Chapter: 1 Introduction Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars, which belong to the Salmonella enterica species. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Salmonella infection occurs via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18+ macrophages present in the intestinal lumen or via a forced entry in the non phagocytic enterocytes. Upon entry Salmonella resides in an intracellular phagosomal compartment called the Salmonella containing vacuole (SCV). The SCV only transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7. It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. The mechanisms by which phagocytes kill the virulent Salmonella are not completely understood, however the role of nicotinamide-adenine dinucleotide phosphate (NADPH) phagocytic oxidase system has been strongly implicated. The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox, and a GTP-binding Rac protein. Further, professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Chapter:2 Resistance to host Nitrosative stress in Salmonella by quenching L-arginine. Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of L-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate L-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor aminoguanidine reverts the attenuation in arginase blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of L-arginine. In the whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner. Chapter:3 Hrg (hydrogen peroxide resistant gene), a LysR type transcriptional regulator confers resistance to oxidative stress in Salmonella LysR type transcriptional regulators are one of the key players that help bacteria adapt to different environments. We have christened STM0952, a putative LysR type transcriptional regulator in Salmonella enterica serovar Typhimurium as the hydrogen peroxide resistance gene (hrg). By generating a knock out of the hrg gene, we demonstrate that the hrg mutant serovar Typhimurium is sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. The hrg mutant is profoundly attenuated in the murine model of infection and shows decreased intracellular proliferation in macrophages. It was also found to induce increased amount of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild type. An overproducing strain of this gene showed a survival advantage over the wild type Salmonella under hydrogen peroxide induced stress condition. Microarray analysis suggested the presence of a Hrg regulon, which is required for resistance to the toxic oxidative products of the reticulo-endothelial system. Chapter:4 Importance of the host oxidative stress in antigen presentation and its modulation by Salmonella: Role of TLR Synthetic CpG containing oligodeoxynucleotide TLR-9 agonist (CpG ODN) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. We report that Salmonella Typhimurium growth can be inhibited by the CpG ODN treatment in the murine dendritic cells. This inhibitory effect was shown to be mediated by an increased reactive oxygen species (ROS) production. We further show that the CpG ODN treatment of the dendritic cells during Salmonella infection leads to a ROS dependent increased antigen presentation. In addition, TLR-9 signaling inhibitor was able to inhibit the CpG ODN mediated increased antigen presentation, ROS production and pathogen killing. These data indicate that CpG ODN can improve the ability of the murine dendritic cells to contain the growth of the virulent Salmonella through ROS dependent killing and could as well be used as an effective adjuvant in vaccines against Salmonella infection.

<p>The interactions between plants and insects are mediated by volatile molecules. Plants respond to stress by biosynthesis of chemical substances which can deter invading insects or pathogens. Some of these substances are volatile and are emitted to the surroundings and may attract or repel insects. Information about the susceptibility of individual plants to infestation, their volatile emissions and chemical defence is of interest, for example in selecting plants for tree breeding programs.</p><p>This research was focused on finding volatile chemical markers of resistance in Norway spruce plants that do influence insects associated to conifers. Collection of headspace volatiles by SPME followed by separation and identification with GC-MS is effective in investigating biological systems with a minimum of disturbance. This method has here been used to investigate Norway spruce plants of different ages and stress conditions as well as trapping semiochemicals like nepetalactone emitted by the spruce shoot aphids. It was even possible to analyse the emission of single needles <i>in vivo</i> and obtain a chemical pattern of the site of the stress reaction. Seedlings of different ages showed differences in chemical composition of emitted volatiles, with the pine weevil repellent (<i>S</i>)-(-)-limonene as one of the main compounds. Wounded phloem of conventional plants emitted high amounts of monoterpenes while the phloem of mini plants emitted (3Z)-hexenal and (3Z)-hexen-1-ol.</p><p>Norway spruce plants did respond to different stress elicitors with similar response, regardless of their genetic origin. The emissions from stressed Norway spruce plants mainly consist of (<i>E</i>)-β-farnesene, (<i>E,E</i>)-α-farnesene, (<i>E</i>)-α-bisabolene, (<i>R</i>)-(-)-linalool and methyl salicylate. Emissions from live spruce shoot aphids were detected during autumn periods, and a method to separate and identify the four diastereomers of nepetalactone by GC-MS and characteristic m/z-fragments was accomplished.</p>

The biogeographic distribution of organisms is determined by physiological characteristics that enable a population to persist in a specific location. Global climate change effects are anticipated to increase the physiological stress experienced by organisms. Consequently, it is important to understand physiological responses to environmental stress and the mechanisms used by animals to cope with variable conditions. I investigated the physiological response to environmental stress in two species of mussel from New Zealand, Perna canaliculus and Mytilus galloprovincialis, using quantitative PCR and ecological field experiments. A series of laboratory and field experiments were done to manipulate stress levels and the expression levels of three heat shock protein genes (hsp24, hsp70, hsp90) were measured. A transcription regulatory gene (elf2) and a cell cycle regulatory gene (tis11d) were also measured. The dynamics of stress response gene expression in response to acute stress and gene expression changes in the natural population due to varying forms of environmental stress were tested. Between-zone translocations of different sized M. galloprovincialis and P. canaliculus were done at two sites in both east and west regions of the South Island of New Zealand. Site was found to be the most important factor in stress response. Apparent low food and high exposure stress interacted to create the particularly elevated stress response at the Timaru site. The adaptive ability of mussels transplanted between sites with varying environmental conditions was also tested. Results suggest that acclimation may be limited under stressful conditions. Furthermore, I found that P. canaliculus, the predominantly low-zone species, had a lower stress response than M. galloprovincialis, which was contradictory to predictions. The investigations described in this thesis suggest that interactive effects of abiotic stress and food limitations are particularly challenging for animals. With the severity of climate change scenarios predicted, changes in water quality and aerial and seawater temperature suggest mussel populations are likely to be negatively affected in the future. This work also illustrates the great potential to utilise molecular techniques for analysis of physiological processes of non-model organisms in a real-world setting.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate, inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2 tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2 accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1. In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using 5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm whether these strains belonged to the Dekkera or Brettanomyces genus. A method to accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2 and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B. bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the same trends as previously found in literature whereas B. bruxellensis strains showed similar trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid, acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly monitored over the time course of each experiment. SO2 had the greatest impact on B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32 of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2 levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism to adapt to ensure survival. In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and metabolism and that SO2 tolerance is highly strain dependent and a far more complicated characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare, maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B. bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik. In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur 5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2- bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei. Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S. cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B. bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur, asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur. ‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer ingewikkelde kenmerk as wat tans verstaan word.

Os fogachos são a queixa mais comum das mulheres peri- e pós-menopausa e estão fortemente relacionados com a diminuição dos níveis de estrogênio. No entanto, a fisiopatologia deste sintoma vasomotor muito desagradável é ainda desconhecido. Por outro lado, o estradiol (E2) apresenta a capacidade de induzir a expressão de HSP72, um membro da família de 70 kDa das proteínas de choque térmico (HSP70), que são citoprotetoras e cardioprotetoras. Sabe-se que a expressão HSP70 é comprometida em doenças inflamatórias relacionadas com o envelhecimento. Por isso, questionamos se a capacidade do organismo de desencadear uma resposta ao choque térmico (Heat Shock Response) robusta, estaria ainda presente após a retirada, via castração, do E2. Para tanto, foram estudados os efeitos do choque térmico (Heat Shock - HS), através de uma sessão de banho de imersão, em ratas Wistar submetidas a ovariectomia bilateral (OVX), após um período de washout hormonal de 7 dias. Doze horas após o HS, os animais foram mortos e o arco aórtico foi excisado cirurgicamente para análises moleculares. Os resultados foram comparados com os marcadores de estresse oxidativo no plasma (superóxido dismutase, catalase e lipoperoxidação), pois é bem estabelecido que a expressão de HSP70 é sensível a regulação redox. A relação entre a iHSP70 (intracelular) e a eHSP70 (extracelular/plasma), proposto como um índice do estado inflamatório sistêmico, também foi investigada. Os resultados mostraram que a Heat Shock Response continua preservada em animais OVX, como inferido a partir da expressão de HSP70 (até 40% de aumento, p <0,01) nas aortas, o que não foi acompanhado por nenhuma outra alteração em marcadores de estresse oxidativo, parâmetros hematológicos e no controle glicêmico. Desta forma, sugerimos que a avaliação periódica do status de HSP70 (iHSP70 vs eHSP70) pode ser de extrema relevância clínica, pois a diminuição da capacidade de defesa do organismo via Heat Shock Response está no centro do aparecimento de disfunções relacionadas com a menopausa. / Hot flashes, the most common complaint of peri- and postmenopausal women, are tightly related to decrease in estrogen levels. However, the pathophysiology of this very unpleasant vasomotor symptom is greatly unknown. On the other hand, estradiol (E2) has been found to induce the expression of HSP72, a member of the 70 kDa family of heat shock proteins (HSP70), which are cytoprotective and cardioprotective. Since it has been noticed that HSP70 expression is compromised in age-related inflammatory diseases, we argued whether the capacity of triggering a robust heat shock (HS) response would be still present after E2 withdrawal. Hence, we studied the effects of HS treatment (hot tub) in female Wistar rats subjected to bilateral oophorectomy (OVX) after a 7 day washout period. Twelve hours after HS, the animals were killed and aortic arches were surgically excised for molecular analyses. The results were compared with oxidative stress markers in the plasma (superoxide dismutase, catalase and lipoperoxidation) because HSP70 expression is sensitive to redox regulation. Extracellular (plasma) to intracellular HSP70 ratio, an index of systemic inflammatory status, was also investigated. The results showed that HS response was preserved in OVX animals, as inferred from HSP70 expression (up to 40% rise, p<0.01) in the aortas, which was accompanied by no further alterations in oxidative stress, hematological parameters and glycemic control either. As a consequence, periodic evaluation of HSP70 status (iHSP70 vs eHSP70) may be of clinical relevance because decreased HS response capacity is at the center of the onset of menopause-related dysfunctions.

O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

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O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

Os fogachos são a queixa mais comum das mulheres peri- e pós-menopausa e estão fortemente relacionados com a diminuição dos níveis de estrogênio. No entanto, a fisiopatologia deste sintoma vasomotor muito desagradável é ainda desconhecido. Por outro lado, o estradiol (E2) apresenta a capacidade de induzir a expressão de HSP72, um membro da família de 70 kDa das proteínas de choque térmico (HSP70), que são citoprotetoras e cardioprotetoras. Sabe-se que a expressão HSP70 é comprometida em doenças inflamatórias relacionadas com o envelhecimento. Por isso, questionamos se a capacidade do organismo de desencadear uma resposta ao choque térmico (Heat Shock Response) robusta, estaria ainda presente após a retirada, via castração, do E2. Para tanto, foram estudados os efeitos do choque térmico (Heat Shock - HS), através de uma sessão de banho de imersão, em ratas Wistar submetidas a ovariectomia bilateral (OVX), após um período de washout hormonal de 7 dias. Doze horas após o HS, os animais foram mortos e o arco aórtico foi excisado cirurgicamente para análises moleculares. Os resultados foram comparados com os marcadores de estresse oxidativo no plasma (superóxido dismutase, catalase e lipoperoxidação), pois é bem estabelecido que a expressão de HSP70 é sensível a regulação redox. A relação entre a iHSP70 (intracelular) e a eHSP70 (extracelular/plasma), proposto como um índice do estado inflamatório sistêmico, também foi investigada. Os resultados mostraram que a Heat Shock Response continua preservada em animais OVX, como inferido a partir da expressão de HSP70 (até 40% de aumento, p <0,01) nas aortas, o que não foi acompanhado por nenhuma outra alteração em marcadores de estresse oxidativo, parâmetros hematológicos e no controle glicêmico. Desta forma, sugerimos que a avaliação periódica do status de HSP70 (iHSP70 vs eHSP70) pode ser de extrema relevância clínica, pois a diminuição da capacidade de defesa do organismo via Heat Shock Response está no centro do aparecimento de disfunções relacionadas com a menopausa. / Hot flashes, the most common complaint of peri- and postmenopausal women, are tightly related to decrease in estrogen levels. However, the pathophysiology of this very unpleasant vasomotor symptom is greatly unknown. On the other hand, estradiol (E2) has been found to induce the expression of HSP72, a member of the 70 kDa family of heat shock proteins (HSP70), which are cytoprotective and cardioprotective. Since it has been noticed that HSP70 expression is compromised in age-related inflammatory diseases, we argued whether the capacity of triggering a robust heat shock (HS) response would be still present after E2 withdrawal. Hence, we studied the effects of HS treatment (hot tub) in female Wistar rats subjected to bilateral oophorectomy (OVX) after a 7 day washout period. Twelve hours after HS, the animals were killed and aortic arches were surgically excised for molecular analyses. The results were compared with oxidative stress markers in the plasma (superoxide dismutase, catalase and lipoperoxidation) because HSP70 expression is sensitive to redox regulation. Extracellular (plasma) to intracellular HSP70 ratio, an index of systemic inflammatory status, was also investigated. The results showed that HS response was preserved in OVX animals, as inferred from HSP70 expression (up to 40% rise, p<0.01) in the aortas, which was accompanied by no further alterations in oxidative stress, hematological parameters and glycemic control either. As a consequence, periodic evaluation of HSP70 status (iHSP70 vs eHSP70) may be of clinical relevance because decreased HS response capacity is at the center of the onset of menopause-related dysfunctions.

Os fogachos são a queixa mais comum das mulheres peri- e pós-menopausa e estão fortemente relacionados com a diminuição dos níveis de estrogênio. No entanto, a fisiopatologia deste sintoma vasomotor muito desagradável é ainda desconhecido. Por outro lado, o estradiol (E2) apresenta a capacidade de induzir a expressão de HSP72, um membro da família de 70 kDa das proteínas de choque térmico (HSP70), que são citoprotetoras e cardioprotetoras. Sabe-se que a expressão HSP70 é comprometida em doenças inflamatórias relacionadas com o envelhecimento. Por isso, questionamos se a capacidade do organismo de desencadear uma resposta ao choque térmico (Heat Shock Response) robusta, estaria ainda presente após a retirada, via castração, do E2. Para tanto, foram estudados os efeitos do choque térmico (Heat Shock - HS), através de uma sessão de banho de imersão, em ratas Wistar submetidas a ovariectomia bilateral (OVX), após um período de washout hormonal de 7 dias. Doze horas após o HS, os animais foram mortos e o arco aórtico foi excisado cirurgicamente para análises moleculares. Os resultados foram comparados com os marcadores de estresse oxidativo no plasma (superóxido dismutase, catalase e lipoperoxidação), pois é bem estabelecido que a expressão de HSP70 é sensível a regulação redox. A relação entre a iHSP70 (intracelular) e a eHSP70 (extracelular/plasma), proposto como um índice do estado inflamatório sistêmico, também foi investigada. Os resultados mostraram que a Heat Shock Response continua preservada em animais OVX, como inferido a partir da expressão de HSP70 (até 40% de aumento, p <0,01) nas aortas, o que não foi acompanhado por nenhuma outra alteração em marcadores de estresse oxidativo, parâmetros hematológicos e no controle glicêmico. Desta forma, sugerimos que a avaliação periódica do status de HSP70 (iHSP70 vs eHSP70) pode ser de extrema relevância clínica, pois a diminuição da capacidade de defesa do organismo via Heat Shock Response está no centro do aparecimento de disfunções relacionadas com a menopausa. / Hot flashes, the most common complaint of peri- and postmenopausal women, are tightly related to decrease in estrogen levels. However, the pathophysiology of this very unpleasant vasomotor symptom is greatly unknown. On the other hand, estradiol (E2) has been found to induce the expression of HSP72, a member of the 70 kDa family of heat shock proteins (HSP70), which are cytoprotective and cardioprotective. Since it has been noticed that HSP70 expression is compromised in age-related inflammatory diseases, we argued whether the capacity of triggering a robust heat shock (HS) response would be still present after E2 withdrawal. Hence, we studied the effects of HS treatment (hot tub) in female Wistar rats subjected to bilateral oophorectomy (OVX) after a 7 day washout period. Twelve hours after HS, the animals were killed and aortic arches were surgically excised for molecular analyses. The results were compared with oxidative stress markers in the plasma (superoxide dismutase, catalase and lipoperoxidation) because HSP70 expression is sensitive to redox regulation. Extracellular (plasma) to intracellular HSP70 ratio, an index of systemic inflammatory status, was also investigated. The results showed that HS response was preserved in OVX animals, as inferred from HSP70 expression (up to 40% rise, p<0.01) in the aortas, which was accompanied by no further alterations in oxidative stress, hematological parameters and glycemic control either. As a consequence, periodic evaluation of HSP70 status (iHSP70 vs eHSP70) may be of clinical relevance because decreased HS response capacity is at the center of the onset of menopause-related dysfunctions.