Dissertations / Theses on the topic 'Stress response'

Mestrado em Biologia Molecular e Celular
Thirty-five Listeria monocytogenes isolates previously collected from food (n=20) and human patients suffering from listeriosis (n=15), with different antibiotic resistance profiles were characterized and compared based on: (i) their ability to survive through sequential conditions that parallel the digestive tract; (ii) their capacity to survive extreme pH values; (iii) the potential relationship, between antibiotic resistance and the resistance of L. monocytogenes isolates to the stress conditions investigated. The response was shown to be strain- and stress-dependent and no relation between food and clinical isolates was observed (p > 0.05). The results showed that L. monocytogenes is able to survive under extreme acid and alkaline conditions and did not survive when submitted to simulated sequential gastro-intestinal transit, i.e. the activity of bile salts after combined action of hydrochloric acid and pepsin. No correlation was observed between antibiotic resistance and response to the stress conditions applied to the isolates investigated.
Trinta e cinco isolados de Listeria monocytogenes provenientes de alimentos (n=20) e pacientes humanos com listeriose (n=15) e com diferentes perfis de resistência a antibióticos foram caracterizados e comparados com base na: (i) sua capacidade de sobrevivência à passagem pelo trato gastrointestinal simulado, (ii) sua capacidade de sobrivência a condições extremas de pH, (iii) potencial relação entre a resistência a antibióticos e a resistência às condições de stresse investigadas. A resposta às várias condições de stresse demonstrou ser estirpe- e stresse-dependente e não foi observada nenhuma relação entre isolados alimentares e clínicos (p > 0.05). Os resultados mostraram que L. monocytogenes sobrevive em condições ácidas e alcalinas extremas e não sobrevive quando submetida à passagem pelo trato gastrointestinal simulado, ou seja, à atividade dos sais biliares após ação conjunta do ácido clorídrico e pepsina. Não foi observada qualquer correlação entre a resistência a antibióticos e a resposta às condições de stresse aplicadas para os isolados estudados.

The heat shock response was studied in the intestinal parasitic protozoan Entamoeba histolytica. Temperature shifts from 37$ sp circ$C to 44$ sp circ$C enhanced the synthesis of five major heat shock (or stress) proteins (HSP) of 100, 50, 42, 37, and 28 kDa. Similarly, exposure of amebae to lymphokine activated macrophages and hydrogen peroxide caused HSP expression. Heat shock caused the reversible inhibition of amebic adherence to Chinese hamster ovary cells and human colonic mucin binding to trophozoites by ${>80 %}$. This was due to a decrease in the surface expression of the Gal/GalNAc adherence lectin and a marked reduction in the lectin mRNA expression. However, the presence of target Chinese hamster ovary cells during recovery at 37$ sp circ$C augmented amebic adherence. These results suggest that E. histolytica trophozoites produce a variety of HSP in response to different stimuli and can modulate the expression of the surface adherence lectin which maybe important in pathogenesis.

Background: Trauma is a major cause of death and disability. Adverse events, such as prolonged prehospital time, hypoxia, hypotension and/or hyperventilation have been reported to correlate to poor outcome. Adequate cortisol levels are essential for survival after major trauma. In hypotensive critically ill patients, lack of sufficient amount of cortisol can be suspected, and a concept of critical illness related corticosteroid insufficiency has been proposed. Corticosteroid therapy has many adverse effects in critically ill patients and should only be given if life-saving. Correct measurement of serum cortisol levels is important but difficult in critically ill patients with capillary leakage. Estimation of the free and biologically active cortisol is preferable. In serum less than 10% of cortisol is free and biologically active and not possible to measure with routine laboratory methods. Salivary cortisol can be used as a surrogate for free cortisol, but salivary production is reduced in critically ill patients. Liver resection could reduce cortisol levels due to substrate deficiency. Aims: 1. Evaluate the occurrence of early adverse events in patients with traumatic brain injury and relate them to outcome. 2. Assess cortisol levels over time after trauma and correlate to severity of trauma, sedative/analgesic drugs and cardiovascular function. 3. Evaluate if saliva stimulation could be performed without interfering with salivary cortisol levels. 4. Assess cortisol levels over time after liver resection in comparison to other major surgery. Results: There was no significant correlation between prehospital time ³60 minutes, hypoxia (saturation <95%), hypotension (systolic blood pressure <90 mmHg), or hyperventilation (ETCO2 <4.5 kPa) and a poor outcome (Glasgow Outcome Scale 1-3) in patients with traumatic brain injury. Cortisol levels decreased significantly over time after trauma, but there was no correlation between low (<200 nmol/L) serum cortisol levels and severity of trauma. Infusion of sedative/analgesic drugs was the strongest predictor for a low (<200 nmol/L) serum cortisol. The odds ratio for low serum cortisol levels (<200 nmol/L) was 8.0 for patients receiving continuous infusion of sedative/analgesic drugs. There was no significant difference between unstimulated and stimulated salivary cortisol levels (p=0.06) in healthy volunteers. Liver resection was not associated with significantly lower cortisol levels compared to other major surgery. Conclusion: There was no significant correlation between early adverse events and outcome in patients with traumatic brain injury. Cortisol levels decreased significantly over time in trauma patients. Low cortisol levels (<200 nmol/L) were significantly correlated to continuous infusion of sedative/analgesic drugs. Saliva stimulation could be performed without interfering with salivary cortisol levels. Liver resection was not associated with low cortisol levels compared to other major surgery.

La frataxina è una proteina mitocondriale, la cui ridotta espressione è responsabile di una malattia neurodegenerativa ereditaria, l’atassia di Friedreich (FRDA). La frataxina è una proteina che lega il ferro ed è coinvolta nella biogenesi dei gruppi ferro-zolfo (ISC), gruppi prostetici che svolgono funzioni cellulari essenziali come la fosforilazione ossidativa, la catalisi enzimatica e regolazione dei geni. La frataxina è richiesta per lo sviluppo, poiché la sua assenza è letale in embrioni di topo e provoca l’arresto nello sviluppo del nematode C. elegans. Una parziale espressione della frataxina permette lo sviluppo e la sopravvivenza dell’organismo, e determina una progressiva degenerazione di tessuti specifici. Sebbene molte evidenze suggeriscano che la frataxina agisca da chaperone nel compartimento mitocondriale, è stato recentemente dimostrato l'esistenza di un pool funzionale di frataxina extramitocondriale in vari tipi di cellule umane. Lo scopo del mio lavoro nella prima parte è stato di indagare sul possibile ruolo fisiologico della frataxina extramitocondriale nel compartimento citoplasmatico studiando l’interazione della proteina con un possibile patner ISC-dipendente. E' stato dimostrato che la forma extramitocondriale della frataxina interagisce direttamente con l’aconitasi citosolica/ proteina regolatoria del ferro-1 (IRP1), una proteina bifunzionale che alterna la funzione enzimatica di aconitasi e la funzione di “RNA-binding” attraverso il meccanismo dello “switch” del cluster ferro-zolfo. Inoltre il difetto dell’aconitasi citosolica e la conseguente attivazione di IRP1 come proteina che lega l’RNA, che si verifica nelle cellule dei pazienti affetti da atassia di Friedreich, viene revertito con l'azione della frataxina extramitocondriale. La frataxina, inoltre, protegge le cellule tumorali dallo stress ossidativo e dall’apoptosi, ma agisce anche da soppressore tumorale. Le basi molecolari di questo apparente paradosso non sono ad oggi note. Nella seconda parte del mio lavoro ho osservato che l'espressione della frataxina è aumentata in diverse linee cellulari tumorali in risposta allo stress ipossico, una condizione spesso associata alla progressione del tumore. Inoltre, l'aumento della frataxina in risposta all'ipossia dipende dai Fattori di espressione Ipossia-Inducibili (HIF) e modula l’attivazione del soppressore tumorale p53. E’ stato mostrato per la prima volta in vivo l’ aumento di frataxina in campioni chirurgici di glioblastoma umano e campioni umani di carcinoma di colon. Questi risultati mostrano che la frataxina partecipa alla risposta allo stress indotto da ipossia nei tumori, ciò implica che la modulazione della sua espressione potrebbe svolgere un ruolo determinante nella sopravvivenza e/o nella progressione delle cellule tumorali.
Defective expression of frataxin is responsible for the degenerative disease Friedreich’s ataxia (FRDA). Frataxin is an iron-binding protein involved in the biogenesis of iron–sulfur clusters (ISC), prosthetic groups allowing essential cellular functions such as oxidative phosphorylation, enzyme catalysis and gene regulation. Frataxin is a protein required for cell survival since complete knock-out is lethal. Partial expression of the frataxin allows the development and survival of the organism, yet results in progressive degeneration of specific tissues. Although several evidence suggest that frataxin acts as an iron-chaperone within the mitochondrial compartment, it was recently demonstrated the existence of a functional extramitochondrial pool of mature frataxin in various human cell types. The aim of my work in the first part was to investigate for a physiological role of extramitochondrial frataxin in the cytoplasmic compartment searching for ISCdependent interaction. The extramitochondrial form of frataxin was demonstrated to directly interact with cytosolic aconitase/iron regulatory protein-1 (IRP1), a bifunctional protein that alternates between an enzymatic and a RNA-binding function through the “iron–sulfur switch” mechanism. Importantly, the cytosolic aconitase defect and consequent IRP1 activation occurring in FRDA cells was found to be reversed by the action of extramitochondrial frataxin. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. The aim of my work in the second part was to investigate the pathways through which frataxin enhances stress resistance in tumor cells. Frataxin expression was found to be upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, frataxin upregulation in response to hypoxia is dependent on HypoxiaInducible-Factors (HIFs) expression and modulates tumor suppressor p53 activation. Importantly, this work shows for the first time an in vivo increase of frataxin in human glioblastoma and colon carcinoma tumor samples. These results show that frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could play a critical role in tumor cell survival and/or progression.

Some aspects of the stress response during acute intensive care for severe burns are described and quantified by measuring hormonal and neuroendocrine patterns and relating these to organ function in the short term. This includes an assessment of whether there are markers for the severity of stress that are better than conventional descriptors of the severity of a burn in predicting failing organ function. P-CgA after a major burn injury is an independent and better predictor of organ dysfunction assessed as SOFA score than the traditionally used TBSA% burned. The results also suggest that the extent of neuroendocrine activation is related to organ dysfunction, and this motivates a more extensive effort to evaluate P-CgA as a prognostic marker with respect to mortality and long-term outcome. P-NT-proBNP exhibited a complex pattern with considerable inter-individual and day-to-day variations. Values of P-NT-proBNP were related to size of burn, water accumulation and systemic inflammatory response. A considerable covariation with trauma response and SOFA scores was observed in day by day analyses, but with weight change only on day 2. Maximum P-NT-proBNP showed a stronger correlation with SOFA score on day 14, with mortality, and with LOS, than did age and TBSA% burned. High values were also independent predictors of all subsequent SOFA scores up to two weeks after injury. P-NT-proBNP and NT-proANP reflect and predict organ function after burn injury similarly, notwithstanding a significantly larger intra-individual variability for P-NT-proBNP. P-NT-proBNP, but not NT-proANP, reflects the systemic inflammatory trauma response. Free cortisol concentration was related to the size of burns, as was the circadian cortisol rhythm. This effect of burn size was, at least in part, related to its effect on organ function. This thesis points to the fact that the stress response is richly interwoven, and cannot be adequately assessed by one biomarker only. All biomarkers studied here can be viewed as representing efferent limbs of the stress reaction, and they would need to be supplemented by biomarkers representing individual physiologic responses that follow the stress signaling.

The cytosolic stress response, also known as the heat-shock response (HSR), is one of the major defense mechanisms activated by cells to maintain the integrity of the cellular proteome under proteotoxic environmental conditions. It is characterized by the increased synthesis of heat-shock proteins (Hsps), mainly molecular chaperones and proteases which prevent the aggregation of misfolded proteins and mediate their refolding or degradation. It is generally accepted that the induction of the HSR is coordinated by the heat-shock transcription factor 1 (HSF1). However, many mechanistic aspects of the HSF1 regulation remain unclear. In the present study, a genome-wide RNA interference screen was combined with an extensive biochemical analysis and quantitative proteomics to better understand the regulation of the HSR upon thermal stress. In the screening experiments novel positive and negative modulators of the stress response were identified, including proteins involved in chromatin remodeling, transcription, mRNA splicing, DNA damage repair, and proteolytic degradation. The diversity of the identified regulators suggests that induction and attenuation of the HSR integrate signals from different cellular pathways and are rather multi-factorial processes than single gene/protein events. The modulator proteins are localized in multiple cellular compartments with the majority having their primary location in the nucleus. A protein-protein interaction analysis revealed a HSR regulatory network, with chromatin modifiers and nuclear protein quality control components occupying hub positions. These observations are supported by quantitative proteomics experiments, which showed specific stress-induced reorganizations of the nuclear proteome, including the transient accumulation of chaperones and proteasomal subunits. The histone acetyltransferase EP300 was shown to specifically control the cellular level of HSF1 by stabilizing it against proteasomal turnover under normal conditions. Moreover, the ubiquitin-proteasome system (UPS) was found to participate in the attenuation of the HSR by degrading stress-activated, hyperphosphorylated HSF1. Since HSF1 competes with stress-denatured proteins for access to the proteasome, the extent of cellular protein damage modulates the rate of HSR attenuation. In addition to thermal stress, various other proteotoxic stresses are known to induce the HSR such as the proteasome inhibitor MG132 and the triterpenoid celastrol, which activates HSF1 by an unknown mechanism. Therefore, the networks regulating HSF1 activation upon thermal stress, proteasome inhibition and celastrol treatment were compared in this study. Whereas there is a large overlap between the sets of regulatory factors activated after heat stress and proteasomal impairment, HSF1 activation after celastrol treatment seems to bypass the HSR regulatory network to a large extent. Nevertheless, comparison of the regulatory networks under different proteotoxic conditions revealed a set of HSR core components, including factors involved in chromatin remodeling, DNA damage repair, RNA transport, transcription, and ion transport. The various cellular functions and localizations of these core components reinforce the multifaceted nature of the HSR regulation. The results obtained in this study can help to identify potential targets for the pharmacologic manipulation of the HSR in the treatment of aggregate deposition diseases and cancer.

Previous transcriptomic experiments have revealed that various Escherichia coli K-12 genes encoding proteins of unknown function are highly expressed during anaerobic growth in the presence of nitrate, or especially nitrite. Products of some of these genes, especially YeaR-YoaG, YgbA, YibIH and the hybrid cluster protein, Hcp, have been implicated in the response to nitrosative stress. The aims of this study were to investigate sources of nitrosative stress, and the possible roles of some of these proteins in protection against nitric oxide. The YtfE protein has been implicated in the repair of iron centres, especially in iron-sulphur proteins. The previously unexplained anaerobic growth defect of the ytfE strain LMS 4209 was shown to be due to a secondary 126-gene deletion rather than to the deletion of ytfE. At the start of the project, the transcription factor, NsrR, was known to respond to low concentrations of intracellular NO, and to repress expression of ytfE, hcp-hcr encoding the hybrid cluster protein and its reductase, and hmp that encodes the flavohaemoglobin, Hmp. In this work, a biochemical assay of hcp promoter activity was developed as a reporter of intracellular NO generation. This assay was used in combination with a range of mutants to show that the major source of intracellular NO is the reduction of nitrite by the cytoplasmic nitrate reductase, NarG. Although the periplasmic cytochrome c nitrite reductase, NrfAB, and the cytoplasmic nitrite reductase, NirBD, decrease nitrosative stress by reducing nitrite to ammonia, at least one additional source of NO production from nitrite remains unidentified. An assay for NO reduction using a Clark-Type electrode was validated. Rates of NO reduction were induced 2-fold in the presence of nitrate. Lowest rates of NO reduction were found in a mutant defective in nsrR. A quadruple mutant defective in Hmp, the flavorubredoxin, NorV, NrfAB and NirBD still reduced NO at more than half the rate of the parent. This residual activity was not due to YibIH, YeaR-YoaG or YgbA. Various hcp-hcr derivatives of strains defective in NO reductases revealed a severe growth defect under conditions of nitrosative stress, but Hcp was eliminated as a possible additional NO reductase. This growth defect was substantially suppressed by a further mutation in ytfE. Growth experiments with isogenic sets of mutant defective in all combinations of hcp and ytfE in addition to deletions in hmp, norVW and nrfAB implicated both YtfE and Hcp in repair of nitrosative damage. However, weaker phenotypes of these strains in absence of nitrate or nitrite are consistent with more general roles for these proteins in the repair of damage to protein iron centres.

Mestrado em Microbiologia
O género Aeromonas é frequentemente associado a uma distribuição ampla no meio ambiente, emergindo como importantes patogénicos de seres humanos. No entanto, a complexidade dos mecanismos de patogenicidade permanece desconhecida e nenhum factor de virulência foi até hoje identificado individualmente como responsável pela patogenicidade. Vários factores de virulência bacteriana podem sofrer uma regulação ambiental por meio de diversos factores abióticos, permitindo a bactérias patogénicas coordenarem a expressão de factores de virulência com a existência de condições favoráveis. Análise de respostas ao stress é fundamental, especialmente no contexto de bactérias patogénicas. No entanto, investigações sobre proteínas e mecanismos associados à resistência ao stress são escassos ou inexistentes para diversas bactérias, principalmente patogénicos incomuns ou oportunistas, como as Aeromonas. Os objectivos deste estudo são a avaliação do proteoma da bactéria, Aeromonas piscicola, em resposta ao stress e desvendar proteínas com variação na expressõa, envolvidas na resposta ao stress. Para atingir estes objectivos procedeu-se à avaliação do proteoma diferencial sob stress causado independentemente por temperatura, pH e salinidade. Através da avaliação do proteoma extracelular foi possível a detecção da presença de 19 proteases nas diferentes condições abióticas e verificar que, 3,5% NaCl e 40 ºC induzem uma evidente redução na expressão de proteases extracelulares. Análise das proteínas secretadas revelou que: a temperatura induz um aumento na expressão de 23 proteínas (4 a 25 ºC, 9 a 37º C e 10 a 40 ºC) para o pH, 8 proteínas (2 a pH 5.0 e 6 a pH 9.0) apresentam um aumento de expressão e para a salinidade, 16 proteínas (7 a 0% e 9 a 3.5% NaCl). Análise das proteínas intracelulares revelou que: a temperatura induz um aumento na expressão de 22 proteínas (5 a 25 ºC, 9 a 37º C e 10 a 40 ºC) para o pH, 8 proteínas (3 a pH 5.0 e 5 a pH 9.0) apresentam um aumento de expressão e para a salinidade, 11 proteínas (4 a 0% NaCl e 7 a 3.5% NaCl). Adicionalmente, verificou-se a expressão de 3 novas proteínas, uma a temperaturas elevadas, outra quando na presença de temperaturas de stress e outra quando exposta ao stress de pH alcalino. Aeromonas piscicola AH-3 revelou a existência de um sistema de proteção geral pré-existente aquando na presença de condições de stress. Uma conjunto de marcadores de stress para cada condição abiótica e marcadores de stress específicos para a modulação de factores abióticos foram detectados. Foi possível concluir que aquando exposta a condições de stress, causadas quer por alterações climáticas ou quando a invasão de um hospedeiro, Aeromonas piscicola AH-3 é capaz de expressar os mecanismos necessários para a sobrevivência e crescimento nas condições abióticas testadas.
Aeromonas are commonly associated with a widely distribution in the environment and as important emerging pathogens for humans. However the complex pathogenesis mechanisms remain poorly understood, and no individual virulence factor has been identified as responsible for the pathogenesis. Several bacterial virulence factors experience an environmental regulation by many abiotic conditions, enabling pathogens to coordinate expression of virulence factors with the existence of favorable conditions. Analysis of stress responses is of fundamental importance, especially in the context of pathogenic bacteria. However, research for proteins and pathways associated with stress tolerance is still lacking for many bacteria and principally for uncommon or opportunistic pathogens, like Aeromonas. The objectives of this study are the evaluation of the proteome response to stress of Aeromonas piscicola, and unravel proteins involved in stress response by the evaluation of differential proteome under temperature, pH and salinity stress. Through the evaluation of the extracellular proteome it was possible to detect the presence of 19 proteases in the different abiotic conditions and verified that, 3.5% NaCl and 40 ºC induces an evident reduction of the expression of extracellular proteases. Analysis of the secreted proteins revealed that: temperature induced the up-regulation of 23 proteins (4 at 25 ºC, 9 at 37º C and 10 at 40 ºC), as for the pH, 8 proteins (2 at pH 5.0 and 6 at pH 9.0) were upregulated and for salinity 16 proteins (7 at 0% and 9 at 3.5% NaCl) were upregulated. Analysis of the intracellular proteome revealed that: temperature induced the up-regulation of 22 proteins (5 at 25 ºC, 9 at 37º C and 10 at 40 ºC), as for the pH, 8 proteins (3 at pH 5.0 and 5 at pH 9.0) were up-regulated, and for salinity 11 proteins (4 at 0% and 7 at 3.5% NaCl) were up-regulated. Additionally, 3 newly expressed proteins were observed, one under high temperatures, another when exposed to global temperature stress and one when exposed to alkaline pH stress. Aeromonas piscicola AH-3 revealed a global pre-existing system of protection, since when in stress conditions, differential expression of proteins occurs in general with the up-regulation of certain proteins. A series of stress markers for each abiotic condition and furthermore some stress markers for specific modulation of abiotic factors were detected. It was possible to conclude that when exposed to stressful conditions, either caused by climate changes or when invading a host, Aeromonas piscicola AH-3 is capable of express the necessary mechanisms for survival and growth in the abiotic conditions tested.

Thesis (B.S.)--California Polytechnic State University, 2009.
Project advisor: Lee McFarland. Title from PDF title page; viewed on Jan. 13, 2010. Includes bibliographical references. Also available on microfiche.

Our lab has shown that the bile salt found in the highest concentration in human fecal water, sodium deoxycholate, induces apoptosis in several cell types including Jurkat cells as well as human colonic epithelial cells. We have also found that cells within the normal appearing flat mucosa of patients with a history of colon cancer are relatively resistant to apoptosis induced by NaDOC. The current studies test the hypothesis that sodium deoxycholate induces multiple stress response pathway s that protect against apoptosis. I have tested this hypothesis by developing and analyzing cell lines that are resistant to sodium deoxycholate-induced apoptosis and focusing on two stress-response proteins known to be activated by sodium deoxycholate, poly(ADP-ribose) polymerase (PARP) and the redo-sensitive transcription factor nuclear factor-kappa B (NF-κB). I found that PARP is protective against NaDOC-induced apoptosis, and by independently inhibiting the individual subunits of NF-κB, I found that the p65 subunit is protective, while the p50 subunit is not. Development and subsequent characterization of the NaDOC-resistant HCT-116 cell lines identified several proteins that may be responsible for the development of apoptosis resistance. These proteins will be further tested in future studies.

The mechanical properties of granular media are investigated using large-scale computer simulations. We probe the relationship between structural and mechanical properties in static, three dimensional granular packings through the development of a coarse graining procedure that connects the microscopic grain scale properties and a macroscopic continuum level description. We study the manner in which local stresses are redistributed in response to localized force perturbations. We first study stress response in frictionless and frictional shallow, ordered, granular arrays confined by solid boundaries for a range of system sizes. Stress response profiles from inside the packing and at the boundaries for frictional packings agree well with the predictions for the semi-infinite half plane of classical isotropic elasticity theory down to boxes of linear dimensions of approximately forty particle diameters and over several orders of magnitude in the applied force. The response profiles for frictionless packings exhibit transitional regime to strongly anisotropic features with increasing box size. The differences between the nature of the stress response are shown to be characterized by very different particle displacement fields. For unconfined overcompressed face centered cubic (FCC) granular arrays of linear dimensions of approximately sixty particle diameters, stress response propagation is studied. Influence of friction coefficient range is the crossover in stress response for a range of the localized force perturbation. The crossover in stress response is from single-peak stress response of the classical isotropic elasticity prediction to double-peak stress response of the anisotropic elasticity prediction for strong anisotropy in material properties. Stress profiles from the bottom layer of these systems indicate crossover from one-peak to double-peak stress response as the magnitude of force perturbation increases for one friction coefficient. Also, these stress profiles show crossover from double-peak to one-peak stress response as the friction coefficient increases for one force perturbation. For most of the systems stress response near the perturbation source is double-peak, for frictional systems as we go further away from the perturbation source a crossover to one-peak stress response occurs. This crossover is associated with length scales. Length scales associated with the material parameters are investigated. Length scales are defined as a distance at which the crossover occurs for a system. The sole effects of friction coefficients on the stress response transition within the systems are expressed in the power laws between length scales and friction coefficients. A multidimensional phase diagram is constructed to show different regions of one-peak and double-peak stress responses that captures the basic mechanical response properties of granular media.

Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第13852号
工博第2956号
新制||工||1436(附属図書館)
26068
UT51-2008-C768
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生
学位規則第4条第1項該当

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Viral infection affects a multitude of cellular processes to facilitate successful replication. Such responses include the formation of stress granules (SGs) and the activation of autophagy. SGs are stalled translational complexes and function to restore cellular homeostasis after stress. Autophagy is a cellular process that recycles misfolded proteins and damaged organelles and plays an important role in various stress responses. We previously demonstrated that infection with Coxsackievirus B3 (CVB3), a common human pathogen for viral myocarditis, disrupts the autophagic process to support effective viral replication. However, the interplay between CVB3 and SGs, and the ability of SGs to regulate autophagy have not been investigated. Here we showed that SGs are formed early and actively disassembled late during CVB3 infection due to viral protease 3Cpro-mediated cleavage of Ras-GAP SH3 domain binding protein 1 (G3BP1), a key nucleating protein of SGs. Overexpression of G3BP1 inhibits CVB3 replication, indicating an anti-viral function of SGs. We further demonstrated that the C-terminal product of G3BP1 has a toxic gain-of-function that further inhibits SG formation. We also examined the interaction between CVB3 and the transactive response DNA-binding protein-43 (TDP-43), an RNA binding protein that mislocates to SGs under cellular stress. We found that TDP-43 is translocated from the nucleus to SGs upon infection through the activity of viral protease 2Apro, followed by cleavage by protease 3Cpro. The C-terminal product of TDP-43 is quickly degraded by the proteasome, whereas the N-terminal truncate acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Knockdown of TDP-43 results in an increase in viral titres, suggesting a protective role for TDP-43 in CVB3 infection. Lastly, we explored the possible role of G3BP1-SGs in regulating autophagy. We showed that G3BP1 inhibits autophagic flux, likely by binding to cytoplasmic signal transducer and activator of transcription 3 (STAT3). Taken together, our results reveal that the host SGs and associated proteins, including G3BP1 and TDP-43, are utilized and modified during CVB3 infection to promote efficient viral replication and induce viral pathogenesis. Moreover, we propose a novel mechanism by which G3BP1 binds cytoplasmic STAT3 to inhibit autophagy.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

The drained loading behaviour of water pluviated Erksak sand is investigated in the triaxial apparatus by varying consolidation history, stress path and loading direction (compression or extension). It is shown that, under identical minor effective principal stress, anisotropically consolidated sand has a higher tangent modulus than the isotropically consolidated sand in the initial stages of the shearing phase. This difference in the tangent modulus reduces as the sand approaches failure. The modified hyperbolic model, in which the increment in the deviator stress after consolidation is considered as the stress variable, is shown to represent satisfactorily the stress-strain response of anisotropically consolidated sand. The small strain response of anisotropically consolidated sand also shows a hyperbolic variation which is different from the large strain one. The elastic tangent modulus, at a given stress state, of water pluviated isotropically consolidated sand is not unique. It varies with stress path and direction of loading. Thus, the incremental elastic modeling based on hyperbola under conventional stress paths is shown not applicable for other stress paths and loading direction. The failure strength of sand is uniquely related to maximum rate of dilatancy d∈[sub v]/d∈[sub a] regardless of the relative density, minor effective principal effective stress at failure and stress path for both compression and extension loading. The failure strength depends only on the normal stresses at failure and relative density and is not affected by consolidation history or stress path. The water pluviated sand yields a higher failure strength under compression loading than under extension loading.
Applied Science, Faculty of
Civil Engineering, Department of
Graduate

Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

\(Escherichia\) \(coli\) encounters nitrosative stress from various sources. It was shown in a previous study that a possible role for Hcp in the nitrosative stress response. The focus of this study was to determine the function of Hcp. The growth of the \(hcp\) mutant lacking all known NO reductases was inhibited by various sources of nitrosative stres. The growth defect was complemented by native Hcp protein, but not by mutated Hcp protein with a disrupted hybrid cluster. The role of Hcp was shown to protect \(E\). \(coli\) from nitrosative stress, and the hybrid cluster is critical for its function. Direct interaction between Hcp and its oxidoreductase Hcr was demonstrated. The \(hcp\)\(^+\)\(hcr\) strain showed that it was still resistant to nitrosative stress. Possibly alternative oxidoreductase of Hcp exists in \(E\). \(coli\). Gas analysis of the headspace of the anaerobic cultures showed that when treated with NO, the Hcp\(^+\) strain lacking all known NO reductases was still capable of reducing sub-micro molar NO into N\(_2\)O, while the further deletion of Hcp completely abolished NO reduction. This provides the first in vivo evidence that Hcp is a high affinity, low capacity NO reductase in \(E\). \(coli\).

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Basidiomycetous yeasts are found in a wide range of geographical areas ranging from tropical forests to desert regions. These yeasts are associated with different habitats such as soil, decaying vegetative debris, living plants and animals. Some may even be opportunistic human pathogens. In most of these habitats the yeasts may periodically be exposed to adverse conditions such as osmotic stress. Forty-one basidiomycetous yeast strains obtained from culture collections and isolated from nature were studied using various methods which includes the determination of different minimum water activities (aw; NaCl or sorbitol) for growth, survival in soil of varying moisture content, intracellular osmolytes accumulated and their release upon hypo-osmotic stress. The growth of most strains showed greater tolerance to NaCl than sorbitol at the same level of water activity. Interestingly, there were no basidiomycetous strains that showed growth below 0.90aw. 13C nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC) was used to analyze the osmolytes accumulated by all the strains of basidiomycetous yeasts when grown at 0.95 aw (NaCl). Glycerol was the major solute accumulated intracellulary by all the yeasts. Arabitol, mannitol or trehalose was accumulated in addition to glycerol in most yeasts whereas a number of yeasts only accumulated glycerol when grown at reduced aw. However, Cryptococcus laurentii US 1F was an exception by accumulating three solutes intracellularly when grown at reduced water activity. When exposed to hypo-osmotic shock all three solutes were rapidly released from the cells. Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 and Cryptococcus neoformans US I1 were further analyzed with 13C NMR spectroscopy to determine whether the type of osmolyte accumulated during different phases of growth at 0.95aw (NaCl) might change. No changes were observed as the same osmolytes were accumulated in all cases. Five yeast strains (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 and Sporidiobolus salmonicolor CBS 5937) were analyzed by HPLC for osmolytes released when exposed to hypo-osmotic shock. The strains differed in the pattern of response of osmolyte release. Only three strains released most of their osmolytes rapidly within 5 min, while C. macerans CBS 2206T and R. muculaginosa CBS 5951 retained most of the osmolytes intacellularly. This suggests that there might be different mechanisms of osmolyte release in basidiomycetous yeasts. A few strains of basidiomycetous yeasts (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F. floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, S. salmonicolor CBS 5937 and Filobasidium capsuligenum CBS 4381) were grown in soil cultures of different field capacity (100%, 25%, 10% and 5%) and evaluated for their survival in this environment. All the strains grew at 100% field capacity. Strains R. mucilaginosa CBS 5951, F. floriforme CBS 6240 and F. capsuligeum CBS 4381 also showed growth in soil at 25% field capacity. However, strains C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and C. laurentii CBS 0139 did not grow at this moisture content but survived up until the end of the experimental period. At lower soil moisture content (5% and 10% field capacity), the yeast strains either showed survival or decreased viability towards the end of the experimental period. Strain C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 and F. floriforme CBS 6240 showed survival at both 5% and 10% field capacity. However, strain F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 and S. salmonicolar CBS 5937 showed a decrease in viability after either 2 or 5 days of incubation. No relationship could be found between the type and number of intracellular osmolytes accumulated when exposed to osmotic stress (0.95aw NaCl) and the ability to grow and survive in soil with lower moisture content. Similarly, the ability of the yeasts to grow and survive in soil with lower moisture content did not correlate with their minimum aw for growth in a liquid medium. It was speculated that other factors, such as the physico-chemical composition of the soil, may also play a role in the survival of a particular yeast species in soil. This study has shown that the responses of basidiomycetous yeasts to reduced aw are physiologically similar to the ascomycetous yeasts. The types of osmolytes accumulated are similar but the basidiomycetous yeasts appear to be more sensitive to reduced aw and they tolerate NaCl better than sorbitol whereas the ascomycetous yeasts tolerate high sugar environments better. This is in agreement with the environments where these yeasts are usually found.
AFRIKAANSE OPSOMMING: Basidiomisete giste word aangetref in ‘n wye reeks geografiese areas, wat strek vanaf tropiese woude tot woestynstreke. Hierdie giste word geassosieer met verskillende habitatte soos grond, verrottende vegetatiewe reste, lewende plante en diere. Sommige mag selfs opportunistiese menslike patogene wees. By meeste van hierdie habitatte mag giste periodies blootgestel word aan moeilike toestande soos osmotiese stres. Een-en-veertig basidiomisete gisrasse, verkry vanaf kultuurversamelings en geisoleer vanuit die natuur, was bestudeer met verskeie metodes, waaronder die bepaling van verskillende minimum water aktiwiteite (aw; NaCl of sorbitol) vir groei, droë massa bepalings, akkumulasie van intrasellulêre osmoliete, asook hul vrystelling met hipo-osmotiese stres. Meeste rasse het meer weerstand teen NaCl as sorbitol gehad by dieselfde vlak van wateraktiwiteit. Dit was interessant om op te let dat geen basidiomisete stamme groei onder 0.90aw getoon het nie. Beide 13C kern magnetiese resonansie (KMR) spektroskopie en hoë uitset vloeistof chromatografie (HUVC) was gebruik om alle opgehoopde osmoliete te analiseer vir alle basidiomisete gisrasse tydens groei tot by 0.95 aw (NaCl). Vir alle giste was gliserol die opgelosde stof wat die meeste intrasellulêr opgehoop het. Arabitol of mannitol of trehalose het saam met gliserol in meeste giste opgehoop, terwyl ‘n aantal giste slegs gliserol opgehoop het tydens groei by verlaagde aw. Cryptococcus laurentii US 1F was daarenteen ‘n uitsondering deurdat dit drie opgelosde stowwe intrasellulêr versamel het tydens groei by verlaagde wateraktiwiteit. Al drie hierdie opgelosde stowwe is uit die selle vrygesel na blootstelling aan hipo-osmotiese skok. Verdere 13C KMR spektroskopie analise was gedoen op Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 en Cryptococcus neoformans US I1 om vas te stel of die tipe opgehoopde osmoliet tydens verskillende fases van groei by 0.95aw (NaCl) mag verander. Geen veranderinge was egter waargeneem aangesien dieselfde osmoliete in alle gevalle opgehoop het. Vyf gisrasse (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 en Sporidiobolus salmonicolor CBS 5937) was geanaliseer deur HUVC vir osmolietvrystelling tydens blootstelling aan hipo-osmotiese skok. Die rasse het verskil in hul responspatroon van osmolietvrystelling. Slegs drie rasse het meeste van hul osmoliete vrygestel binne die eerste 5 minute, terwyl ander, C. macerans CBS 2206T en R. muculaginosa CBS 5951 meeste van hul osmoliete intrasellulêr teruggehou het. Dit dui daarop dat daar dalk verskillende meganismes van osmolietvrystelling in basidiomisete giste mag voorkom. Etlike basidiomisete gisrasse (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, (B) S. salmonicolor CBS 5937, Filobasidium capsuligenum CBS 4381) was opgegroei in grondkulture van verskillende veldkapasiteit (100%, 25%, 10% en 5%) en ondersoek vir hul oorlewing in hierdie omgewing. Al die rasse kon groei by ‘n 100% veldkapasiteit. Die rasse R. mucilaginosa CBS 5951, F. floriforme CBS 6240 en F. capsuligeum CBS 4381 kon ook groei in grond met 25% veldkapasiteit. Alhoewel rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T en C laurentii CBS 0139 nie kon groei by hierdie voginhoud nie, het hulle nog steeds oorleef tot aan die einde van die eksperimentele tydperk. By verlaagde grond voginhoud (5% en 10% veldkapasiteit) het die gisrasse of oorleef of ‘n verlaagde lewensvatbaarheid openbaar teen die einde van die eksperimentele prosedure. Die rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 en F. floriforme CBS 6240 het oorleef by beide 5% en 10% veldkapasiteit. Die rasse F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 en S. salmonicolar CBS 5937 het egter ‘n verlaging in lewensvatbaarheid getoon na ‘n periode van of 2 of 5 dae inkubasie. Geen ooreenstemming kon gevind word tussen die tipe en aantal intrasellulêre opgehoopde osmoliete nie na blootstelling aan osmotiese druk (0.95aw NaCl), asook hul vermoë om te groei en oorleef in grond met ‘n laer voginhoud. Terselfdertyd het die giste se vermoë om te groei en oorleef in grond met ‘n laer voginhoud nie ooreengestem met hul minimum aw vir groei in vloeibare groeimedium nie. Ter spekulasie kan ander faktore, soos die fisio-chemiese samestelling van die grond ook ‘n rol speel in die oorlewing van ‘n spesifieke gisspesie in grond. Hierdie studie het getoon dat basidiomisete giste se respons tot verlaagde aw fisiologies dieselfde is as vir askomisete giste. Alhoewel die tipe versamelde osmoliete identies was, wil dit voorkom asof basidiomisete giste meer sensitief teenoor ‘n verlaagde aw is, tesame met ‘n beter bestandheid teen NaCl as sorbitol, terwyl askomisete giste hoë suiker omgewings beter kan verdra. Dit is in ooreenstemming met die tipe omgewings waar hierdie giste gewoonlik voorkom.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Previous studies have indicated that fermentation with yeast strains whose cell walls contain higher chitin levels may lead to reduced wine haze formation. In order to adjust cell wall chitin levels, more information on the regulation of chitin synthesis in wine-relevant yeast is required. Yeast cells are known to increase chitin levels when subjected to certain environmental changes such as an increase in temperature. The main aim of this project was to investigate chitin accumulation and synthesis in wine yeast strains when exposed to environmental change. This was achieved by subjecting the strains to various environmental conditions and comparing chitin levels. The information gained may aid future selection and/or manipulation of yeast strains for the production of higher chitin levels. Three Saccharomyces cerevisiae strains and two Saccharomyces paradoxus strains were subjected to conditions that had been linked to a change in chitin synthesis in past studies in laboratory yeast strains. Of the conditions used in this study, the addition of calcium to a rich media led to the highest cell wall chitin levels. The data also show that chitin synthesis is largely strain dependant. Two conditions which resulted in increased chitin deposition were chosen for gene expression analyses, using strains with strongly diverging average chitin levels. Results showed that an increase in chitin levels correlates with an increase in expression of GFA1, the gene encoding for the first enzyme of the chitin synthesis pathway. Overall, this study provides novel insights into chitin synthesis in Saccharomyces cerevisiae wine yeast strains as well as Saccharomyces paradoxus strains, with possible future implications on haze prevention studies.
AFRIKAANSE OPSOMMING: Vorige studies het aangetoon dat fermentasie met gisrasse waarvan die selwande hoë chitienvlakke bevat, kan lei tot verminderde wynwaasvorming. Om selwandchitienvlakke aan te pas, word daar meer inligting rakende die regulering van chitienvlakke in wyn gisrasse verlang. Dit is bekend dat gisselle chitienvlakke verhoog wanneer die selle onderwerp word aan sekere veranderinge in die omgewing soos ’n verhoging in temperatuur. Die hoofdoel van hierdie projek was om die chitienopbou en -sintese in wyngisrasse te ondersoek waar gis blootgestel word aan omgewingsveranderinge. Dit is bereik deur die selle aan verskeie omgewingstoestande bloot te stel en chitienvlakke met mekaar te vergelyk. Die inligting hieruit verkry kan toekomstige gisraskeuses asook die manipulering van gisrasse met die oog op hoër vlakke van chitienproduksie vergemaklik. Drie Saccharomyces cerevisiae rasse en twee Saccharomyces paradoxus rasse is onderwerp aan toestande wat in vorige studies gekoppel is aan ’n verandering in chitienvorming in laboratorium-gisrasse. Van die toestande toegepas in hierdie studie, het die toevoeging van kalsium tot ’n nutrientryke medium gelei tot die hoogste chitienvlakke in selwande. Die data toon ook aan dat chitiensintese hoofsaaklik rasverwant is. Twee toestande wat gelei het tot verhoogde chitienafsetting is gekies vir geen-uitdrukkingsanalise, terwyl rasse gebruik is met gemiddelde chitienvlakke wat wyd uiteenlopend is. Die resultate het getoon dat ’n verhoging in chitienvlakke ooreenstem met ’n verhoging in die uitdrukkingsvlakke van GFA1, die geen wat kodeer vir die eerste ensiem in die chitiensintesebaan. Oor die algemeen verskaf hierdie studie nuwe insigte oor chitiensintese in Saccharomyces cerevisiae wyngisrasse en Saccharomyces paradoxus rasse en verskaf dit belangrike inligting vir moontlike toekomstige studies oor waasvoorkoming.

Francisella tularensis is capable of infecting numerous cell types, including professional phagocytes. Upon phagocytosis, F. tularensis resides within the phagosome before escaping into the cytosol to replicate. Phagocytes constitute a hostile environment rich in ROS, which are employed as a means of killing pathogens. ROS interact with and disrupt the function of vital molecules such as DNA, proteins and bacterial structures. Iron potentiates the danger of ROS through the Fenton reaction where ferrous iron reduces H2O2 causing the formation of highly reactive hydroxyl radicals and anions. Low levels of ROS are formed during normal aerobic metabolism and pathogens thus have a need for defense mechanisms to handle the ever present levels of ROS but even more so to combat the onslaught of ROS experienced within a host. This thesis was focused on the investigation of the iron status and oxidative stress response of F. tularensis; thereby identifying key players controlling the bacterial iron content, its adaptation to oxygen-rich environments and defense against ROS. We identified subspecies-specific differences in iron content, where F. tularensis subsp. tularensis was found to contain significantly less iron than strains of subsp. holarctica. The reduced iron content resulted in an increased tolerance to H2O2, despite simultaneously causing a decrease in the activity of catalase - the iron-dependent enzyme responsible for degrading H2O2 in F. tularensis. This strongly suggests that the restricted iron uptake and storage by subsp. tularensis strains is beneficial by rendering the bacteria less susceptible to H2O2, thereby evading the toxic effects of the iron-driven Fenton reaction. This evasion is likely to be an important part of the higher virulence displayed by subsp. tularensis as compared to subsp. holarctica. We further identified that the global regulator, MglA, is important for the adaptation of LVS to oxygen-rich environments. Deletion of mglA from LVS resulted in a mutant, ΔmglA, with impaired defense to oxidative stress, as manifested by an inability to grow to wild-type levels under aerobic conditions, an accumulation of proteins with oxidative damage, a suppressed expression of iron-uptake related genes, an increased catalase activity, and an increased tolerance to H2O2. This phenotype was reversed in a microaerobic environment. We therefore conclude that MglA is an important factor for the defense of LVS to oxidative damage under aerobic conditions and speculate that MglA is of greatest importance in oxygen-rich foci. We also studied the role of OxyR in LVS by creating a ΔoxyR mutant as well as a double mutant, ΔoxyR/ΔkatG. The in vitro response of these mutants, as well as of ΔkatG, to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS as was ΔkatG, with the exception of O2- Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages whereas ΔoxyR/ΔkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver, but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis, and further reveal overlapping roles of OxyR and catalase in the defense against ROS. The results thus shed new light on the complexity of ROS defense in F. tularensis.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7412号
農博第996号
新制||農||763(附属図書館)
学位論文||H10||N3158(農学部図書室)
UT51-98-G341
京都大学大学院農学研究科水産学専攻
(主査)教授 坂口 守彦, 教授 内田 有恆, 教授 宮本 元
学位規則第4条第1項該当

The response of plants to environmental stress spans several orders of magnitude in time and space, causing system-wide changes. These changes comprise of both protective responses and adverse reactions in the plant. Stresses like water deficit or drought cause a drastic effect in crop yield, while concomitantly agriculture consumes 1/3rd of the fresh water available to us and there is widespread water scarcity around the world. It is, hence, a fundamental goal of modern biology and applied biotechnology to unravel this complex stress response in laboratory model plants like Arabidopsis and crop models like rice. Such an understanding, especially at the cellular level, will aid in informed engineering of stress tolerance in plants. We have developed and used integrative functional genomics approaches to characterize environmental stress response at various levels of organization including genes, modules and networks in Arabidopsis and rice. We have also applied these methods in problems concerning bioenergy. Since the poor knowledge of the cellular roles of a large portion of plant genes remains a fundamental barrier to using such approaches, we have further explored the problem of 'gene function prediction'. And, finally, as a contribution to the community, we have curated a large mutant resource for the crop model, rice, and established a web resource for exploratory analysis of abiotic stress in this model. All together, this work presents insights into several facets of stress response, offers numerous novel predictions for experimental validation, and provides principled analysis frameworks for systems level analysis of environmental stress response in plants.
Ph. D.

Background: The last two decades have seen several new methods for analysing gene expression data. One such method is pathway analysis in which it’s possible to decipher response patterns between pathways (groups of interacting genes) and external stress factors. A critical issue in pathway analysis is often limited sample sizes resulting in undesirable batch affects. A method for reducing such effects, called Centralization Within Sub-Experiments (CSE), has recently been developed. This method makes it possible to predict pathways using data from experiments with a diversity of external conditions. Aims: We propose a method for identifying stress-responsive pathways predicted using CSE pre-processed expression data. Method: 27 CSE predicted pathways were analysed and tested for stress-responsiveness associated with specific external stress factors. Firstly, we screened the complete gene list for DE genes. Secondly, we analysed the occurrences of DE genes within each pathway and finally, an over-representation analysis was performed with the aim of identifying pathways with significantly larger portions of differently expressed genes categorised as stress-responsive. Results: The analysis resulted in a list of pathways with significantly larger proportions of DE genes. This was only when screened for salt-responsive pathways. We also found that these pathways contained several mitochondrial genes confirmed to be associated with salt stress. Conclusion: The results show great promise in using the method for extracting information regarding the stress responsiveness amongst pathways predicted using CSE-pre-processing expression data.

Mestrado em Bioquímica - Bioquímica Clínica
Iron is a key element for basic cellular functions. If iron homeostasis is not maintained it may lead to iron overload. Patients with Hereditary Hemochromatosis (HH) and with the C282Y HFE mutation have a progressive severe iron overload that, if it is not treated, may lead to tissue damage, that mostly culminate in hepatic cirrhosis and carcinoma. Having in mind that tissue damage in HH may be related with oxidative stress (OS) caused by iron toxicity, it is important to understand in what way the OS defense is acting in cells from HH patients with severe forms of iron overload. Few studies have been performed concerning the eventual prooxidant state in blood cells, which bear a major source of OS. Nevertheless, in a recent study it was shown that cultured lymphocytes (LY) from HH, when compared with cultured LY from controls and patients with secondary forms of hemochromatosis, have an increased protection against chromosome instability (CI) induced by 1,2:3,4 diepoxybutane (DEB) – an OS-related alkylating agent. This suggests an adaptive response of HH cells to the high level of OS. However, it is not known yet if the same response can be observed with other sources of iron toxicity, namely in the presence of bleomycin (BLM), that acts forming a complex with non-transferrin bound iron (NTBI). In order to better understand the oxidant status of HH blood cells and the putative adaptive response of HH cells to iron toxicity, a study was performed to characterize two selected OS parameters: evaluation of reduced glutathione (GSH) depletion and of lipid peroxidation (LPO). The study was performed in red blood cells (RBC) and lymphocytes (LY), either basal and after 36h in culture, with and without induction of OS. Induction of OS was performed with DEB and with BLM. A second objective of the present work was to test if the previously observed adaptive response of HH cells to DEB-induced OS can also be observed after induction with BLM. Characterization of the OS parameters was performed in RBC and LY from 5 HH patients with severe iron overload and 6 healthy donors (HD), at day 0 and after 36h of culture, non-treated and treated with DEB or BLM. Studies of CI were performed in BLM-induced LY from the same 5 HH patients and 6 HD. The results show that RBC from HH patients, compared with those from HD, have a larger GSH depletion and more LPO, either at day 0 and after 36h in culture medium. This suggests an increased level of OS in HH RBC. On the contrary, LY from HH patients present less GSH depletion after 36h of culture than LY from HD, being this effect more pronounced in DEB and BLM-treated cultures. Additionally, LPO levels were decreased in LY from HH patients after 36h of culture when compared with LY from HD. This result suggests that HH cultured LY, either non-treated or treated with DEB and BLM, have a still not completely understood mechanism of defense against OS. BLM-induced CI in cultured LY from HH patients was not different from the observed in cultured LY from HD. Therefore, we can postulate that toxicity induced by BLM did not increased CI in cells from HH patients with severe iron overload.
O ferro é um dos elementos chave para as funções celulares básicas. Se a sua homeostasia não for corretamente mantida, poderá ocorrer uma sobrecarga de ferro no organismo. Os doentes com Hemocromatose Hereditária (HH), com a mutação C282Y no gene HFE, possuem uma progressiva e severa sobrecarga de ferro que, se não for tratada, pode levar a dano nos tecidos, podendo mesmo culminar em cirrose hepática e carcinoma. Tendo em conta que o dano tecidular pode estar associado ao stress oxidativo (OS) causado pela sobrecarga de ferro, é importante perceber de que modo atua o sistema de defesa contra o OS nas células dos doentes HH com forma severa de sobrecarga de ferro. Poucos estudos foram realizados sobre o potencial estado oxidante nas células do sangue, onde se encontra uma das maiores fontes de reações oxidativas. Contudo, num estudo recente foi demonstrado que linfócitos de doentes com HH, quando comparados com linfócitos de controlos e pacientes com formas secundárias de hemocromatose, apresentam uma maior proteção relativamente à instabilidade cromossómica (CI) induzida por 1,2:3,4 diepoxibutano (DEB) – um agente alquilante que provoca OS. Este estudo sugere uma resposta adaptativa das células HH a níveis elevados de OS. No entanto, ainda não se sabe se esta mesma resposta pode ser observada com outras fontes de toxicidade do ferro, nomeadamente na presença de bleomicina (BLM) cuja atividade depende da formação de complexos com o ferro não ligado à transferrina (NTBI). Para compreender melhor o estado oxidante das células do sangue dos doentes HH e a suposta resposta adaptativa das células dos doentes de HH à toxicidade do ferro, foi feita a análise de dois parâmetros de OS selecionados: avaliação da depleção da glutationa reduzida (GSH) e da peroxidação lipídica (LPO). Esta análise foi efetuada em eritrócitos (RBC) e linfócitos (LY), tanto no tempo 0 como passadas 36h em cultura, com ou sem indução de OS. O segundo objetivo deste trabalho foi testar se a BLM promove uma resposta adaptativa à CI comparável à que foi observada com o DEB. Tanto a caracterização dos parâmetros de OS como os estudos de CI foram efetuados em células de 5 doentes com HH, com elevada sobrecarga de ferro, e em células de 6 dadores saudáveis (HD). Os resultados mostraram que os RBC dos doentes com HH, comparativamente com os dos HD, apresentam uma maior depleção de GSH e maior LPO, quer ao dia 0 quer após 36h em meio de cultura. Estes resultados sugerem um aumento de OS nos RBC dos doentes. Contrariamente, os LY dos doentes de HH apresentaram menor depleção de GSH após 36h de cultura, sendo esta mais notória nas culturas induzidas com DEB e BLM. Adicionalmente, os níveis de LPO são menores em LY dos doentes de HH, após 36h de cultura, comparativamente com os dos HD. Isto sugere que culturas de LY, quer não-tratadas quer tratadas com DEB ou BLM, têm um algum tipo de mecanismo de defesa contra o OS, ainda não compreendido. A frequência de CI induzida por BLM em LY de doentes com HH não é significativamente diferente da observada em LY de HD, não se observando assim uma diferença na capacidade de resposta à BLM, entre células de doentes e controlos. Pode-se então concluir que a toxicidade induzida por BLM não aumenta a CI em células de doentes com HH com forma severa de sobrecarga de ferro.

Introduction Laparoscopic colorectal surgery and enhanced recovery programs have been shown to improve patient outcomes and reduce length of stay following surgery. The use of regional analgesia is usually a fundamental element of an enhanced recovery program. A proposed benefit of regional analgesia in colorectal surgery is suppression of the post-operative stress response. No data is available to indicate if this is applicable in laparoscopic colorectal surgery. In addition there is no direct evidence addressing whether there is an appropriate type of fluid, crystalloid or colloid, to use in goal-directed fluid therapy. The aim of this study was to examine the effects the choice of analgesia and intravenous fluid had on physiological and biochemical outcomes following laparoscopic colorectal surgery in patients within an enhanced recovery program. Methods A randomized clinical trial (NCT 01128088) was conducted between 2010-2011 at a single institution. All patients underwent laparoscopic colorectal surgery for benign or malignant conditions within an established enhanced recovery program. Patients were randomly assigned to receive either a spinal or morphine PCA as their primary post-operative analgesia. In addition, patients were randomly allocated to receive either 6% Volulyte or Hartmann’s solution, which was administered as directed by an oesophageal Doppler monitor in order to achieve stroke volume optimisation. Blood was taken to measure aspects of the stress response at pre-op, 3, 6, 12, 24 and 48 hour time intervals. Various other physiological and patient outcomes were measured. Results One hundred and twenty patients were analysed in the study. There was no significant difference in patient characteristics between the groups. No significant difference was seen between the analgesia groups at pre-op, 3, 6, 12, 24 or 48 hours in the levels of insulin, IL-2, 4, 6, 8, 10, 12, TNF-α, VEGF or IFN-γ. Median cortisol (468 nmol/l (IQR: 329-678) vs 701 nmol/l (IQR: 429-820); p=0.004) and glucose (6.1 mmol/l (IQR: 5.4-7.5) vs 7 mmol/l (IQR: 6-7.7); p=0.012) levels were significantly lower at 3 hours post-op in the spinal group and thereafter the same. Patients receiving Hartmann’s solution received significantly greater volumes of fluid in comparison to those receiving 6% Volulyte (20.98ml/kg (IQR: 16.68-25.73) vs 13.95ml/kg (IQR: 11.76-18.1); p<0.0005). There was no significant difference in the median length of stay between either fluid (6% Volulyte 2.75 days [IQR: 2.08-3.6] vs Hartmann’s 2.29 days [IQR: 2.01-3.59]; p=0.807) or analgesia (spinal 2.25 days [IQR: 1.89-3.13] vs PCA 2.9 days [IQR: 2.09-3.93]; p=0.059) groups. The number of complications was no different between the two types of fluids but patients receiving spinal analgesia (20% vs 37%, p=0.013) had a significantly reduced number. Conclusion Following laparoscopic colorectal surgery within an enhanced recovery program the use of spinal analgesia in comparison to morphine PCA significantly reduces the levels of cortisol and glucose at 3 hours only. This difference does not translate to a reduction in length of stay. There is no effect on other aspects of the stress response at various post-operative time intervals. A significantly greater quantity of Hartmann’s solution is required to achieve stroke volume optimisation in comparison to 6% Volulyte. This, however, did not make any difference to the incidence of post-operative complications or length of stay. Either fluid is acceptable in the quantities given in this study.

The pathogen Streptococcus pneumoniae encounters different levels of oxygen during the infection cycle including colonisation, pneumonia, bateraemia and meningitis. These different anatomical niches require high levels of genome changes to sense and respond to those external environmental stimuli. The bacterial gene expression is known to be affected by oxygen, and it must react properly for survival and for developing invasive pneumococcal desiseases (IPDs). Microarray techniques have allowed scanning the whole pneumococcal genome during growth in different tensions of oxygen mimicking in vivo conditions. It was found that oxygenated growth conditions have significantly elevated several key virulence genes. This was further confirmed with qRT-PCR for a selection of genes implicated in pathogenicity. Moreover, post-transcriptional stages have been also investigated such as protein production, biofilm formation, biological activities and adherence assays for several virulence factors performed under the effect of presence or absence of oxygen. The data illustrate that 420 out of 2,236 genes (17 % of the entire TIGR4 genome) were differentially expressed in the presence of oxygen compared to its absence. 262 genes (11 %) were over-expressed when pneumococci were grown in oxygenated conditions relative to transcriptional profile in anaerobic growth conditions, indicating the magnitude of roles played by oxygen on pneumococcal gene expression. Anaerobic growth of TIGR4 showed down-regulation of 158 genes (7 %). Oxygen modulates induction of ply, pspC and other seven genes involved in pili structuring subunits (rlrA, rrgA, rrgB and rrgC) and assembling enzymes (srtB, srtC and srtD). This may suggest that the pneumococcal population grown under atmospheric environment is equipped with greater capability to progress IPDs compared to anaerobically grown bacteria. In addition to this, pneumococcal adhesion in vitro for TIGR4 grown in oxygenated or anaerobic growth conditions revealed a significant increase in those grown in oxygenated growth conditions, indicating that oxygen may play a key role in bacterial-host attachment. Interestingly, ablation of pspC has resulted in similar adhesion percentages of TIGR4 grown under both conditions, oxygenated and anaerobic. Furthermore, several genes involved in metabolism were up-regulated in oxygenated environment, particularly transporters, which are considered highly important for a bacterium that lacks an electron transport chain, catalase and tricarboxylic acid. Additionally, the results showed phenotypic characterisation and changes in cells morphology from pneumococcal growth curves for several strainswith different genome backgrounds grown under different levels of oxygen concentrations. Further investigation of the pathogen biology revealed differences in pneumolysin production and activity. These findings highlight that virulence genes expression is induced once the micro-organism is exposed to oxygenated environment, and data analysis has demonstrated potential links between pneumococcal metabolism and their ability to cause diseases.

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that 13C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained to a remarkable extent during an ischemic time course. These findings suggested an unexpected source of glucose in HIF-1α hearts during global ischemia. Accordingly, the presence of gluconeogenesis in hearts was evaluated. Indeed, gluconeogenic intermediates (i.e. m+3) including glucose-6-phosphate [m+3], fructose-6-phosphate [m+3], and fructose 1,6-bisphosphate [m+3] were observed at significantly elevated levels in the ischemic HIF-1α heart. Collectively, these data establish the surprising finding that HIF-1α supports active gluconeogenesis in the heart during ischemia. As less is known regarding the effects of CTRP3 we first tested whether CTRP3 overexpression would protect the ischemic heart. Our data indicate that CTRP3 failed to confer ischemic tolerance in heart ex vivo. However,we were able to show that CTRP3 protected the liver from lipid-induced stress and prevented hepatic lipid accumulation. To further investigate the mechanisms of hepatic protective effect mediated by CTRP3, we identified the receptor and established that CTRP3 increases oxygen consumption in response to lipid overloaded. Lysosomal-associated membrane protein 1 (LAMP-1), In summary, these data indicate that targeted metabolic rearrangements within cardiomyocyte/hepatocyte holds promise for the alleviation of common pathological conditions.

Human cells are continuously subject to conditions that cause breaks to DNA. This project explored the interaction between molecules involved in repairing this damage, called hSSB1 and THOC4. Novel discoveries were made that expand our understanding of this important process and identified THOC4 as a potential novel therapeutic target in lung and breast cancer.

A recent evolutionary theory hypothesizes that there are two primary biobehavioral profiles of stress responding. Labeled "hawk" and "dove," each is characterized by divergent patterns of autonomic nervous system and neuroendocrine system activations in response to stress as well as distinct affective and behavioral tendencies. These profiles are prominent in a number of species, and it has been hypothesized that hawk-like and dovelike responses to stress may, in part, explain variability in stress-related health outcomes. This study is a preliminary investigation of hawk and dove biobehavioral profiles in humans. Participants included 73 Midwestern university students recruited from undergraduate-level psychology classes. Upon completion of a stressor task, participants answered questions regarding their psychological experiences during and immediately following the task and reported their emotions and health-related behaviors over the past several weeks. Physiological measures of cortisol and high frequency heart rate variability reactivity were used to identify relatively hawk-like and dove-like responders. Associations between patterns of physiological responding and emotional and behavioral responses were tested. The results showed mixed support for the existence of hawk and dove biobehavioral profiles in humans.

Coral reef biodiversity is threatened by rapidly changing anthropogenic activities and natural perturbations, leading to massive ecological and economic consequences ranging from the loss of fisheries to coastal erosion. It is necessary to understand corals responses to environmental changes in order to determine management programs on appropriate spatial and temporal scales to address these issues. Coral larvae are the product of sexual reproduction, have the potential to recruit to new areas, and are fundamental in maintaining genetic diversity. These larvae are subjected to variations in local environmental conditions until they settle, inducing specific larval molecular response patterns. One factor that influences coral health is salinity. Low salinities can alter cell homeostasis creating stress in cells. In the natural environment larvae may be exposed to low salinities due to heavy rainfall or run-off. This study investigated larvae responses to low salinity and characterized gene expression in the reef-building coral Porites astreoides using a coral stress-focused microarray. Nine batches of 250+ larvae from three different colonies were collected and immediately exposed in an acute hyposalinity experiment. Samples from two treatments of 25 and 30 ppt, and a control at 35 ppt were used in this study. After experimental exposure these samples were stored in RNAlater® and molecular analysis was performed. The RNA from the samples was extracted, purified and hybridized to a coral stress-focused microarray. Statistical analysis indicates 72 genes were differentially expressed across treatments (p<0.003, analysis of variance). The hierarchical cluster analysis groups together the larvae exposed to salinities of 30 and 35 ppt indicating both treatments induced similar patterns of gene expression. Larvae responses to 30 ppt are minimal, suggesting larvae can tolerate acute exposures to 30 ppt salinity levels. In contrast, the lower salinity (25 ppt) induced a strong response in both the coral and zooxanthellae. The coral larvae up-regulated stress response genes and down-regulated genes associated with normal cell functioning. Additionally, the zooxanthellae down-regulated genes associated with photosynthesis. These results suggest larvae may be vulnerable to bleaching, which may affect the ability of larvae to successfully undergo metamorphosis and survive at low salinities. However, this has yet to be confirmed with complementary techniques. Long-term studies are recommended to examine the effects of hyposalinity on larvae at different time scales and life history stages.

Mathematical modelling of gene regulatory networks is a relatively new area which is playing an important role in theoretical and experimental investigations that seek to open the door to understanding the real mechanisms that take place in living systems. The current thesis concentrates on studying the animal stress-response gene regulatory network by seeking to predict the consequence of environmental hazards caused by chemical mixtures (typical of industrial pollution). Organisms exposed to pollutants display multiple defensive stress responses, which together constitute an interlinked gene network (the Stress-Response Network; SRN). Multiple SRN reporter-gene outputs have been monitored during single and combined chemical exposures in transgenic strains of two invertebrates, Caenorhabditis elegans and Drosophila melanogaster. Reporter expression data from both species have been integrated into mathematical models describing the dynamic behaviour of the SRN and incorporating its known regulatory gene circuits. We describe some mathematical models of several types of different stress response networks, incorporating various methods of activation and inhibition, including formation of complexes and gene regulation (through several known transcription factors). Although the full details of the protein interactions forming these types of circuits are not yet well-known, we seek to include the relevant proteins acting in different cellular compartments. We propose and analyse a number of different models that describe four different stress response gene networks and through a combination of analytical (including stability, bifurcation and asymptotic) and numerical methods, we study these models to gain insight on the effect of several stresses on gene networks. A detailed time-dependent asymptotic analysis is performed for relevant models in order to clarify the roles of the distinct biochemical reactions that make up several important proteins production processes. In two models we were able to verify the theoretical predictions with the corresponding laboratory experimental observations that carried out by my coworkers in Britain and India.