at 4 h, but not at 24 h post-heat shock. To more specifically determine how cortisol affected the release of Hsp70, in vitro studies using primary cultures of hepatocytes demonstrated that cortisol significantly decreased eHsp70 levels in the medium at 24 h when compared with untreated controls, and this response was abolished in the presence of a GR antagonist, mifepristone (RU486). This result for the first time established a link between cortisol signaling and eHsp70 release in any animal model. When hepatocytes were exposed to LPS in vitro, eHsp70 levels were significantly lower in the LPS (30 µg/ml) group; however, heat shock abolished this effect at 24 h. Though eHsp70 levels in the heat shocked hepatocytes treated with low-dose LPS (10 µg/ml) was similar to untreated control levels, high-dose LPS treated hepatocytes showed significant elevation of eHsp70 levels above the low dose group. The ability of LPS to modulate eHsp70 release was not observed to be further regulated by cortisol. While this work suggests the modulation of eHsp70 by LPS, the physiological role remains to be elucidated. Finally when hepatocytes were exposed to exogenous Hsp70, there was no effect on key immunoregulatory genes (IL-1β and IL-8) transcript levels; however, the effect of this protein remains to be tested using other cell systems, including immune cells in fish. Overall, eHsp70 concentration was measured in trout plasma using a competitive ELISA and demonstrates for the first time that stressor exposure affects plasma eHsp70 levels in fish. Furthermore, cortisol, the primary corticosteroid in teleosts, modulates eHsp70 release in trout hepatocytes and this is action is mediated by GR signaling. Also, while trout hepatocytes secrete eHsp70 in response to endotoxin shock, a role for eHsp70 in eliciting an immune response is not clear in lower vertebrates. Taken together the results from this study suggest a role for eHsp70 in acute stress adaptation in fish, but the target tissues involved and the physiological responses remain to be elucidated. Further work on the effects of eHsp70 on target tissues effects, and the mechanisms involved, may have important implications in our understanding of the role of this stress protein in cell signaling and stress adaptation in fish.

Ebola virus (EBOV) causes severe disease in humans characterized by high case fatality rates and significant immune dysfunction. A hallmark of EBOV infection is the formation of viral inclusions in the cytoplasm of infected cells. These inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV gene expression and genome replication, little is known about their role in the host response to infection. The cellular stress response is an antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Related to this response is the post-transcriptional regulation of RNA mediated by stability elements called AU-rich elements (AREs) and their associated binding proteins (ARE-BPs), many of which are found in SGs. Because these processes have antiviral implications, many viruses have evolved strategies to interfere with SG formation, or appropriate ARE-BPs to benefit viral replication. However, it is unknown if EBOV interacts with these cellular systems. Here, we show that SG proteins were sequestered within EBOV inclusions where they formed distinct granules that colocalized with viral RNA. The inclusion-bound aggregates were not canonical SGs, and did not lead to translational arrest in infected cells. EBOV did not induce cytoplasmic SGs at any time post infection, but was unable to overcome SG formation induced by additional stressors. Despite the sequestration of SG proteins, canonical SGs did not form within inclusions. At high levels of expression, viral protein 35 (VP35), the viral polymerase co-factor that also mediates various immune evasion functions, disrupted SGs formation independently of eIF2α phosphorylation. Finally, we found that the cellular ARE-BP tristetraprolin (TTP) specifically targeted the 3’untranslated region (UTR) of the viral nucleoprotein (NP) mRNA and promoted its degradation. Interestingly, TTP was not found within viral inclusions, leading us to speculate that inclusions might serve to prevent viral RNA from encountering TTP. These results indicate that EBOV interacts with the cellular stress response and associated RNA regulatory proteins in ways that promote viral replication.

abstract: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative enteric pathogen that causes self-limiting gastroenteritis in healthy individuals and can cause systemic infections in those who are immunocompromised. During its natural lifecycle, S. Typhimurium encounters a wide variety of stresses it must sense and respond to in a dynamic and coordinated fashion to induce resistance and ensure survival. Salmonella is subjected to a series of stresses that include temperature shifts, pH variability, detergent-like bile salts, oxidative environments and changes in fluid shear levels. Previously, our lab showed that cultures of S. Typhimurium grown under physiological low fluid shear (LFS) conditions similar to those encountered in the intestinal tract during infection uniquely regulates the virulence, gene expression and pathogenesis-related stress responses of this pathogen during log phase. Interestingly, the log phase Salmonella mechanosensitive responses to LFS were independent of the master stress response sigma factor, RpoS, departing from our conventional understanding of RpoS regulation. Since RpoS is a growth phase dependent regulator with increased stability in stationary phase, the current study investigated the role of RpoS in mediating pathogenesis-related stress responses in stationary phase S. Typhimurium grown under LFS and control conditions. Specifically, stationary phase responses to acid, thermal, bile and oxidative stress were assayed. To our knowledge the results from the current study demonstrate the first report that the mechanical force of LFS globally alters the S. Typhimurium χ3339 stationary phase stress response independently of RpoS to acid and bile stressors but dependently on RpoS to oxidative and thermal stress. This indicates that fluid shear-dependent differences in acid and bile stress responses are regulated by alternative pathway(s) in S. Typhimurium, were the oxidative and thermal stress responses are regulated through RpoS in LFS conditions. Results from this study further highlight how bacterial mechanosensation may be important in promoting niche recognition and adaptation in the mammalian host during infection, and may lead to characterization of previously unidentified pathogenesis strategies. / Dissertation/Thesis / Masters Thesis Biology 2016

Made available in DSpace on 2014-12-17T14:05:19Z (GMT). No. of bitstreams: 1 DelanneCSSF_TESE.pdf: 5733269 bytes, checksum: db8ba3b543c1bd1c178d56cb6e6335bb (MD5) Previous issue date: 2011-09-08 / The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III ? subunit). In this way, we propose that the C. violaceum ? subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., ?S subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress. / O seq?enciamento do genoma da esp?cie Chromobacterium violaceum identificou um cromossomo simples circular de 4.8 Mb, no qual aproximadamente 40% das ORFs encontradas s?o classificadas como hipot?ticas conservadas ou hipot?ticas. Algumas regi?es g?nicas de interesse biotecnol?gico e biol?gico v?m sendo caracterizadas, como por exemplo, genes de detoxifica??o ambiental e genes de reparo de DNA, respectivamente. Diante disso, o objetivo deste trabalho foi identificar genes de C. violaceum envolvidos com a resposta ao estresse, como por exemplo, mecanismos de reparo de DNA e/ou manuten??o da integridade gen?mica. Para tanto, foi constru?da uma biblioteca gen?mica de C. violaceum na cepa DH10B de Escherichia coli (RecA-), a qual foi testada para clones resistentes a UVC, resultando na sele??o de cinco clones candidatos. Foram identificadas quatro ORFs (CV_3721 a 3724) no clone PLH6A. Das quais, as ORFs CV_3722 e CV_3724, foram subclonadas e uma atividade sin?rgica de complementa??o foi observada. A ocorr?ncia de um operon foi confirmada usando cDNA de C. violaceum em ensaio de RT-PCR. Adicionalmente, foi observada a indu??o do operon ap?s tratamento com UVC, dessa forma, esse operon foi relacionado ? resposta ao estresse em C. violaceum. Ensaios de mutag?nese com rifampicina ap?s tratamento com luz UVC mostraram alta freq??ncia de mutagenicidade para a ORF CV_3722, subunidade ? da Polimerase III. Assim, propomos que esta subunidade de C. violaceum pode agir em DH10B na s?ntese transles?o utilizando Pol IV em uma via RecA independente. Em ensaios de viabilidade outros quatro clones (PLE1G, PLE7B, PLE10B e PLE12H) foram capazes de complementar a fun??o na dose de 5 J/m2. E em ensaios de mutagenicidade PLE7B, PLE10B e PLE12H apresentaram freq??ncias de muta??o com diferen?as significativas em rela??o ao controle (DH10B), demonstrando que de alguma forma eles est?o envolvidos na resposta ao estresse em C. violaceum. Estes clones parecem estar inter-relacionados, provavelmente, regulados por mol?cula mensageira (como o nucleot?deo c-di-GMP) e/ou mol?cula regulat?ria global (como a subunidade ?S da RNA Polimerase). Os resultados obtidos contribuem para um melhor conhecimento da gen?tica desta esp?cie e de seus mecanismos de resposta ao estresse ambiental. / 2020-01-01

In our study, we analyzed four stress markers (cortisol, cortisone, DHEA and DHEAS) in blood serum in young sows using minimally invasive heart catheterisation as the stress factor. The marker levels were assessed in four defined periods of the experiment, beginning with the baseline level on the day before intervention (1), the second period was after the introduction of anaesthesia (2), the third was after conducting tissue stimulation or ablation (3), and the final period was after the end of the catheterisation (4). Cortisol and cortisone were detected using HPLC method, DHEA(S) by commercial kits. For statistical analyses non-parametric tests were used (due to non standard Gaussian data distribution). In our study we arranged these experiments: 1. Diurnal variability in these markers concentration during heart catheterisation was tested. 2. Are there differences between stress markers concentration the day before experiment (sampling 1) and in the day of the catheterisation (samplings 2, 3, 4)? 3. Are there differences between these markers concentration during operation (the first sampling was excluded)? 4. The cortisol/cortisone ratio was calculated. We found only minimal statistical differences in studied markers between the morning and afternoon group (p > 0.05) in experiment 1. For tested...

A YakA é uma proteína quinase necessária para a regulação da resposta a diversos estresses em Dictyostelium e é uma efetora chave da transição do crescimento para desenvolvimento nesse organismo. O gene keaA foi isolado como um supressor do mutante yakA- em uma busca para revelar genes envolvidos na sobrevivência a estresse nitrosoativo. O gene keaA codifica uma proteína com seis repetições kelch na porção C-terminal, um domínio zf-C3HC4 na porção N-terminal, também chamado RING-finger, e uma sequência rica em cisteínas localizada na porção mediana da proteína. Mutantes deficientes em keaA foram avaliados revelando-se um papel para esse gene também no processo de desenvolvimento. A expressão de mRNA de keaA é induzida quando células selvagens crescem e a fonte de alimento começa a esgotar. A indução do mRNA de keaA também ocorre durante o desenvolvimento. Essa indução não é observada em células yakA- indicando que YakA regula KeaA. Células deficientes em keaA expressam baixos níveis de mRNA de pkaC, acaA e carA durante a agregação em baixa densidade celular, o que pode explicar porque as células deficientes em keaA apresentam o processo de desenvolvimento mais lento em baixa densidade celular. Células deficientes em keaA são mais resistentes a estresses nitrosoativo e oxidativo e keaA é necessário para a produção e detecção de AMPc. A análise da agregação de células deficientes em keaA durante o desenvolvimento multicelular indica que KeaA é necessário para que as células participem eficientemente desse processo. Células que super-expressam o domínio rico em cisteínas levam o mesmo tempo que as células selvagens para atingir o estágio de agregação. No entanto, essas células apresentam agregados, culminantes e corpos de frutificação menores. Células que super-expressam o domínio Kelch expressam altos níveis de acaA e carA depois de 8 horas de desenvolvimento, mas os níveis de pkaC são similares aos observados em células selvagens. Isso poderia indicar que o domínio Kelch induz a ativação da PKA. No entanto, essa interação não foi observada em experimentos de duplo híbrido. Adicionalmente, a expressão gênica em resposta a compostos que geram estresse oxidativo e nitrosoativo foi estudada utilizando-se microarranjos de cDNA. Os resultados revelaram um papel de keaA na resposta à pré-carência e no controle do ciclo celular / The YakA is a protein kinase required for the regulation of several stress responses in Dictyostelium and is a key effector of the transition from growth to development in this microorganism. The gene keaA was isolated as suppressor of the yakA- mutant in a screen targeted to reveal genes involved in the survival to nitrosoative stress. The keaA gene codes a protein with six kelch domain repeats at the C-terminus, a zf-C3HC4 domain at the N-terminus, also called RING-finger, and a cysteine-rich sequence located in the mid-portion of the protein. The analysis of mutants deficient in keaA revealed a role for this gene also in the development process. keaA mRNA expression is induced when wild type cells grow and the food source becomes scarce. An induction of keaA mRNA expression also occurs during development. This induction is not observed in yakA- cells, indicating that YakA regulates KeaA. keaA deficient cells express low levels of pkaC, acaA and carA mRNA during aggregation in low cell densities, which may explain why the keaA deficient cells present a delay in development in low cell densities. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. The analysis of agreggation of keaA deficient cells during multicellular development indicated that KeaA is required for the cells to efficiently participate in the process. Cells over-expressing the cisteine-rich domain took the same time as the wild-type cells to reach the aggregation stage. However, these cells present smaller aggregates, culminants and fruiting bodies. Cells over-expressing the Kelch domain express high levels of acaA and carA after 8 hours of development, but the levels of pkaC are kept similar to those in wild-type cells. This could indicate that the Kelch domain induces the activation of PKA. However, this interaction was not observed when we conducted tests in a two-hybrid system. Additionally, gene expression in response to compounds that generate redox stresses was studied using cDNA microarrays. The results revealed a role of keaA in response to pre-starvation and control of cell cycle

Several studies have highlighted the possibility that radio-frequency electromagnetic fields (RF-EMF) used in mobile phone technology could influence DNA integrity of male germ cells as well as sperm motility. Current knowledge concerning the influence of RF-EMF on male germ cells is extremely limited. In the present study the hypothesis that 900 MHz GSM radiation could induce the activation of stress response in human spermatozoa was investigated. Ejaculated, density purified, human spermatozoa from donors were exposed to 900 MHz GSM mobile phone radiation at specific absorption rate (SAR) levels of 2.0 and 5.7 W/kg and examined at various time points post exposure. Sperm motility and morphology were evaluated by computer-aided sperm analysis (CASA). The ability of RF-EMF exposed sperm to undergo the acrosome reaction was evaluated by flow cytometry. Sperm binding to the zona pellucida of human oocytes was determined by the hemi-zona (HZA) assay. Apoptotic markers, phosphatidylserine (PS) externalization, change in mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, caspase activation and DNA fragmentation were analysed using flow cytometry. Heat shock protein (Hsp) 27 and 70 expression and activity were analyzed using specific antibodies with flow cytometry and Western blot methods. Stress fibre stabilization (F-actin polymerization) was visualized using fluorescent dye labelled phalloidin. No effect was seen on kinematic parameters assessed at SAR 2.0 W/kg, however straight line velocity (VSL) and beat cross frequency (BCF) were significantly altered after exposure at SAR 5.7 W/kg. Sperm shrinkage (decrease in surface area) was observed at both exposure levels. RF-EMF did not influence exposed spermatozoa’s ability to undergo the acrosome reaction. A significant decrease in sperm-zona binding was observed at both exposure levels. RF radiation did not have an effect on any apoptotic markers. ROS generation increased significantly with an increase in SAR (5.7 W/kg). RF-EMF did not induce a stress response in exposed sperm (no activation of Hsp70 and 27 activity). These results cannot be ascribed to heating, as the temperature did not increase by more than 0.2 - 0.3ºC during exposure. The decrease in sperm-zona binding is the result of an alternative non-stress inducible pathway. This study should be replicated at lower SAR levels that would simulate the radiation absorption from carrying the cell phone in a pocket close to the testes. / Thesis (PhD (Reproductive Biology))--University of Pretoria, 2008. / Obstetrics and Gynaecology / unrestricted

It is understood that in order for athletes to perform to their potential consistently they must learn to optimally self-regulate their psychological and physiological states. Yet, the process by which this is accomplished is not well understood. The purpose of this doctoral dissertation was to explore the concept of self-regulation in the Olympic athlete population through the use of biofeedback and neurofeedback. To address this purpose, two studies were conducted. Study One (Article 1) used a quantitative methodology to explore the relationship between Olympic athletes’ overall self-regulation ability and world ranking. Fifteen Olympic level athletes underwent a 9-stage psychophysiological stress assessment to determine each athlete’s ability to return to baseline after a stress load was applied. Findings revealed that there was a significant correlation between the athletes’ overall self-regulation ability and their ranking at the world level, meaning the better the overall self-regulation ability of the athlete the better the world ranking. Study Two (Articles 2 and 3) employed a qualitative methodology and explored what and how five Olympic level athletes learned from participating in a 20 session biofeedback and neurofeedback training intervention. Data was collected from post-intervention interviews with the athletes. In Study Two, Olympic athletes perceived that the biofeedback and neurofeedback training intervention assisted them in learning to improve self-awareness and self-regulation of their physical and mental states enabling them to feel more in control during sport performances. Engaging in active learning exercises, receiving real-time formative feedback, and utilization of the intervention exercises in training and competition environments were how athletes perceived they learned to self-regulate. Together, the findings from the two studies highlight the relevance and intricacies of self-regulation in high performance sport. Overall, the present dissertation makes a contribution to the sport psychology literature particularly with regard to our understanding of the use of biofeedback and neurofeedback for enhancing self-regulation with Olympic athletes. Thus, learning to improve self-regulation skills using biofeedback and neurofeedback training should be an integral part of a comprehensive and holistic approach used by sport psychology practitioners in assisting athletes to perform to their potential.

Orientador: Paulo Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:19:31Z (GMT). No. of bitstreams: 1 Kiyota_Eduardo_D.pdf: 12985637 bytes, checksum: 423c7614185847e8e3a4c43acbef92fe (MD5) Previous issue date: 2015 / Resumo: O amino ácido lisina é catabolizado em plantas e animais pela via da sacaropina. Nesta via, a lisina é convertida a ?-aminoadipato-?-semialdeído (AASA) pela ação da enzima bifuncional lisina-cetoglutarato redutase/sacaropina desidrogenase (LKR/SDH). O intermediário AASA é então convertido a ?-aminoadipato (AAA) pela enzima ?-aminoadipato-?-semialdeído desidrogenase (AASADH). A LKR/SDH já foi bem caracterizada em plantas e animais, mas a atividade enzimática bem como o possível papel fiiológico da AASADH ainda não foi demonstrada em plantas. A via da sacaropina, além do seu importante papel na regulação dos níveis de lisina, está também envolvida em processos de resposta a estresses. Este trabalho está dividido em dois capítulos. No capítulo I descrevemos a identificação do gene que codifica a AASADH em milho e a caracterização da atividade enzimática da enzima em endosperma imaturo de milho. Mostramos que a AASADH é codificada pelo gene Aldh7b1, um gene muito conservado em eucariotos. A enzima codificada pelo gene Aldh7b1 foi parcialmente purificada de endosperma imaturo de milho e através de eletroforese em condições denaturantes e cromatografia em coluna de gel filtração mostramos que a enzima, na sua forma nativa, apresenta-se como um tetrâmero constituído por quatro subunidades de 55 kDa. A AASADH isolada de endosperma imaturo de milho converte o semi-aldeido AASA em AAA. O produto da reação catalisada pela AASADH foi confirmado por cromatografia em camada delgada. No capítulo II discutimos o papel da via sacaropina no desenvolvimento da semente e na resposta de planas jovens de milho a estresses abióticos. As enzimas LKR/SDH e AASADH são co-expressos nas células das camadas da sub-aleurona do endosperma de milho nas fases intermediarias do desenvolvimento. No entanto, embora a proteína AASADH seja produzida no endosperma e no embrião de sementes imaturas e nos tecidos de plantas jovens, a proteína LKR/SDH é detectada unicamente nas células da sub-aleurona das sementes imaturas. A AASADH mostrou atividade máxima a pH 7,4 e Kms para AASA e NAD+ na ordem de micromolar. Em endosperma imaturo a via da sacaropina é induzida por lisina e reprimida por estresse salino, enquanto prolina e ácido pipecólico são significativamente reprimidos por lisina. Em coleóptiles jovens as enzimas LKR/SDH e AASADH são induzidas transcricionalmente por estresses salino, osmótico e oxidativo, mas enquanto que a proteína AASADH acumula nos tecidos sob estresse, a proteína LKR/SDH não é detectada. Nossos resultados indicam que os genes que codificam as enzimas LKR/SDH e AASADH são co-expressos a nível transcricional, mas não a nível traducional. A ausência da proteína LKR/SDH em plantas jovens sob estresses e os altos níveis do seu transcrito serem detectados mostra um desacoplamento transcrição/tradução que podem ter consequências regulatórias ainda desconhecidas / Abstract: Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into ?-aminoadipate-?-semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into ?-aminoadipate (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). LKR/SDH was characterized in higher eukaryotes, but AASADH has not been demonstrated in plants. Furthermore, studies have shown that besides the degradation of lysine, the saccharopine pathway is involved in stress response processes in plants, animals and bacteria. This work was divided into two chapters. Chapter I describes the identification of the gene encoding AASADH and the partial purification and characterization of the enzyme from developing maize endosperm. The enzyme AASADH is encoded by the Aldh7b1 gene, a gene highly conserved among eukaryotes. The enzyme partially purified from developing endosperm and analyzed by SDS-PAGE and gel filtration chromatography behaved, in its native form, as a tetramer constituted by four monomers of 55 kDa. The enzymatic convertion of AASA into AAA was verified by thin layer chromatography. In Chapter II the role of the saccharopine pathway in seed development and stress responses is discussed. LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing seeds. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD+ in the micromolar range. In the developing endosperm the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in stressed tissues, LKR/SDH does not. Our results indicate that the genes encoding the LKR/SDH and AASADH enzymes are co-expressed at the transcriptional level, but not the translational level. The absence of LKR/SDH protein in young plants under stress despite of the high levels of transcripts being detected suggests a decoupling transcription/translation that may have regulatory consequences yet unknown / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

ABSTRACT A review of annual monitoring reports for stream restoration projects on surface coal mines in the central Appalachian Mountains found that the criteria used for judging the success of the projects was generally based on visual assessments of habitat structure which were evaluated using the Rapid Bioassessment Protocol (RBP) (Palmer and Hondula, 2014). In recent years the Hydrogeomorphic Approach (HGM), which was originally developed to evaluate wetlands, has been adapted for stream evaluations as well (Summers, et al., 2017). Both of these methods are primarily a means to determine if suitable habitat structure and riparian growth are present to support aquatic life. It is assumed that if habitat structure is suitable then macroinvertebrate and other life will be present. However, each of these two methods place emphasis on different aspects of habitat and riparian structure. The primary purpose of this project is to compare and contrast how effective these two methods are at evaluating reconstructed streams on surface coal mines. A secondary objective is to determine if macroinvertebrate assemblages in reconstructed streams is significantly different from that of reference streams not impacted by mining. Research on benthic community structure downstream of coal mining activities suggests that even after many decades taxa richness and abundance still have not recovered from indirect impacts (Petty, et al., 2010). Information on reconstructed streams directly impacted is lacking. This project evaluates streams that were reconstructed five years prior using the RBP and HGM methods, and compares them to local reference streams that have minimal to no mining impacts. Multiple benthic metrics are also used to evaluate community structure. REFERENCES Petty, J. Todd, Jennifer B. Fulton, Michael P. Strager, George T. Merovich Jr., James M. Stiles, and Paul F. Ziemkiewicz. 2010. Lanscape indicators and thresholds of stream ecological impairment in an intensely mined Appalachian watershed. Journal of the North American Benthological Society, 29(4): 1292-1309. Palmer, Margaret A., and Kelly L. Hondula. 2014. Restoration as mitigation: analysis of stream mitigation for coal mining impacts in southern Appalachia. Environmental Science and Technology 48: 10552-10560. Summers, Elizabeth A., Chris V. Noble, Jacob F. berkowitz, and Frank J. Spilker. 2017. Operational Draft Regional Guidebook for the Functional Assessment of High-Gradient Headwater Streams and Low-Gradient Perennial Streams in Appalachia. ERDC/EL TR-17-1.