Mesmo com o constante avanço no estudo da sibilância e asma, existem inúmeras controvérsias sobre a participação da exposição à endotoxina, background genético e ativação celular. Investigamos a participação da exposição à endotoxina ambiental e o papel do LPS no desenvolvimento dos fenótipos de sibilância e asma. Para tanto selecionamos crianças sibilantes e não sibilantes, e crianças asmáticas e não asmáticas, sendo seu sangue coletado e as PBMC cultivadas com LPS. O sobrenadante foi colhido para análise de citocinas por ELISA, e analisamos os polimorfismos nos genes de CD14 e TLR4 por PCR-RFLP. Não encontramos relação entre a exposição à endotoxina ambiental e o quadro de sibilância. Observamos que PBMC estimuladas ou não com LPS de crianças sibilantes e asmáticas produzem baixos níveis de IL-12 e IFN-<font face=\"Symbol\">&#947 quando comparado com crianças não sibilantes e não asmáticas. Os polimorfismos de TLR4 e CD14 não tiveram associação com sibilância ou asma. Nossos dados sugerem que não somente a polarização Th2 é importante para desenvolver essas patogenias, mas também uma diminuição na resposta Th1. / Although the great advance in the study of asthma and wheezing, there are numerous controversies about the involvement of endotoxin exposure, genetic background and cellular activation. We investigated the involvement of environmental endotoxin exposure and the role of LPS in the development of phenotypes wheezing and asthma. For experiments we selected wheezing and non-wheezing, and asthmatic and non-asthmatic children, and their blood collected and the PBMC cultured with LPS. The supernatant was collected for analysis cytokines by ELISA, and analyzed CD14 and TLR4 polymorphisms by PCR-RFLP. There was no relationship between environmental endotoxin exposure and the framework of wheezing. We observed that LPS-stimulated PBMC of wheezing and asthmatic children produce lower levels of IL-12 and IFN-<font face=\"Symbol\">&#947 when compared with non-wheezing and non-asthmatic children. The polymorphisms TLR4 and CD14 were not associated with wheezing or asthma. Our data suggest that not only Th2 polarization is important to develop these diseases, but also a decrease in Th1 response.

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:03:01Z (GMT). No. of bitstreams: 1 Herrera_DanielRodrigo_D.pdf: 1815873 bytes, checksum: c8ecc16aeec2193dae476030ea7ba24d (MD5) Previous issue date: 2014 / Resumo: O principal fator de virulência das bactérias Gram-negativas é representado pela liberação de seus subprodutos [Lipopolissacarídeos (LPS) ¿ endotoxinas] presentes na membrana externa do envelope celular bacteriano. O acumulo destes componentes bacterianos no canal radicular e a sua saída para os tecidos periapicais estimula o sistema imune do hospedeiro produzindo uma reação antígeno-anticorpo que gera uma resposta inflamatória a nível periapical. Esta reação é caraterizada pela expressão de mediadores químicos e enzimas, tais como as citocinas pró-inflamatórias e as metaloproteinases (MMPs). Assim, foram objetivos do presente estudo: 1) avaliar a influência do conteúdo infeccioso diante as diferentes etapas do tratamento endodôntico de dentes com infecção primária, na resposta imune do hospedeiro para a produção de interleucina 1 alfa (IL-1?), 1 beta (IL-1?), fator de necrose tumoral alfa (TNF-?), prostaglandina E2 (PGE2), MMP-2, MMP-3, MMP-8, MMP-9 e MMP-13 correlacionando esses níveis com os sinais e sintomas clínicos (capitulo 1); 2) avaliar o efeito da ativação do EDTA 17% com ultrassom na redução do conteúdo infeccioso de dentes com infecção primária (capitulo 2); 3) investigar os níveis de endotoxinas de dentes com infecção primária, antes e após o preparo químico-mecânico (PQM) e determinar seu potencial antigênico contra fibroblastos 3T3 através da atividade gelatinolítica de MMPs (capitulo 3). Método: Foram selecionados 24 pacientes com necessidade de intervenção endodôntica por necrose do tecido pulpar e presença de lesão periapical. Amostras do conteúdo do canal radicular foram coletadas antes do PQM (S1), depois do PQM (S2) e depois do uso de EDTA 17% (S3) com e sem ativação com ultrassom (G1 e G2, respectivamente). Amostras para quantificação de citocinas e MMPs foram coletadas passando um cone de papel 2 mm além do ápice radicular depois do PQM. As amostras microbiológicas foram processadas por cultura para contagem de unidades formadoras de colônia (UFC) e identificação. PCR foi realizado utilizando primers espécie-específicos. As amostras de LPS foram analisadas pelo método Limulus Amoebocyte Lysate (LAL). As coletas de citocinas e MMPs foram quantificadas utilizando kits específicos pelo ensaio imunoenzimático de absorção (ELISA). A atividade gelatinolítica foi avaliada por zimografia. Os níveis de produção das citocinas e MMPs foram correlacionados individualmente com os sinais e sintomas clínicos [dor à percussão (POP), dor à palpação (TOP), presença de exudato (EX)]. O teste de Pearson foi utilizado para avaliar a correlação entre endotoxinas e a produção de citocinas e MMPs. Os testes de Friedman e Wilcoxon compararam os níveis de endotoxina e carga microbiana em cada tempo operatório. Os dados obtidos pela atividade gelatinolítica foram analisados pelos testes de ANOVA e Tuckey. O nível de significância foi estabelecido em 5% (p<0,05). Resultados: IL-1?, IL-1?, TNF-?, PGE2, MMP-2, MMP-3, MMP-8, MMP-9 e MMP-13 foram detectados em todas as amostras. Foi encontrada correlação positiva entre os níveis de endotoxinas e de todas as citocinas e MMPs avaliadas (p<0,05). EX foi correlacionado positivamente com TNF-?, enquanto os níveis de IL-1?, PGE2 e MMP-8 foram correlacionados com sintomatologia dolorosa (POP/TOP) (p<0,05). O PQM reduziu significativamente os níveis de endotoxina e da carga bacteriana (p<0,05). Maiores níveis de redução de endotoxinas foram registrados quando a irrigação com EDTA foi ativada com ultrassom. Não foram encontradas diferenças significativas na redução da carga bacteriana entre G1(99.98%) e G2 (99.93%) (p>0,05). Foi encontrada correlação entre os níveis de entotoxinas (S1) e a expressão de MMP-2 por fibroblastos. Não foi observada atividade gelatinolítica para MMP-9. Conclusão: 1) O conteúdo infeccioso/endotóxico é um potente estímulo para a resposta imune do hospedeiro na produção de IL-1?, IL-1?, TNF?, PGE2, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13. 2) O PQM consegue reduzir significativamente a carga microbiana e os níveis de endotoxinas. Adicionalmente, a ativação do EDTA com ultrassom promove uma redução maior dos níveis residuais de endotoxinas. 3) O conteúdo infeccioso/endotóxico é um potente estímulo para a expressão gênica de MMP-2 por fibroblastos 3T3 / Abstract: Lipopolysaccharides (LPSs, known as endotoxins) present on the outer layers of Gram-negative bacterial envelope, and released during bacteria multiplication and death, can egress into periradicular tissues, acting as one of the most potent stimuli for immunocompetent cells in the release of inflammatory mediators and matrix metalloproteinases (MMPs). Thus, the objectives of this study were: 1) To evaluate the influence of primarily infected root canal contents (bacterial and endotoxin contents) on host-immune response by interleukin (IL)-1?, IL-1?, tumor necrosis factor ? (TNF-?), prostaglandin E2 (PGE2), matrix metalloproteinases (MMP) MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 production and to correlate their levels with clinical features (chapter 1); 2) To investigate the influence of 17% ethylenediaminetetraacetic acid (EDTA) ultrasonic activation after chemo-mechanical preparation (CMP) on eliminating/reducing oral bacterial lipopolysaccharide (LPS, known as endotoxins) and cultivable bacteria in teeth with pulp necrosis and apical periodontitis (chapter 2); 3) To investigate the endotoxin levels from primary endodontic infection before and after CMP, and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of MMPs (chapter 3). Methods: Root canal content samples were taken from 24 primarily infected root canals with apical periodontitis by using sterile/apyrogenic paper points. Samples were taken at different clinical times: S1- before CMP; S2- after CMP; S3- after EDTA: G1- with ultrasonic activation (n=12) and G2- without ultrasonic activation (n=12). PCR technique (16S rRNA) was used for the detection of the target bacteria. Culture techniques were used to determine the number of colony-forming units (CFU). Limulus Amebocyte Lysate (LAL) was used to measure endotoxin. Cytokines and MMPs levels were measured by enzyme-linked immunosorbent assay (ELISA) from samples that were taken passing 2 mm through the root apex after CMP. The levels of MMP-2 and MMP-9 gelatinolytic activity were measured using the zymography technique. The Pearson coefficient was used to correlate the amount of endotoxins with cytokine and MMP levels. Clinical data were set as dependent variables and correlated by individual correlation with each cytokine and MMP level. Friedman¿s and Wilcoxon tests was used to compare the amount of bacteria and endotoxin contents at each clinical time. Data obtained from gelatinolytic activity was analysed using Anova and Tukey¿s tests. The significance levels always were set at 5% (p<0,05). Results: IL-1?, IL-1?, TNF-? and PGE2, as well as, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 were detected in all samples. A correlation between endotoxin levels with cytokines and MMPs production was found (p<0.05). Root canal exudation was positively correlated with high levels of TNF-?, and symptomatic teeth were correlated with IL-1?, PGE2 and MMP-8 (p<0.05). CMP were effective in reducing endotoxins and bacterial load (p<0.05). Higher values of endotoxin reduction were achieved when EDTA received ultrasonic activation compared with the no-activation group (p<0.05). No differences were found in the bacterial load reduction after EDTA when comparing G1 (99.98%) and G2 (99.93%) (p>0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (p<0.05). No gelatinolytic activity of MMP-9 was observed. Conclusion: 1) Primarily infected root canal infection contents are potent stimuli factor for host-immune response by the production of IL-1?, IL-1?, TNF-?, PGE2, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13. 2) Although CMP was effective in reducing bacteria and endotoxins, it was not able to eliminate them from all root canals analyzed. The ultrasonic activation of EDTA was efficient in reducing even more the endotoxins levels in the root canals of teeth with pulp necrosis and apical periodontitis. 3) Root canal content from primary endodontic infection showed gelatinolytic activity for MMP-2 / Doutorado / Endodontia / Doutor em Clínica Odontológica

M.Sc. / Plant disease can be naturally suppressed by plant growth promoting rhizobacteria and endophytic / endorhizosphere bacteria. Apart from direct antagonism against pathogenic organisms, these plant growth promoting bacteria and endophytes can induce a form of systemic resistance (ISR) in plants. The main bacterial inducing component has been suggested to be the outer membrane lipopolysaccharides (LPS), found in the cell walls of Gramnegative bacteria. Burkholderia cepacia (Pseudomonas cepacia) is a bacterial endophyte that has potential as a biocontrol agent. Although a few studies have indicated that LPS from, certain Pseudorrionads has a protective effect in plants against disease, a controlled investigation has not been attempted previously with a purified preparation of LPS. LPS was isolated from the bacterial cell wall, prepared and characterized by denaturing electrophoresis. Characterization of the LPS also included the determination of 2-keto-3-deoxyoctonate, carbohydrate —, as well as the protein content. The purified LPS was found to possess activity as an elicitor of plant defence responses in tobacco where the induction of pathogenesisrelated (PR) proteins were investigated and electrophoretically analysed. An optimum LPS concentration range of 50-150 14/m1 was determined by studying cell death using the Evans blue procedure. Time and concentration ranges for LPS induced responses were established in cell suspensions, leaf discs, whole leaves and whole plants. It was determined that the PR-protein response could be optimally induced after four days following elicitation with 100 fag/ml LPS. Systemic induction of resistance was tested by treatment of the lower leaves and following the response in the upper leaves; as well as bacterial inoculation of the plant roots followed by PR-protein extraction of the leaves. Treatment of tobacco plants with LPS protected the plants against subsequent infection by the pathogen Phytophthora nicotianae, thereby suggesting a role for LPS as activators of systemic acquired resistance (SAR). It can be concluded from this study that the lipopolysaccharides from Burkholderia cepacia, that were used in this study, are effective local as well as systemic inducers of the defense PR-proteins in Nicotianae tabacum cv Samsun NN. The fact that protection is associated with PR-protein induction distinguishes it from the protection induced by rhizobacteria.

Abstract Prematurity is the main cause of morbidity and mortality in infants. In 25–40% of the cases preterm birth is associated with intrauterine inflammation. Surfactant proteins (SPs) A, C, and D have roles in innate immunity. In the female reproductive tract and in amniotic fluid (AF), these proteins may modulate the inflammatory responses leading to preterm birth. The aim of the present study was to establish a mouse model of lipopolysaccharide (LPS)-induced preterm birth of live-born pups and to study the activation of the innate immune system. By using mice overexpressing either rat SP-A (rSP-A) or rSP-D the roles of SP-A and SP-D in inflammatory responses were investigated. In addition, the expression of SP-C in gestational tissues was analyzed. The association of SP-C single nucleotide polymorphism (Thr138Asn) with spontaneous preterm birth and preterm premature rupture of membranes (PPROM) was investigated in a homogenous northern Finnish population of mothers and infants. Wild-type (WT) mice were injected with a single dose of intraperitoneal LPS at 16 or 17 days of gestation (term 19–20 days) leading to preterm delivery of live-born fetuses. After LPS, cytokine levels increased rapidly in maternal serum and in the uterus. This maternal inflammatory response was followed by the modest inflammatory activation in fetal and feto-maternal compartments. In fetal lung the expression of SP-A and SP-D was downregulated. The overexpression of SP-A or SP-D was evident in gestational tissues of rSP-A or rSP-D mice, respectively. In addition, excess of these proteins was detected in AF. Overexpression of either rSP-A or rSP-D modulated the LPS-induced inflammatory response related to preterm birth. Most notably, the expression of IL-4 and IL-10 in uteri and IL-10 in fetal membranes was lower in overexpressing animals. SP-C was detected in mouse and human placentas, fetal membranes, and in uteri of pregnant mice. The fetal SP-C polymorphism strongly associated with the duration of PPROM. The present study provides new information about the molecular events in inflammation induced preterm birth, particularly about the roles of cytokines and SPs in this process. Understanding of the mechanisms involved in preterm parturition may provide means for prevention and management of preterm births in the future. / Tiivistelmä Ennenaikaisuus on suurin vastasyntyneiden terveyttä ja henkeä uhkaava vaara. Kohdunsisäiset tulehdusreaktiot ovat ennenaikaisten synnytysten yleisimpiä aiheuttajia. Surfaktanttiproteiinit (SP:t) A, C ja D osallistuvat synnynnäisen immuniteetin säätelyyn. Voidaan olettaa, että synnytyskanavassa ja lapsivedessä surfaktanttiproteiinit säätelevät ennenaikaiseen synnytykseen johtavia tulehdusreaktioita. Tutkimuksen tavoitteena oli luoda hiirimalli, jossa ennenaikainen synnytys saadaan aikaan lipopolysakkaridin (LPS:n) injektiolla vatsaonteloon. Hiirimallin avulla tutkittiin puolustusjärjestelmän aktivaatiota sekä äidin että sikiön kudoksissa. Rotan SP-A:ta (rSP-A:ta) tai rSP-D:tä yli-ilmentävien hiirten avulla selvitettiin, muuttavatko nämä proteiinit ennenaikaiseen syntymään johtavaa vastetta. Lisäksi määritettiin SP-C:n ilmentyminen hiiren ja ihmisen kohdussa, sikiökalvoilla ja istukassa. SP-C-geenin yhden emäksen polymorfian (Thr138Asn) liittymistä ennenaikaiseen synnytykseen tai sikiökalvojen puhkeamiseen tutkittiin homogeenisessä pohjoissuomalaisessa tutkimuspopulaatiossa. Villin tyypin hiirille raskauden 16. tai 17. päivänä annettu LPS-annos sai aikaan elävien poikasten ennenaikaisen syntymisen. Emon seerumissa havaittiin sytokiinipitoisuuksien nopea nousu LPS:n vaikutuksesta. Emon tulehdusvaste johti synnynnäisen immuniteetin aktivaatioon istukassa, sikiökalvoilla ja kohdussa, kun taas muutokset sikiön kudoksissa olivat pieniä. Sikiön keuhkoissa SP-A:n ja SP-D:n ilmentyminen väheni. SP-A:ta tai SP-D:tä yli-ilmentävillä hiirillä havaittiin lisääntynyt SP-A:n tai SP-D:n määrä kohdussa, sikiökalvoilla, istukassa ja lapsivedessä. SP-A:n tai SP-D:n yli-ilmentyminen muutti LPS:n aiheuttamaa tulehdusvastetta. Erityisesti IL-4:n ja IL-10:n ilmentyminen kohdussa ja IL-10:n ilmentyminen sikiökalvoilla vähenivät. SP-C:n ilmentyminen havaittiin hiiren ja ihmisen istukassa ja sikiökalvoilla sekä hiiren kohdussa raskauden aikana. Sikiön SP-C-geenin polymorfia liittyi sikiökalvojen ennenaikaisen puhkeamisen kestoon. Tutkimus antaa lisätietoa tulehduksen aiheuttaman ennenaikaisen synnytyksen mekanismista sekä sytokiinien ja SP-A:n SP-D:n osuudesta synnytystapahtumassa. Mekanismin ymmärtäminen on erittäin tärkeää, jotta ennenaikaiset synnytykset voitaisiin tulevaisuudessa ehkäistä tehokkaammin.

Pyrogens are substances that may induce fever in the human body. They can be parts of bacteria, virus or fungi and due to the reaction they may cause in the body, they are routinely looked for in the medical technology industries. A method called in vitro pyrogen test (IPT) has been developed to detect these pyrogens. It is based on the fever reaction in the human body and only requires blood in combination with a solution believed to contain pyrogens. If the result is positive, the production of cytokines is started. The cytokines of interest in the IPT method are those involved in the fever process and two of them are IL-1β and TNF-α, which are the cytokines used as markers of infection in this study. Since the production of cytokines is in proportion to the amount of pyrogens, the inflammation-inducing potential of the sample can be decided. Due to problems in standardizing the method, mainly because it handles with living blood cells, focus is still pointed at improving it. The aim of this study was to optimize parameters within the IPT method by analysing air samples taken in indoor surroundings believed to contain pyrogens. The different parameters included extraction of the filter from the air sampling, incubation of whole blood and sample extract and analysis of the incubation with ELISA (enzyme linked immunosorbent assay). More specific, some of the issues concerned extraction media, time and shaking intensity for the extraction, blood ratio for the whole blood incubation and cytokines suitable for the method. A possible approach for the IPT method, when analysing air samples containing pyrogens, was reached. / Pyrogener kallas ämnen som framkallar feber och de kan exempelvis bestå av hela eller delar av bakterier, virus eller svamp (fungi). En metod som kallas för in vitro pyrogen test (IPT) har utvecklats för att detektera dessa pyrogener. Metoden bygger på att en lösning som misstänks innehålla pyrogener får komma i kontakt med blod från en människa. Efter en inkubering på mellan 4-24 timmar har blodet reagerat på eventuella pyrogener och bildat cytokiner, där mängden cytokiner är proportionell mot mängden pyrogener. De intressanta cytokinerna i den här studien var IL-1β och TNF-α, som båda är involverade i feberprocessen. Det har varit svårigheter med att standardisera metoden, mycket beroende på att det är levande celler som hela metoden bygger på, så syftet med den här studien var att förbättra in vitro pyrogen test. Luftprover tagna i inomhusmiljöer som misstänks innehålla pyrogener har använts i försöken att optimera varje steg i processen. De olika stegen inkluderade extraktion av filter som använts vid luftprovtagningen, inkubering med helblod och provextrakt och analys av inkuberingen med ELISA (enzyme linked immunosorbent assay). Några av de parametrar som undersöktes gällde extraktionsmedium, skaktid och skakintensitet under extraktionen, blodförhållande under helblodsinkuberingen och lämpliga cytokiner för metoden. Studien resulterade i att en metodik, för att analysera luftprov innehållande pyrogener med in vitro pyrogen test, kunde tas fram.

M.Sc. / Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.

M.Sc. / LPS, as a pathogen associated molecular pattern (PAMP) molecule can interact with eukaryotic host cells. Interaction occurs by either direct contact or due to the release of micelles containing LPS from bacterial cell surfaces. LPS activates innate host defence systems in both invertebrate and vertebrate animal/insect cells via analogous pathways, where the lipid A component,is responsible for the activities. LPS from several plant pathogens have been shown to activate a number of defence-related responses in plants. Initial concentration studies and cell viability assays were conducted to assess isonitrosoacetophenone (INAP) and LPS as elicitors of defensive responses in tobacco (Nicotiana tabacum cv. Samsun) cell suspensions. The effective concentrations were found to be 100vM INAP and 100μg/ml LPS. RNA was isolated, quantified and analysed to confirm the quality of the starting material for differential display analysis. The DDRT-PCR technique was successfully applied in order to obtain comparative "displays" of PCR amplicons derived from three sub-divided mRNA pools (i.e. each of the three different anchor primers, per treatment). Significant differences in the profiles of control, INAP and LPS treated cells were observed, indicating that the eliciting agents had prominent effects on cellular homeostasis, resulting in an altered gene expression profile. DDRT-PCR can be technically challenging at a number of steps. Modifications were incorporated to initially obtain differentially expressed transcripts (DETs), as well as reamplify the DETs. 223 Putative DETs were isolated from denaturing polyacrylamide sequencing gels. 172 Putative DETs were re-amplified, of which 126 appeared as good candidates for further analysis. Finally, 96 putative DETs were chosen for reverse Northern analysis. DDRT-PCR has been reported to be plagued by false positives. Reverse Northern analysis confirms the presence of the putative DET from the subdivided RNA pool, as well as affirming the differential expression, compared between the control and inducer blots. 26 DETs were selected for cloning, of which 16 were sequenced. Homologies between the DETs and known sequences were determined using BLASTN and BLASTX alignments, DNAssist software, as well as MIPS alignments to the Arabidopsis genome. Five of the DETs were assigned putative functions in plant signal perception, transduction and the defence response, based on their respective sequence homologies to sequences involved in innate immunity. It is proposed that the DET, HAP3-15, represents the plant equivalent of a component of the innate immunity pathway in mammals and Drosophila. It is further proposed that HAP3-15 represents a S-Receptor kinase protein (SRK), with a defensive role in distinguishing self from potential pathogens. Therefore, as a SRK, HAP3-15 would function as a transmembrane receptor able to conduct an external signal through the membrane to the cytoplasm as a form of signal perception. Subsequently HAP3-15 could ii play a role in phosphorylation cascades through the kinase domain and, consequently, be responsible for signal transduction. In addition, LPS would then represent the ligand creating the signal perceived by the SRK, HAP3-15, with oligosaccharide binding ability. HAP3-15 was also identified as a true positive by the INAP probe in reverse Northerns, implying that both the biological and chemical inducers used, activated the same receptor kinase. Whether the same signalling pathway was followed during the phosphorylation cascades has not been determined. Further analysis will require Northern blots in a time study to investigate the kinetics of induction. In addition, longer sequence information for each of the five DETs needs to be obtained to identify the corresponding genes in order to investigate their roles in innate immunity in plants.

Heat shock proteins (HSP), although not considered classical defence proteins, have general cytoprotective properties, which promote survival of cells and organisms. Hsp70, in particular, provides resistance to the harmful consequences of various forms of otherwise damaging or even lethal stress including pathogen infection. Increased levels of Hsp70, due to stable transfection of cells with hsp70 genes, or elevated expression in response to stress, generally correlate with the hindrance of cell death processes triggered by a variety of noxious stimuli or toxic agents. The effect of lipopolysaccharides (LPS), the major constituent of the outer membrane of the cell wall (envelope) of almost all Gram-negative bacteria, on Hsp70/Hsc70 expression in plants is unknown. In various mammalian systems, LPS has been shown to induce Hsp70 accumulation, along with programmed (apoptotic) cell death. Contrary to the effects of LPS on animal hosts however, LPS does not elicit cell death in plants, but rather pre-treatment with LPS fraction can prevent or delay the so-called hypersensitive response (HR), thus sensitizing plant tissue to respond more rapidly, or to a greater extent, to subsequently inoculated phytopathogenic bacteria. Elevated levels of reactive oxygen species (ROS) reportedly contribute to stress sensing and hsp gene activation, and subsequent Hsp70 induction, during the stress response. Increased ROS production can also trigger cell death via either programmed cell death (PCD), an active (i.e., energy-dependent) physiological suicide mechanism that is genetically regulated, or uncontrolled necrosis, an accidental, lytic form of cell destruction passively triggered by severe trauma or injury. In plants specifically, ROS may be involved in PCD activation during the HR. As a pathogen-associated molecular pattern (PAMP) or general elicitor of defence or resistance-related responses, LPS may trigger some defence-related responses, including an oxidative burst (manifest as increased levels of reactive oxygen species or ROS) in certain plant cells as it does in animal systems. However, there is conflicting evidence that shows that LPS treatment does not elicit an oxidative burst in plants. The aim of this study was to determine the effect of LPS isolated from Ralstonia solanacearum, an extremely harmful soil-borne bacterium that causes Southern wilt in over 200 plant species by infecting the host’s roots and invading the xylem vessels, on Hsp70/Hsc70 accumulation, cell death and ROS production in suspension-cultured tobacco (Nicotiana tabacum) cells, in order to gain a better understanding of the inter-relationship between these three phenomena in plant cells responding to LPS(Ralstonia). Western (immuno)blot analysis was used to study the unknown effect of LPS(Ralstonia) on Hsp70/Hsc70 accumulation in tobacco cell suspensions. LPS(Ralstonia) (all concentrations and time periods studied) generally suppressed Hsp70/Hsc70 accumulation. However, only exposure to 100 μg LPS/ml for 3 h caused a significant reduction (P < 0.05). Therefore, there was an early suppression of Hsp70/Hsc70 accumulation in response to 100 μg LPS(Ralstonia)/ml. To determine whether the observed LPS-mediated attenuation in Hsp70/Hsc70 accumulation was due to an increase in cell death in these cells, we investigated the effect of LPS(Ralstonia) on i) the general viability of the cells, and ii) the integrity of nuclear or genomic DNA extracted from LPS-treated suspension-cultured tobacco cells. The AlamarBlue™ (AB) assay was used to investigate the general cell viability in response to LPS(Ralstonia) treatment. LPS(Ralstonia) (all concentrations and time intervals studied) did not significantly affect the overall viability of the cells. Because treatment of tobacco cell suspensions with LPS(Ralstonia) did not result in a significant decrease (P < 0.05) in AB reduction, it was presumed that LPS(Ralstonia) did not appreciably compromise metabolic activity and was therefore not particularly toxic to these cells. Genomic DNA from cells undergoing PCD-associated internucleosomal DNA fragmentation (IDF) typically runs as a ladder of internucleosomal-sized DNA fragments corresponding to multimers of ca. 180 bp in agarose gels. In contrast, random DNA cleavage, usually manifest as smearing of nuclear DNA following agarose gel electrophoresis, is a token of uncontrolled necrosis. Therefore, if so-called “DNA laddering” is observed following agarose gel electrophoresis of genomic DNA extracted from suspension-cultured tobacco cells exposed to LPS(Ralstonia) then it can be assumed that LPS(Ralstonia) induced PCD. Alternatively, if a long, continuous “necrotic smear” is evident after electrophoretic separation of nuclear DNA from LPS-treated cells then LPS(Ralstonia) clearly induced uncontrolled necrosis. Whether or not LPS(Ralstonia) induced PCD-associated IDF or necrotic smearing was determined by investigating genomic DNA fragmentation (or DNA integrity) in response to LPS(Ralstonia) iii treatment. Although no typical DNA ladders were detected following electrophoresis of DNA isolated from LPS-treated cells, PCD may still have transpired. However, this is highly unlikely. No necrotic smearing was evident in LPS-treated samples either, which verifies the hypothesis that LPS(Ralstonia) (25–100 μg/ml) did not induce uncontrolled necrosis in suspension-cultured tobacco cells. In fact, these concentrations of LPS(Ralstonia) did not seem to significantly compromise DNA integrity given that LPS(Ralstonia) (25–100 μg/ml) generally had no appreciable effect on genomic DNA fragmentation (compared to untreated control samples). Incidentally, 24-h exposure of tobacco cell suspensions to higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) may have resulted in partial DNA cleavage and/or degradation. Exposure of tobacco cell suspensions to 400 μg LPS(Burkholderia)/ml for 7 days may also have evoked partial DNA cleavage and/or degradation. Whether this cleavage and/or degradation occurred deliberately by means of a fixed or predetermined mechanism or randomly by an uncontrolled mechanism remains uncertain. Finally, the H2DCF-DA (2′, 7′-dihydrodichlorofluorescein-diacetate) fluorescence assay was used to investigate the effect of LPS(Ralstonia) on ROS production, a common factor in the regulation of HSP expression and cell death activation. LPS(Ralstonia) treatment (25–100 μg/ml) generally increased ROS levels in suspension-cultured tobacco cells (compared to untreated control cells). Exposure to 75 μg LPS(Ralstonia)/ml resulted in a particularly prominent elevation in ROS levels almost instantaneously. Incidentally, higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) resulted in decreased ROS levels at some point during the assay. Although LPS(Ralstonia) (100 μg/ml for 3 h) significantly decreased Hsp70/Hsc70 accumulation in tobacco cell suspensions, cell death did not appear to be induced. In fact, LPS(Ralstonia) had no effect on general cell viability and appeared to be ineffective at causing PCD-associated IDF (DNA laddering) or necrotic smearing regardless of concentration or time of exposure. Despite these findings, treatment of suspension-cultured tobacco cells with LPS(Ralstonia) (≤ 100 μg/ml) resulted in a mild increase in ROS production. Although the exact mechanism(s) by which LPS(Ralstonia) suppressed Hsp70/Hsc70 accumulation is elusive, our results suggest that the suppression is not related to excessive LPS-mediated injury caused by excessively high ROS levels or increased cell death. We speculate that the prevention of HR-related PCD often observed in plants that are pre-treated with LPS and subsequently inoculated with phytopathogenic bacteria may be dependent on the LPS-mediated suppression of cytosolic Hsp70 expression. / Dr. M.J. Cronje

Lipopolysaccharides from Gram-negative bacteria are amphipathic, tripartite molecules consisting of a hydrophobic lipid A portion, a core hetero-oligosaccharide and a repetitive hydrophilic O-antigen polysaccharide region. Through cell : cell interactions, plants can come into contact with LPS originating from root-associated rhizobacteria, bacterial endophytes as well as bacterial pathogens. Biologically active LPS molecules have been shown to act as determinants of bacterial virulence but also as determinants of induced systemic resistance (ISR) and activators of the phenotypically related systemic acquired resistance (SAR), characterised by accelerated and enhanced defence responses. LPS as a ¡¥pathogen associated molecular pattern, PAMP¡¦ molecule, has the ability to activate the innate mammalian immunity system and to act as an immunomodulator of immune ¡V and inflammatory systems via the conserved lipid A region. It is thus believed that LPS is able to promote plant disease resistance through activation of ISR and/or SAR; however here, the O-antigen region is also implicated to play a pivotal role in the signal perception and transduction in response to elicitation by this bio-active lipoglycan. LPS was isolated from the cell walls of the endophyte, Burkholderia cepacia, characterised by denaturing electrophoresis and compared to the equivalent from the pathogen Ralstonia solanacearum. When dissolved in the presence of Ca2+ and Mg2+, the LPS attained its biologically active micellar state through complex formation. The former LPS strongly induced the activation of two MAPKs following treatment of Nicotiana tabacum cv Samsun leaves, while comparative inductions with the R. solanacearum counterpart were extremely weak and might be ascribed to it lacking an extensive O-antigen region. No previous reports on LPS-responsive MAP kinases in plant tissues exist in the literature. The time- and dose dependent activation of the two kinases were therefore investigated and their physico-chemical properties compared. A novel 32 kDa MAP kinase was transiently activated in response to exposure to LPS with optimal activation at 7 min post-elicitation with 100 ƒÝg.ml-1 LPS. Its identity as an ERK (extracellular signal-related) MAPK was confirmed by immunodetection with a pTEpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylated association of activation and inhibition of activation by U0126, an inhibitor of upstream MAPKKs. The kinase did not utilise casein, histone or myelin basic protein as substrates and no endogenous substrate could be identified. The activated MAP kinase exhibited a pI of 6.3, but two charge isomers of 32 kDa respectively were found upon two-dimensional electrophoresis. Although loss of the dual-phosphorylated epitope during purification attempts prevented extensive purification, 30% ammonium sulphate fractionation significantly (33 fold) enriched the MAPK. A second, distinct, 30 kDa MAP kinase was transiently activated in response to 125 ƒÝg.ml-1 LPS at 40 min post-elicitation, and its identity as a p38 MAPK, to date not yet found in plants, was confirmed by immunodetection with a pTGpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylation associated with activation and inhibition of activation by SB203580, a direct inhibitor of p38 MAPKs. The kinase did not utilise casein, histone or MBP as substrates and no endogenous substrate could be identified. The kinase displayed a pI of 6.0, but two charge isomers of 30 kDa respectively were found following two-dimensional electrophoresis. Loss of the dual-phosphorylated epitope again prevented significant purification, but the protein was found to be significantly (83 fold) enriched by 30% ammonium sulphate fractionation. Although LPS has been reported to be capable of altering Ca2+ permeability and perturbation of Ca2+ homeostasis across plasma membranes, Ca2+ did not appear to potentiate or reduce the activation of either the 30 or the 32 kDa kinases. To date other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates (ROI) produced during the oxidative burst. It was found that peroxide and concomitant ROI is either not generated in leaf tissue in response to LPS elicitation, or if generated, do not trigger the activation of the two kinases. The identification and partial characterisation of these two novel tobacco MAPKs in the signal perception and transduction response to LPS, significantly contributes to understanding the biochemical basis of the mechanism of action of LPS as a ¡¥resistance elicitor¡¦ involved in the triggering of effective plant defence responses and contributes towards relating the activation of mammalian innate immunity to similar responses in plants. / Prof. I.A. Dubery

Plants respond to pathogen attack by inducing a coordinated resistance strategy, which results in the expression of defense gene products. When a plant-pathogen interaction results in disease establishment, parasite colonization is caused by a delayed plant defense response, not due the absence of any response. Thus, the speed and intensity of the plant response and intracellular signalling determines the outcome of a plant-pathogen interaction. The acceleration of plant responses by the application of resistance inducers could provide a commercially, biologically and environmentally feasible alternative to existing pathogen control methods. Lipopolysaccharides are amphipathic lipoglycans that are attached to the outer bacterial membrane by a lipidic entity inserted into the bacterial phospholipid monolayer, with the saccharidic part oriented towards the exterior. The general structure of this compound is comprised of an anchor named lipid A associated with a core polysaccharide, which bears an O-antigen domain. LPS has been described as one of the pathogen-associated molecular patterns (PAMPs) capable of eliciting the activation of the plant innate immune system. LPS present in the outer membranes of plant growth-promoting rhizobacteria (PBPR) are major determinants of induced systemic resistance (ISR). In addition, LPS may function as an activator of systemic acquired resistance (SAR), providing non-specific immunization against later infection. Evidence suggests that LPS may advance plant disease resistance using the mechanism of ISR or SAR through its application to plants as a sensitizing agent, priming them to respond more effectively to subsequent pathogen attack. Phosphorylation plays a major role during the plant defense response, exemplified by its phosphorylation of transcription factors, required for the expression of defense-related genes. One of the most extensively documented phosphorylation responses is that of MAP kinase activation by phosphorylation in response to elicitation by race-specific and non-racespecific elicitors in various plant species.Proteins that undergo differential phosphorylation as a result of elicitation could be components of signal transduction pathways which connect pathogen perception with defense responses. Thus the identification of protein kinases, protein phosphatases and their substrates is essential in the elucidation of plant defense responses. The hypothesis behind this dissertation is that LPS elicitation results in alterations in the phosphorylation profile of Arabidopsis thaliana proteins. In this study, LPS was extracted from the cell walls of Burkholderia cepacia, a bacterial endophyte, and characterized by SDS-PAGE. The exposure of Arabidopsis callus culture cells to LPS resulted in distinctive changes in the phosphoprotein profile of the cells. Radioactive phosphorous labelling of proteins provided evidence that phosphorylation occurs in Arabidopsis following LPS perception, as part of a defense response related to LPS elicitation. Further investigation of differential protein phosphorylation via immunoblotting with antiphosphotyrosine antibodies revealed that tyrosine phosphorylation of Arabidopsis proteins occurs in response to LPS. One of the tyrosine-phosphorylated proteins was found to be a 42 kDa kinase, activated in response to LPS elicitation. The identity of the kinase as a mitogen-activated protein (MAP) kinase was confirmed by immunoblotting with anti-active MAP kinase antibodies. In addition, an assay of MAP kinase activity demonstrated the ability of the LPS-responsive MAP kinase to phosphorylate the ERK-MAP kinase substrate Elk1. In terms of the global phosphoproteome of Arabidopsis in response to LPS, phosphopeptides were purified from a crude protein digest by immobilized metal affinity chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LCMS/ MS). While LC indicated both quantitative and qualitative differences resulting from LPS elicitation, no peptides could be positively identified as phosphopeptides by MS analysis. This work can however be repeated with further precautions to prevent the loss of phosphate groups prior to analysis. The results obtained in this study indicate that LPS causes specific alterations in Arabidopsis protein phosphorylation as a post-translational modification in response to the perception of LPS during a plant-pathogen interaction, proving the original hypothesis. / Prof. I.A. Dubery