Dissertations / Theses on the topic 'Bacterial hormones'

Soil and root microbial communities have been studied for decades, and the incorporation of high-throughput techniques and analysis has allowed the identification of endophytic/non-culturable organisms. This has helped characterize and establish the core microbiome of many model plant species which include underground and aboveground organs. Unfortunately, the information obtained from some of these model plants is not always transferable to other agronomic species. In this project, we decided to study the microbiome of the Nicotiana genus because of its importance in plant physiological and plant-microbe interactions studies. The data obtained was used as baseline information that allowed us to better understand the effect of microbial inoculums on the assembly of the microbiome of the plant. We analyzed 16s rRNA amplicons to survey the microbiome in different plant organs and rhizosphere from four different species. Bacterial strains evaluated were screened for a consistent reduction or improvement in plant growth. Four bacterial strains were tested and used as seed inoculum (Lf-Lysinobacillus fusisormis, Ms –Micrococcus sp., Bs–Bacillus sp., Bc–Bacillus cereus). Bs and Bc inoculants caused plant growth promotion, and in contrast Ms caused retarded growth, while Lf acted as a neutral or non-inducing phenotype strain. Data supported that microbial inoculum used as seed treatment caused systemic changes in the host plant microbiome. Functionality of the inoculum was studied and the response in plant growth was linked to hormonal changes (evaluated in the plant and in the bacterial strains). Gene expression analysis using a genome-scale approach revealed that genes that could possibly be involved in stress response are down-regulated for Bc and Bs treatments and up-regulated for Ms. Flexibility variability of the inoculum was also evaluated to have a better understanding of the main factors involved in the promotion or suppression of growth, and possibly its effect in following generations. In summary, the findings of this project support that the plant functional microbiome responds to exogenous stimulation from abiotic and biotic factors by adapting endogenous hormone responses.

Bacteria in most environments exist and grow in association with surfaces, leading to formation of biofilms. In the medical context, biofilms are particularly significant for human health because of their high resistance to antimicrobial and immune system attack. In the health care setting biofilms associated with indwelling devices such as intravenous catheters and endotracheal tubes is a major clinical problem. It has been shown in previous reports that catecholamine stress hormones such as epinephrine, norepinephrine and structurally similar inotrope drugs used to treat heart and kidney problems in seriously-ill patients, are able to promote growth and virulence of certain bacteria. In this thesis, the role of catecholamine inotropes and stress hormones as an environmental factor for the induction of biofilm formation by infectious bacteria relevant to medical devices is considered. In vitro phenotypic characterisation was investigated by mainly using microbiological techniques, microscopy and proteomic analysis. The first section of this thesis shows how clinically attainable levels of catecholamine inotropes stimulated Pseudomonas aeruginosa growth and biofilm formation. The mechanism by which growth stimulation occurs was found to be via delivery of iron from the serum Fe-binding transferrin. P. aeruginosa growth, biofilm formation and motility were all significantly enhanced by catecholamine inotropes (P<0.05). Inotropes may be a risk factor for ventilator associated pneumonia as they stimulated biofilm formation on endotracheal tubing. In the second section which focuses on Escherichia coli and Salmonella Typhimurium, it was found that catecholamine stress hormone effect on growth, motility and biofilm formation was independent of the putative QseC and QseE catecholamine sensing receptors. In the third section, factors related to catecholamines inotropes Staphylococcus epidermidis biofilm stimulation were considered. Collectively, these findings show that levels of catecholamine inotropes found within critically ill patients can promote bacterial biofilm formation, and so contribute to bacterial pathogenesis within the hospital setting.

Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases. GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G6LRPGGKR13, and correctly cleaves the R13-D14 bond to yield bioactive GAP and a three residue extension of GnRH. We and others have postulated that the recognition site for GAP-releasing enzyme forms a defined structural element at the surface of the substrate protein and that this structural element helps mediate limited endoproteolysis. In the work reported here, hundred mg quantities of pro-GnRH/GAP were prepared by novel methods of both chemical synthesis and bacterial expression. Large amounts of pure protein are required for enzymatic and biophysical studies of pro-GnRH/GAP, which are intended to establish whether or not the processing site within the prohormone exists as a defined structural element that plays a central role in endoproteolytic processing. Synthetic pro-GnRH/GAP was prepared in high yield but proved difficult to purify to homogeneity. Recombinant pro-GnRH/GAP was prepared in sufficient yield and purity to perform all subsequent experiments. An immunoassay was developed against a processing site epitope within pro-GnRH/GAP. Both synthetic and recombinant pro-GnRH/GAP proteins are immunoreactive, consistent with the idea that the epitope, and, thus, the processing site, is located on the surface of the molecule. Proteolysis of synthetic or recombinant pro-GhRH/GAP by trypsin or kallikrein caused immediate loss of immunoactivity, showing that the processing site is susceptible to proteolysis and that the integrity of the processing site is required for immunoactivity. One of the kallikreih hydrolytic products was identified as GAP. Therefore, kallikrein cleaves at the R13-D14 bond. The intrinsic fluorescence yield of the Trp residue near the processing site region of pro-GhRH/GAP is sensitive to changes in pH, but not to changes in ionic strength or calcium concentration; its fluorescence yield is maximal at neutral pH. This suggests that the processing site displays maximum structure at neutral pH. This finding is coincident with the fact that GAP-releasing enzyme is optimally active at neutral pH. However, the relative contribution of secondary structural elements, as discerned by circular dichroism, remains constant over the range of pH 5.2-10.7. Only at pH Thermal denaturation of pro-GnRH/GAP follows a simple two-state transition at neutral pH, as assessed by differential scanning calorimetry. This shows that pro-GnRH/GAP assumes a protein—like tertiary structure at neutral pH. 1D NMR data obtained at variable pH showed changes in resonance position and spectral resolution which are consistent with pH mediated conformational change and with the assumption of organized structure at neutral pH. However, the lack of through space correlations in the 2D NOESY experiment indicates that determination of the three-dimensional structure of pro-GnRH/GAP at neutral pH may be problematic.

Sepsis is the leading cause of death among critically ill patients and the 10th most common cause of death overall in the United States. The mortality rates increase with severity of the disease, ranging from 15% for sepsis to 60% for septic shock. Patient with sepsis can present varied clinical symptoms depending on the personal predisposition, causal microorganism, organ system involved, and disease severity. To facilitate sepsis diagnosis, the first sepsis consensus definitions was published in 1991 and then updated in 2001. Early recognition of a sepsis patient followed with timely and appropriate treatment and management strategies have been shown to significantly reduce sepsis-related mortality, and allows care to be provided at lower costs. Despite the rapid progress in the knowledge of pathophysiological mechanisms of sepsis and its treatment in the last two decades, identifying patient with sepsis and therapeutic approaches to sepsis and its complications remains challenging to critical care clinicians. Hence, the objectives of this thesis were to 1) evaluate the test characteristics of the two sepsis consensus definitions and delineate the differences in patient profile among patients meeting or not meeting sepsis definitions; 2) determine the relationship between the changes in several physiological parameters before sepsis onset and sepsis, and to determine whether these parameters could be used to identify sepsis in critically ill adults; 3) evaluate the effect of corticosteroids therapy on patient mortality. Data used in this thesis were prospectively collected from an electronic medical record system for all the adult patients admitted into the seven critical care units (ICUs) in a tertiary medical center. Besides analyzing data at the ICU stay level, we investigated patient information in various time frames, including 24-hour, 12-hour, and 6-hour time windows. In the first study of this thesis, the 1991 sepsis definition was found to have a high sensitivity of 94.6%, but a low specificity of 61.0%. The 2001 sepsis definition had a slightly increased sensitivity but a decreased specificity, which was 96.9% and 58.3%, respectively. The areas under the ROC curve for the two consensus definitions were similar, but less than optimal. The sensitivity and area under the ROC curve of both definitions were lower at the 24-hour time window level than those of the unit stay level, though the specificity increased slightly. At the time window level, the 1991 definitions performed slightly better than the 2001 definition. In the second study, minimum systolic blood pressure performed the best, followed by maximum respiratory rate in discriminating sepsis patients from SIRS patients. Maximum heart rate and maximum respiratory rate can differentiate sepsis patients from non-SIRS patients fairly well. The area under ROC of the combination of five physiological parameters was 0.74 and 0.90 for comparing sepsis to non-infectious SIRS patients and comparing sepsis to non-SIRS patients, respectively. Parameters typically performed better in 24-hour windows compared to 6-hour or 12-hour windows. In the third study, significantly increased hospital mortality and ICU mortality were observed in the group treated with low-dose corticosteroids than the control group based on the propensity score matched comparisons, and multivariate logistic regression analyses after adjustment for propensity score alone, covariates, or propensity score (in deciles) and covariates. This thesis advances the existing knowledge by systemically evaluating the test characteristics for the 1991 and 2001 sepsis consensus definitions, delineating physiological signs and symptoms of deterioration in the preceding 24 hours prior to sepsis onset, assessing the prediction performances of single or combined physiological parameters, and examining the use of corticosteroids treatment and survival among septic shock patients. In addition, this thesis sets an innovative example on how to use data from electronic medical records as these surveillance systems are becoming increasingly popular. The results of these studies suggest that a more parsimonious set of definitional criteria for sepsis diagnosis are needed to improve sepsis case identification. In addition, continuously monitored physiological parameters could help to identify patients who show signs of deterioration prior to developing sepsis. Last but not least, caution should be used when considering a recommendation on the use of low dose corticosteroids in clinical practice guidelines for the management of sepsis.

The plant hormones salicylic and jasmonic acid (SA and JA, respectively) both play a crucial role in the induction of plant defense system pathways and long-term pathogen resistance. Plants, which do not have an active cellular immune system like animals, instead rely on the release of specific molecules to mediate defense. In general, the SA pathway is activated by biotrophic pathogens and primarily induces antimicrobial responses, while JA is activated by necrotrophic pathogens and herbivory, and induces separate chemical responses. SA and JA are reciprocally antagonistic: activation of one pathway inhibits activation of the other. Here we explore how SA-JA inhibitory crosstalk is used by pathogens or herbivores to combat plant defense. We study the effects of hormone crosstalk on bacterial growth in two plant models: Cardamine cordifolia and Arabidopsis thaliana, which we treated to induce defense pathways. These plants were inoculated with endophytic bacteria isolated from field C. cordifolia plants, and the effects of hormone treatment on bacterial growth rates were measured. We show that JA-induced defenses, which are commonly associated with necrotrophic pathogens, affect varied biotrophic Pseudomonas strains both positively and negatively. Notably, we show that JA-induce defenses affect wild P. fluorescens strains more negatively than SA-defenses.

Orientador : Prof. Dr. Aleksander Roberto Zampronio
Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 29/04/2016
Inclui referências : f. 91-116
Área de concentração
Resumo: A febre pode ser definida como um aumento controlado da temperatura corporal interna de um organismo para níveis acima dos normais, decorrente de uma alteração do ponto de termorregulagem hipotalâmico (set point). Existem diferentes maneiras de mimetizar a febre em animais de laboratório, porém o modelo de administração intraperitoneal de lipopolissacarídeo (LPS) é amplamente utilizado e estável. Uma vez injetada, essa substância se liga à receptores do tipo Toll-Like 4 em células do sistema imune como macrófagos, que passam a sintetizar e liberar diversos pirogênios endógenos, tais como interleucina(IL) -1?, IL-1?, IL-6, IL-8, proteína inflamatória de macrófago (MIP-1?) e o ligante do receptor ativador de fator nuclear ?B (RANKL) entre outros. Estes mediadores induzem a formação e liberação de diversos mediadores centrais como prostaglandinas (PG), endotelina-1 (ET-1), fator liberador de corticotrofina (CRF), substância P (SP), opióides endógenos e endocanabinóides que por sua vez irão atuar em respostas complexas culminando na resposta febril. Entretanto, esses estudos foram todos realizados em animais de experimentação machos. Considerando-se que há evidências de diferenças na resposta inflamatória e febril relacionadas ao gênero em animais experimentais e que mulheres possuem maior incidência de patologias inflamatórias e relacionadas à dor, o objetivo deste trabalho foi avaliar a resposta febril induzida por LPS e por diferentes mediadores que são liberados por este pirogênio em ratas, bem como a influência de hormônios sexuais femininos nestas respostas. Em complementação, investigamos a expressão de ciclooxigenase-2 (COX-2) e de diversos receptores destes mediadores centrais em ratas e a influência da ovariectomia sobre esta expressão. Verificamos que fêmeas ovariectomizadas (OVX) apresentaram temperaturas basais maiores que fêmeas falso-operadas. A injeção de LPS (50 ?g/kg,i.p.) em animais falso-operados induziu uma resposta febril significativamente menor quando comparado com ratas OVX. Estas apresentaram um perfil de resposta febril similar ao observado em machos. O tratamento subcrônico com 17?-estradiol (1 mg/kg, s.c.) em animais OVX reduziu a febre induzida pelo LPS. Fêmeas OVX apresentaram uma redução nos níveis de estradiol plasmático. A administração de LPS aumentou os níveis de testosterona em animais falso-operados e OVX. A administração intracerebroventricular (i.c.v.) de TNF-? (250 ng) e MIP-1? (500 ng) promoveram um aumento da temperatura corporal em animais falso-operados que foi abolida em ratas OVX. Já a administração de IL-1? (3.1 pg) em animais falso-operados e OVX induziu uma resposta febril similar em ambos os grupos. O tratamento com antagonista para RANK, osteoprotegerina, aboliu a resposta febril induzida pelo LPS em animais OVX, porém não modificou essa resposta em animais falso-operados. A administração de LPS aumentou a expressão de receptores RANK em neurônios hipotalâmicos de ratas falso-operadas e este aumento foi reduzido após a ovariectomia. A fim de avaliar o envolvimento de prostaglandinas nestas respostas, observamos que o tratamento com o inibidor não seletivo de ciclooxigenase indometacina reduziu a resposta febril induzida pelo LPS tanto em animais falso-operados e OVX. A injeção de LPS aumentou a concentração de PGE2 no fluido cerebroespinhal tanto em animais falso-operados como em OVX mas os níveis deste eicosanoide foi significativamente maior em animais OVX quando comparado com falso-operados. A administração de LPS também aumentou a expressão de COX-2 no hipotálamo em ambos os grupos mas a expressão desta enzima foi significativamente maior em OVX quando comparado com animais falso-operados. A administração i.c.v. de PGE2 (250 ng) também induziu uma resposta febril maior em animais OVX quando comparado com animais falso-operados. No mesmo sentido, a expressão de receptores de prostaglandinas EP3 aumentou em neurônios hipotalâmicos de ambos os grupos após a administração de LPS mas foi significativamente maior em animais OVX quando comparada à animais falso-operados. A injeção i.c.v. de CRF (2,5?g), ET-1 (1 pg), do agonista opiodérgico morfina (10 ?g) e de substância P (500 ng) induziu febre em animais falso-operados. A ovariectomia reverteu a resposta febril induzida por CRF, ET-1 e substância P mas não aquela induzida por morfina. Por outro lado, a administração de LPS aumentou a expressão de receptores CRFI/II, ?-opioidérgicos, e NK1 sem alterar significativamente a expressão de receptores ETB em animais falso-operados. A ovariectomia não modificou o aumento promovido pelo LPS de receptores de CRF e de opióides, mas um aumento significativo na expressão de receptores ETB e NK1 foi observado em fêmeas OVX que receberam LPS quando comparadas a falso-operadas. No presente estudo discutimos as relações entre estes diferentes mediadores e seus receptores na resposta febril e a modulação destas respostas por hormônios ovarianos. Os dados obtidos sugerem que a resposta febril em fêmeas, embora menor do que em machos envolve mediadores similares. No entanto, a ação de alguns destes mediadores é modificada pela na ausência dos hormônios gonadais apresentando-se mais dependente de prostaglandinas nesta condição que simula a menopausa do que de outros mediadores centrais. Palavras-chave: Febre, Lipopolissacarídeo, Endotoxina, Hormônios sexuais.
Abstract: Fever can be defined as a controlled increase in internal body temperature resulting from a change in the hypothalamic thermoregulatory set point. There are different ways to mimic this phenomenon in laboratory animals, but the model of intraperitoneal administration of lipopolysaccharide (LPS) is widely used and stable. Once injected, LPS binds to Toll-Like type 4 receptor in immune cells such as macrophages, and induces the synthesis and release various endogenous pyrogens such as interleukin (IL) -1?, IL-1?, IL-6, IL -8, macrophage inflammatory protein (MIP-1?) and the receptor-activator of nuclear factor ?B ligand (RANKL), among others. These mediators induce formation and release of several central mediators such as prostaglandins (PG), endothelin-1 (ET-1), corticotrophin-releasing factor (CRF), substance P (SP), endogenous opioids and endocannabinoids which in turn will act in complex ways culminating in the febrile response. However, these studies were all performed in male experimental animals. Considering that there is evidence of differences in inflammatory and febrile response related to gender in experimental animals and that women have a higher incidence of inflammatory disorders and related pain, the aim of this study was to evaluate the fever induced by LPS and different mediators that are released by this pyrogen in female rats as well as the influence of female sex hormones in these responses. Additionally, we investigated the expression of cyclooxygenase-2 (COX-2) and various receptors of these central mediators in female rats and the influence of ovariectomy on this expression. We found that ovariectomized (OVX) female rats had higher baseline temperatures than sham-operated female rats. The injection of LPS (50 ?g/kg, i.p.) in sham-operated animals induced a significantly lower febrile response when compared to OVX rats. OVX female rats had a febrile response similar to that observed in males. The subchronic treatment with 17?-estradiol (1 mg/kg, s.c.) in OVX animals reduced the LPS-induced fever. OVX females showed a reduction in plasma oestradiol levels. The LPS administration increased testosterone levels in sham-operated and OVX animals. The intracerebroventricular (i.c.v.) administration of TNF-? (250 ng) and MIP-1? (500 ng) promoted an increase in body temperature sham-operated animals was abolished in OVX rats. However, the administration of IL-1? (3.1 pg) in sham-operated and OVX animals induced a similar febrile response. The treatment with RANK receptor antagonist, osteoprotegerin, abolished the fever induced by LPS in OVX animals but did not modify this response in sham-operated animals. The LPS administration increased expression of RANK receptors on hypothalamic neurons of sham-operated rats, and this increase was reduced after ovariectomy. In order to assess the involvement of prostaglandins in these responses, we observed that the treatment with the nonselective cyclooxygenase inhibitor indomethacin reduced the fever induced by LPS in both sham-operated and OVX animals. The injection of LPS increased the PGE2 concentration in the cerebrospinal fluid in both sham-operated and OVX animals but the levels of this eicosanoid was significantly higher in OVX rats compared to sham-operated ones. The LPS administration also increased COX-2 expression in the hypothalamus in both groups but the expression of this enzyme was significantly higher in OVX female rats compared to sham-operated animals. The i.c.v. administration of PGE2 (250 ng) also induced a higher febrile response in OVX compared to sham-operated animals. Similarly, the expression of prostaglandin EP3 receptors was increased in hypothalamic neurons of both groups after administration of LPS but was significantly higher in OVX compared to sham-operated animals. The i.c.v. injection of CRF (2,5?g), ET-1 (1 pg), opiod receptor agonist morphine (10 g) and substance P (500 ng) induced fever in sham-operated animals. The ovariectomy reversed the febrile response induced by CRF, ET-1 and substance P but not that induced by morphine. Moreover, LPS administration increased the expression of CRFI/II, ?-opioid and NK1 receptors without significantly altering the expression of ETB receptors sham-operated animals. Ovariectomy did not affect the increase in the expression of CRF and ?-opioid receptors promoted by LPS but a significant increase in the expression of NK1 and ETB receptors was observed in OVX females which received LPS as compared to sham-operated rats. In the present study we discuss the relationship between these different mediators and their receptors in the febrile response and modulation of these responses by ovarian hormones. The data suggest that the febrile response in females, although lower than in males involves similar mediators. However, the action of some of these mediators is modified by the absence of gonadal hormones, being more dependent on prostaglandin synthesis in this condition that simulates menopause than other central mediators. Key-words: Fever, Lipopolysaccharide, Endotoxin, Sexual Hormones.

Bypass or resection of the stomach and oesophagus, has long been recognised to result in profound changes in the handling of ingested nutrients. This results in significant morbidity after radical surgery for oesophago-gastric cancer, in particular post-prandial hypoglycaemia, altered appetite, early satiety and noxious post-prandial symptoms. By profiling and challenging the gut hormone axis in healthy volunteers and patients who had undergone total or subtotal gastrectomy, or oesophagectomy, this thesis explores the possible causative mechanisms for the challenges faced by this patient population. In the surgical groups, an oral glucose tolerance test (OGTT) resulted in enhanced secretion of satiety and incretin gut hormones (GLP-1, GIP, PYY) and insulin, followed by hypoglycaemia in a cohort of patients. Continuous glucose monitoring of gastrectomy participants over two weeks of normal lifestyle identified an increased incidence of day and night time hypoglycaemia. RNAseq and mass spectrometry based peptidomics of human and murine enteroendocrine cells in the pre- and post-operative populations revealed no significant change in the underlying cellular pathways for nutrient sensing and gut hormone secretion, indicating that the altered hormone secretion is primarily driven by accelerated nutrient transit, rather than adaptive changes in the gut. Finally, specific blockade of the GLP-1 receptor in post-gastrectomy patients using Exendin 9-39 normalised insulin secretion and prevented reactive hypoglycaemia after an OGTT. In conclusion, profound changes in gut hormone secretion as a result of enhanced nutrient transit after foregut surgery likely underlie the early and late post-prandial symptoms seen in this group, and therapies specifically targeting the gut hormone axis, and GLP-1 in particular, could be the first targeted treatments for post-gastrectomy syndromes.

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Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

O sistema de expressão baseado nos promotores PL ou PR do fago lambda que usualmente é regulado pelo repressor termo lábil (cIts) é amplamente utilizado para produzir proteínas recombinantes em células procarióticas. No entanto, o aumento da temperatura requerido neste sistema para promover a inativação do repressor apresenta algumas limitações, como o aumento da expressão de proteínas de HSP (Heat Shock Proteins), por exemplo, proteases, que dependendo da natureza da proteína expressa podem ser prejudiciais ou não. Uma outra limitação é a ativação da resposta SOS, resultando na parada da replicação do DNA celular ou dependendo da cepa pode ocorrer lise celular. Nesse trabalho nós descrevemos o uso do promotor λPL para expressão constitutiva, isto é, sem a regulação do repressor. Nós otimizamos diferentes condições de cultura para aumentar a secreção no espaço periplásmico de Escherichia coli de cinco proteínas: o hormônio do crescimento humano (hGH), que tem sido amplamente utilizado no tratamento de crianças com deficiência e/ou resistência ao hGH, síndrome de Turner, entre outras desordens; prolactina humana (hPRL), um hormônio polipeptídico conhecido por estimular a lactação e por exercer ação regulatória no crescimento e na diferenciação da glândula mamária, dois antagonistas de hPRL, estudados como potenciais fármacos para o tratamento de alguns tipos de cânceres e por fim o interferon α2a (IFN-α2a), que é uma citocina produzida pelas células, em resposta a diferentes estímulos, incluindo ácidos nucléicos virais, células estranhas (particularmente as neoplásicas), antígenos de bactérias, protozoários e vírus. No caso do IFN-α2a, essa citocina de alto valor agregado e de importante aplicação terapêutica, foi desenvolvida em nosso laboratório como parte desse trabalho, incluindo o desenvolvimento e a validação da metodologia de análise por HPLC de fase reversa para determinação do IFN presente no fluído periplásmico bacteriano ou na sua forma pura. As principais estratégias utilizadas para melhorar a expressão foram iniciar a indução junto à densidade óptica máxima do crescimento bacteriano e otimizar a temperatura de indução para controlar a expressão da proteína heteróloga. Essa metodologia pode ser utilizada nos casos onde o produto não será tóxico para a célula hospedeira ou quando a instabilidade do plasmídeo não é problema. A possibilidade de cultivo em temperaturas mais baixas, já que o repressor termo-sensível não se encontra presente, colaborou para o aumento significativo da expressão, mesmo para proteínas menos sensíveis à temperatura de cultivo, como o hGH.
The expression system based on the PL or PR promoters of the lambda phage that is usually regulated by the term labile repressor (clts) is widely used to produce recombinant proteins in prokaryotic cells. However, the temperature increase required in this system to promote the repressor inactivation shows some limitations, like the increase of the HSP (Heat Shock Proteins) proteins expression, proteases e.g., that depending on the nature of the expressed protein can be harmful or not. Another limitation is the activation of SOS response, resulting on the stop of the DNA cell replication or depending on the strain can occur cell lysis. In this paper we describe the use of the λPL promoter for constitutive expression, without the repressor regulation. We optimized different cultivation conditions to increase the secretion in the periplasmic space of Escherichia coli of five proteins: the human growth hormone (hGH), that is being widely used in the treatment of children with disabilities and/or resistance to hGH, Turner syndrome, within another disorders; human prolactin (hPRL), a polypeptide hormone known for stimulating the lactation and for exercising regulatory action on growth and on the differentiation of the mammary gland; two hPRL antagonists, studied as potential medicine to the treatment of some kinds of cancers and finally the interferon α2a (IFN-α2a), that is a cytokine produced by the cells, in response to different actions, including viral nucleic acids, neoplastic cells, antigens of bacteria, protozoa and viruses. In the case of IFN-α2a, this high value added and important therapeutic application, was developed in our laboratory as a part of this paper, including the development and the validation of the analysis methodology by reversed-phase HPLC to determine the IFN present in the bacterial periplasmic fluid or in its pure form. The main strategies used to improve the expression were to start the induction with the maximum optical density of bacterial growth and optimize the induction temperature to control the expression of heterologous protein. This methodology can be used in cases where the product wont be toxic to the host cell or when the instability of the plasmid is not a problem. The possibility of cultivation in lower temperatures, since the heat-sensitive repressor is not present, contributed to the significant increase of the expression, even to proteins that are less sensitive to the cultivation temperature, like the hGH.

Les plantes se développent et évoluent en interaction avec les organismes du sol. L'impact des vers de terre sur la croissance des plantes, généralement positif, a été attribué à des modifications physiques, chimiques ou biochimiques du sol, souvent sans démonstration rigoureuse. Dans ce travail, les techniques développées en sciences du végétal (culture in vitro, utilisation de mutants et transcriptomique) ont été utilisées afin de comprendre les mécanismes à l'origine de l'effet des vers de terre sur les plantes. Nos résultats apportent de nouvelles connaissances fondamentales: (1) la production de molécules-signal à l'intérieur des déjections de vers de terre a un impact significatif sur la croissance d'Oryza sativa et Lolium perenne. (2) Ces molécules agissent sur la voie de signalisation fortement liée à l'auxine, comme suggéré par l'effet significatif du ver de terre sur la croissance du double mutant d'A. thaliana aux1-7;axr4-2. (3) L'abondance de ces molécules-signal en présence de vers de terre pourrait être liée à la stimulation de certaines populations bactériennes capables de synthétiser de l'auxine. (4) Le ver de terre induit une accumulation de transcrits pour des gènes sous contrôle de l'acide jasmonique et de l'éthylène. Ces hormones sont notamment impliquées dans un mécanisme de résistance systémique induite (ISR), connu pour être induit par certaines rhizobactéries promotrices de la croissance des plantes. Enfin, (5) le piétin échaudage, maladie due à un champignon pathogène, déclenche chez le blé (Triticum aestivum) une réaction d'hypersensibilité et une modification de la signalisation hormonale, qui sont considérées comme des mécanismes de contrôle du métabolisme de la plante qui facilitent l'infection du pathogène. La sévérité de cette maladie est réduite en présence de vers de terre. La synthèse de ces résultats indique que les vers de terre, comme d'autres organismes du sol, modifient l'équilibre hormonal de la plante. L'homéostasie hormonale apparaît comme un élément incontournable pour prédire l'issue des interactions multiples que les plantes entretiennent avec les organismes du sol
Plants develop and evolve in interaction with soil organisms. The impact of earthworms, likely positive, has been attributed to modifications of physical, chemical or biochemical soil properties, without rigorous demonstration. In this work, techniques developed in plant science (in vitro culture, use of mutant plants and trancriptomic analysis) were used to understand the mechanism involved in the effect of earthworms on plants. Our results bring new fundamental knowledge: (1) production of signal-molecules within earthworm dejections has a positive impact on the growth of Oryza sativa and Lolium perenne. (2) These molecules act on auxin signaling, as suggested by the positive impact of the earthworm on the growth of A. thaliana double mutant aux1-7;axr4-2. (3) The abundance of these signal-molecules in presence of the earthworms could be related to the stimulation of bacterial communities able to produce auxin. (4) Earthworms induce an accumulation of gene transcripts known to be under control of jasmonic acid and ethylene. These two hormones are most notably involved in the defense mechanism called induced systemic resistance (ISR), known to be induced by plant growth promoting rhizobacteria. Finally, (5) Take-all disease, due to a pathogenic fungus, induced in wheat (Triticum aestivum) a hypersensitive response and a modification on hormone signaling, which are known as manipulations of plant metabolism in a way that facilitates pathogen infection. The severity of take-all disease was alleviated in the presence of earthworms. Synthesis of these results showed that earthworms, like other soil organisms, modify the hormone balance in the plant. Hormone homeostasis appeared to be an important element to predict the issue of the multiple interactions that plants established with soil organisms

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The maize is a cereal with high economic importance due to its magnitude forms of use. Increasing productivity levels, promoting substances plant development are highlighted role in contemporary agriculture, arising from biological or chemical origin. However, the lack of results regarding the joint use of these promoters leads to the need for information on their simultaneous use. In this context, this work aimed to evaluate the effects of joint use of thiamethoxam combined with seed inoculation and foliar spray Azospirillum brasilense on physiological quality of corn seeds and its agronomic performance in two environments in the growing field. Initially, we tested bacterial growth in culture medium containing increasing concentrations of thiamethoxam. It was also evaluated the initial development and physiological quality of seedlings by morphometric measurements, emergency speed index and SPAD index through factorial 2 x 3 with seed inoculation with A. brasilense (100 mL per 25 kg seeds) thiamethoxam and three doses (0, 28 and 42 mg per 60,000 seeds). In the field, factorial 2 x 4 was composed by the presence and absence of chemical seed treatment with bioactivator of thiamethoxam plants (28 mg per 60,000 seeds), while the second factor comprised seeds without inoculation; seed inoculation with A. brasilense (100 mL per 60,000 seeds), foliar spray inoculant base A. brasilense (300 mL ha-1) and associated with the seed inoculated with A. brasilense foliar spray, tested in high ambient and applying average production technology. The treatments were evaluated by morphometric analysis, nutrient content in plant tissues, components production and crop yield. The bacterial population showed a linear decrease with increasing doses of thiamethoxam. The dose of 42 mg of thiamethoxam caused reduction in emergence speed, SPAD index, shoot length and root, root volume, root/shoot and dry matter of shoots, while inoculation showed activity positive physiological quality of seedlings. Thus, combined with inoculation dose of 28 mg thiamethoxam demonstrates synergistic effect in the early development of maize seedlings. In the field, thiamethoxam, as well as seed inoculation + foliar spray promote increase in plant length, stem diameter, leaf and mass accumulation area throughout the plant cycle in both environments. Reduction in moisture content, impurities and discounts of grain mass, as well as increases in leaf phosphorus levels are the result of inoculation, yet without deterioration in corn yields. Thiamethoxam also reduced the moisture of the grain mass. Productivity was increased by the presence of thiamethoxam on average productive technology environment
O milho é um cereal de elevada importância econômica devido à sua magnitude de formas de utilização. Incrementando os níveis de produtividade, substâncias promotoras de desenvolvimento vegetal ganham papel de destaque na agricultura contemporânea, advindas de origem biológica ou química. Contudo, a escassez de resultados a respeito da utilização conjunta desses promotores leva à necessidade de informações sobre sua utilização simultânea. Neste contexto, esta obra buscou avaliar os efeitos da utilização conjunta de tiametoxam aliado à inoculação via semente e pulverização foliar de Azospirillum brasilense na qualidade fisiológica de sementes de milho e seu desempenho agronômico em dois ambientes de investimento em cultivo a campo. Inicialmente, testou-se o crescimento bacteriano em meio de cultura contendo concentrações crescentes de tiametoxam. Avaliou-se também o desenvolvimento inicial e qualidade fisiológica de plântulas por medidas morfométricas, índice de velocidade de emergência e índice SPAD, por meio de arranjo fatorial 2 x 3, com inoculação de sementes com A. brasilense (100 mL por cada 25 kg de sementes) e três doses de tiametoxam (0, 28 e 42 mg por 60.000 sementes). À campo, esquema fatorial 2 x 4 foi composto pela presença e ausência de tratamento químico de sementes com o bioativador de plantas tiametoxam (28 mg por 60.000 sementes), enquanto o segundo fator englobou sementes sem inoculação; inoculação de sementes com A. brasilense (100 mL por 60.000 sementes), pulverização foliar de inoculante a base de A. brasilense (300 mL ha-1) e inoculação de sementes associado à pulverização foliar com A. brasilense, testados em ambientes de alta e média aplicação de tecnologia de produção. Os tratamentos foram avaliados por análises morfométricas, teor de nutrientes nos tecidos vegetais, componentes da produção e produtividade da cultura. A população bacteriana mostrou redução linear de acordo com o aumento das doses de tiametoxam. A dose de 42 mg de tiametoxam causou redução na velocidade de emergência, índice SPAD, comprimento de parte aérea e de raiz, volume de raiz, relação raiz/parte aérea e massa da matéria seca de parte aérea, enquanto que, a inoculação evidenciou atividade positiva na qualidade fisiológica das plântulas. Deste modo, a inoculação combinada com dose de 28 mg de tiametoxam demonstra efeito sinérgico no início do desenvolvimento de plântulas de milho. À campo, o tiametoxam, assim como a inoculação via semente + pulverização foliar, promoveram incrementos no comprimento das plantas, diâmetro de colmo, acúmulo de massa e área foliar ao longo do ciclo vegetal em ambos os ambientes. Redução no teor de umidade, impurezas e descontos da massa de grãos, assim como incrementos nos níveis foliares de fósforo são resultados da inoculação, contudo sem apresentar alterações na produtividade do milho. Tiametoxam também reduziu a umidade da massa de grãos. A produtividade foi incrementada pela presença do tiametoxam em ambiente de média tecnologia produtiva

碩士
國立虎尾科技大學
光電與材料科技研究所
101
This paper studies the plasma enhanced chemical vapor deposition (PECVD) Preparation of titanium dioxide photocatalyst film using argon (Ar) as the carrier gas, the source of titanium (4-isopropyl titanium, Titanium (IV) isopropoxide, TTIP) delivered to the mixinggroove and do mixed with oxygen (O2) reacts then into the body cavity, and deposited on glass and stainless steel substrate and by modulating the plasma power to explore the influence of the type of plasma power on the film quality, this catalyst Photocatalyticdegradation of methylene blue and methyl orange reaction to study the catalytic properties. UV - visible spectrometer (UV-Vis Spectrum) measurement structure by X-ray diffraction (XRD) analysis of film properties and the degradation of methylene blue and methyl orange light absorption rate. The results showed that the stainless steel substrate in a process temperature of 25 ° C, the process pressure of 150 mTorr, plasma power of 300 W, the TTIP flow of 100 sccm, oxygen flow rate 15 sccm, annealing temperature of 500 ℃ under 30 minutes, preferably anatase (anatase) crystalline phase peak and the efficiency of the photocatalytic degradation XRD FIG respectively in A (101) (104) (200) (105) position anatase peak in the high-power UV LED light (wavelength 395 nmvoltage 50.2 v current of 1.48 mA), uniform light, titanium dioxide thin films deposited on stainless steel substrate degradation rate of degradation of methylene blue in 1 hour up to 62.8%, the degradation rate of up to 9 hours degradation of methyl orange67.1%. In addition, the decomposition of water environmental hormone phenol and inhibit the growth of E. coli experimental part, due to the general industrial wastewater containing organic pollutants - phenol, traditional processing method is difficult to be removed, the study by to deploy different concentrations from 5ppm, 10 ppm to 15 ppm of phenol solution, titanium dioxide photocatalyst film photocatalytic degradation of phenol compounds in the catalytic time 25 hours later, the ultimate success of the phenol mineralization into carbon dioxide and water. E. coli part of the BCRC 11634 strain test, culture placed in 30 ml LB liquid culture medium and cultured at 37 ° C oven take 3 ml culture 16 hours after the titanium dioxide photocatalyst antibacterial experiments, the results show the high power UV LED Light (wavelength 395 nmvoltage of 47.7 V, current of 1.14 mA), we can see uniform irradiation the titania catalyst irradiated with UV light photocatalytic reaction is triggered by the count after E. coli significantly reduce the number of successfully inhibited the growth of E. coli.

碩士
高雄醫學大學
牙醫學研究所
82
Gingivitis is one of the most common oral diseases, which is caused by dental plaque and by the factors produced/released from it. The black pigmented bacteria in subgingival dental plaque is thought to be the pathogens of the gingivitis. Prevotella intermedia and Porphylomonas gingivalis have been shown to be closely associated with human gingivitis. These two kinds of black pigmented bacteria can use female sex hormones such as progesterone or estradiol as a source of nutrients. In pregnant women, the concentrations of progesterone and estradiol are markely increased in serum and both of them are accumulated and found in the gingival tissue. The purpose of this study was to test levels of female sex hormones in gingival crevicular fluid and to observe the relationship between hormones and black pigmented bacteria in subgingival plaque. The results showed that the level of progesterone found in the gingival crevicular fluid of pregnant women was markedly higher than that of women in postpartum stage. The percentage of black pigmented bacteria in subgingival plaque were also higher than that in postpartum stage, and both results were statistically significant. Percentage of black pigmented bacteria was positively correlated with both the progesterone level, pregnancy and the severity of the gingivitis. Severity of the gingivitis was positively correlated with both the plaque index and the percentage of black pigmented bacteria in subgingival plaque.

The increasing development of drugs such as antidepressants and hormones led to serious environmental and health problems, becoming mandatory to study different ways to remove a selective serotonin re-uptake inhibitor like fluoxetine (FLX) and estrogens like 17α- ethinylestradiol (EE2) during the wastewater treatment process. The study was carried out by using activated sludge samples from the Faro Northwest wastewater treatment plant (WWTP) to isolate bacteria under aerobic conditions in order to test their resistance in the presence of FLX and EE2 and investigate the biodegradability of chosen pharmaceuticals. In the present study, the identification of bacterial isolates was performed by sequencing the 16S rRNA gene of the obtained resistant bacteria. The obtained isolates which showed the ability to grow in the presence of 20 and 50 mg/L FLX were Pseudomonas putida, Enterobacter ludwigii, Pseudomonas nitritireducens, Alcaligenes faecalis, Pseudomonas aeruginosa, Pseudomonas nitroreducens and in the presence of 15 mg/L EE2 were Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans Shinella zoogloeoides. These isolates were identified as FLX and EE2 degraders for the first time. FLX and EE2 biodegradation assays was performed in liquid medium. The results showed that for 20 mg/L FLX the bacterial isolate Pseudomonas nitroreducens displayed the highest removal efficiency of 55 ± 1% and for 15 mg/L EE2 Pantoea agglomerans removed 64 ± 4% of the drug. This research shows the importance of the involved bacteria for the effectiveness of the removal of these drugs in a wastewater treatment plant. A complete removal of pharmaceutical compounds from wastewater will stimulate water reuse, directing attention worldwide increasing requirements for clean and safe fresh water.

碩士
國立中興大學
獸醫學系暨研究所
94
Pyometra is one of the common reproductive system diseases associated with the estrus status in bitches. Among sex hormones, progesterone plays an important role in initiating the pathogenesis of cystic endometrial hyperplasia. The most likely source of the bacteria that establish a uterine infection is the resident bacteria of the vaginal vault. These bacteria have the potential of ascending through the relatively dilated cervix, into the uterus, during proestrous and estrus. Dogs with pyometra frequently have clinical signs of renal dysfunction, in which polyuria and polydipsia as prominent clinical signs. However, the actually mechanism of polyuria is still unknown. With further progression, renal failure in some bitches can subsequently develop in the later stage of pyometra. Metrix metalloproteinases (MMPs) are secreted by cells and are able to digest proteins of the extracellular matrix. These enzymes have been involved in many normal remodeling processes as well as in some disease. In order to realize the effect of hormones in pyometra, chemiluminescence immunoassay and radioimmunoassay were used to determine reproductive hormones (estrogen and progesterone) and vasopressin (VP) concentration in plasma, respectively. The seriousness of subsequent renal function disorder was studied by determining blood urea nitrogen (BUN) and creatinine value in pyometra dogs. The causative bacteria was isolated from uterine fluid and identified according to bacterial morphology and biochemical reaction. The identified bacteria proceed to test the antibiotics susceptibility. MMP-2 and MMP-9 expression in plasma were analyzed by 6% SDS-PAGE containing 0.2% gelatine. In results, the concentration of reproductive hormones in plasma was no significantly difference from control group. As the respect of renal function, there were 50% (25/50) of pyometra dogs with azotemia (BUN> 20 mg/dL), and 22% (11/50) of pyometra dogs had renal function disorder (creatinine> 1.5 mg/dL). VP secretory ability in pyometra dogs was not reduced, but the group of pyometra subsequent renal dysfunction had significantly higher VP concentration compared with control group (p=0.02). In identification of causative bacteria, gram-negative species is the major causative agent in this study (81%, 46/57), among which Escherichia coli was the predominant bacteria (41%, 24/57). In antibiotic susceptibility test, sulfamethoxazole had the highest resistance (100%), gentamicin was the lowest (29%). In MMPs expression in plasma, MMP-2 and MMP-9 were all expressed significantly higher in pyometra than in control dogs. According to this study, when pyometra was found, the most important thing was maintain renal function. Secondly, because of insufficient antidiuretic function, dogs with pyometra subsequent renal failure may have nephrogenic diabetes insipidus. In the end, when veterinary choose antibiotics in the therapy of dogs with pyometra needed further consideration before using to prevent the more bacteria have high antibiotic resistance.

碩士
國立交通大學
生物科技系所
94
Human follicle-stimulating hormone (hFSH) plays an essential role in mammalian reproduction and ovarian folliculogenesis through interaction with its specific receptor, FSHR. However, epidemiologic data have implicated higher expression of follicle-stimulating hormone receptor (FSHR) in ovarian cancerous tissues compares to normal tissues and reproductive FSH as a probable risk factor for ovarian cancer development. On the other hand, the previous studies have showed that site-directed mutagenesis identified the specific roles of the individual carbohydrate chains of FSH in signal-transducing activity and receptor-binding affinity. The binding affinity of FSH lacking any one of the oligosaccharides was increased over wild-type FSH, while the signal-transducing activity of FSH lacking the oligosaccharide at αAsn52 was markedly reduced, and that of FSH lacking either β oligosaccharide was slightly reduced. According to these previous studies, the objective of the study is to express and reconstitute Non-glycosylated hFSH in Escherichia coli and evaluate the effect of NG-hFSH on ovarian cancer cell proliferation. First, hFSHα cDNA (hFSHA) and hFSHβ cDNA (hFSHB) were amplified by PCR. Then, the products of PCR were inserted into pET30a and pACYCDuet-1, respectively. Escherichia coli NovaBlue (DE3) and BL21 (DE3) were transformed for expression hosts of hFSHA-pET30a and hFSHB-pACYCDuet-1, respectively. Through purification, refolding and dimerization, expected NG-hFSH variants were reconstituted. Using MTT and TMB assay, the proliferative and receptor-binding effects of wild-type hFSH and NG-hFSH variants on ovarian cancer cell line, SKOV-3, were identified. Finally, NG-hFSH variants have been examined that they could indeed suppress the proliferation of SKOV-3 and significantly increase the receptor binding affinity on the SKOV-3.

Plants regulate responses to their environment through complex hormone signaling; these hormones can be categorized broadly into two categories: growth and defense, though many have roles in both. Much remains to be understood about the complexity of hormone signaling in relation to environmental responses, especially species- and genotype-specific differences. Unraveling this complexity of hormone signaling will lead to the development of resilient crops that are able to respond appropriately to their environment. In this dissertation, I hypothesize novel roles for growth and defense hormones in Solanum spp. responses to 1) biochar, a black carbon soil amendment (Chapter 2), 2) infection with Ralstonia solanacearum, an economically important soilborne pathogen causing bacterial wilt (Chapter 3), and 3) endophytic colonization by the soil bacterial community (Chapter 4). In Chapter 2, I showed that biochar upregulates GA signaling and affects GA-related traits in a species- and cultivar-specific manner. Biochar amendment also downregulates defense signaling. In Chapter 3, I demonstrated a novel role for auxin in resistance against R. solanacearum, including differential expression of auxin signaling genes in resistant genotype H7996 compared to susceptible WV in response to R. solanacearum infection. In addition, I observed stronger and faster upregulation of defense hormone marker genes for SA and ET in H7996 compared to WV. In Chapter 4, I showed that SA and ET are required for normal tomato root microbial community assembly, affecting the colonization of a few key taxa in order to promote alpha diversity. H7996 and WV root communities differ in alpha diversity, and a panel of H7996 x WV RILs showed quantitative variation in alpha diversity that correlated negatively with the abundance of these key taxa. In conclusion, I elucidated novel roles for hormones in responses to the soil environment, pathogen infection, and root community colonization. These findings are important for developing resilient, sustainable crops.