Os peptídeos antimicrobianos são importantes ferramentas do sistema imune inato para controle das infecções e recentemente tem sido investigado seu potencial como estratégia terapêutica nas infecções e sepse. Estes peptídeos apresentam diversas atividades decorrentes de mecanismos de ação antimicrobianas e imuno estimuladoras. A indução de tolerância com a administração de pequenas doses de LPS reduz a mortalidade em modelos animais de sepse, modulando diversos aspectos do sistema imune. Nossa hipótese neste estudo foi que o efeito protetor da tolerância ao LPS está relacionado com a modulação da produção dos peptídeos antimicrobianos na sepse. A tolerância ao LPS foi induzida em camundongos C57bl/6 por injeção de lipopolissacarídeo de E. coli na dose de 1mg/kg por cinco dias. No oitavo dia os animais foram eutanasiados por overdose anestésica ou submetidos ao modelo de ligadura e punção cecal. Após seis horas os animais foram eutanasiados por overdose anestésica e o sangue, baço, intestino e pulmões foram coletados para determinação das concentrações de citocinas e peptídeos antimicrobianos. Nossos resultados mostram que o efeito da tolerância ao LPS sobre a produção de peptídeos antimicrobianos é tecido específica. Sistemicamente (plasma) a tolerância aumenta a concentração plasmática de beta defensina-3 e CRAMP somente em animais submetidos à CLP. No baço ocorre redução de beta defensinas 1 e 7 pela CLP e também pela tolerância. No pulmão há elevação de beta defensinas 1 e 3 pela CLP e esta é revertida pela tolerância. No intestino ocorre redução de defensinas pela tolerância tanto em animais controle quanto em animais submetidos à CLP. Observamos em nosso estudo um padrão invertido entre as concentrações de peptídeos antimicrobianos e citocinas no intestino e baço dos animais, principalmente nos submetidos à CLP. Quanto maior a concentração da citocina no tecido, menor a concentração da defensina em questão. No intestino podemos observar que a tolerância e a CLP aumentam a concentração de IL-6 e IL-10 e diminuem as concentrações de beta defensinas 1 e 7. No baço observamos esse padrão entre beta defensina-7 e IL-6 e entre beta defensina-1 e TNF-alfa. Não foi possível encontrar esse tipo de correlação no pulmão. Concluímos que os efeitos protetores da tolerância ao LPS estão relacionados com uma menor produção de defensinas no baço, pulmão e intestino de animais submetidos à sepse, e com maior concentração de peptídeos antimicrobianos no plasma / Antimicrobial peptides are important tools of the innate immune system to control infections and its potential as a therapeutic strategy in infections and sepsis has recently been investigated. These peptides present both antimicrobial and immuno-stimulatory activities. Lipopolysaccharide (LPS) tolerance is a defense mechanism against invading microorganisms also widely distributed in nature. Induction of tolerance with the administration of small doses of LPS reduces mortality in animal models of sepsis, modulating several immune system aspects. Our hypothesis was that the protective effect of LPS tolerance is related to the modulation of antimicrobial peptide production in sepsis. LPS tolerance was induced in C57bl / 6 mice by injection of E. coli LPS at 1mg / kg for five days. On the eighth day the animals were submitted to the sepsis model of cecal ligation and puncture. After six hours the animals were euthanized by anesthetic overdose and blood, spleen, intestine and lungs were collected for the determination of cytokines and antimicrobial peptides concentrations. Our results show that the effect of LPS tolerance on the production of antimicrobial peptides is tissue specific. LPS tolerance increases the plasma concentration of beta-defensin-3 and CRAMP only in animals undergoing CLP. In the spleen, there is reduction of ? defensins 1 and 7 by CLP and also by tolerance. In the lung, there is elevation of beta defensins 1 and 3 by PLC and this is reversed by tolerance. In the intestine, there is reduction of defensins by tolerance in both control and CLP animals. In our study, we observed an inverted pattern between the concentrations of antimicrobial peptides and cytokines in the intestine and spleen of animals, especially those submitted to CLP. The higher the cytokine concentration in the tissue, the lower the concentration of defensin in question. In the gut we can observe that tolerance and CLP increases IL-6 and IL-10 concentration and decreases ? defensins 1 and 7 concentrations. In the spleen, we observed this pattern between ?-defensin-7 and IL-6 and between alpha-defensin-1 and TNF-alpha. It was not possible to find this type of correlation in the lung. We conclude that the protective effects of LPS tolerance are related to a lower production of defensins in the spleen, lung and intestine of animals submitted to sepsis, and with a higher concentration of antimicrobial peptides in plasma

Fong, Oi Ning. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 170-195). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Contents --- p.viii / List of Abbreviations --- p.xii / Chapter CHAPTER ONE --- Introduction --- p.1 / Chapter 1.1 --- Bacterial Infection in Neonates --- p.1 / Chapter 1.2 --- Gram-positive and Gram-negative Bacterial Cell Wall --- p.3 / Chapter 1.2.1 --- Gram-negative Bacterial Cell Wall Component - Lipopolysaccharide --- p.3 / Chapter 1.2.2 --- Gram-positive Bacterial Cell Wall Component - Peptidoglycan --- p.4 / Chapter 1.3 --- Differential Host Response against Gram-specific Bacterial Infection --- p.6 / Chapter 1.4 --- Role of Neutrophils in Host Defense against Bacterial Infection --- p.8 / Chapter 1.4.1 --- Recognition of Bacterial Components --- p.9 / Chapter 1.4.2 --- Neutrophil Functions --- p.10 / Chapter 1.5 --- Expression Profiling of Activated Neonatal Neutrophils --- p.15 / Chapter CHAPTER TWO --- Objectives --- p.26 / Chapter CHAPTER THREE --- Materials and Methodology --- p.27 / Chapter 3.1 --- Overview of the Experimental Procedure --- p.27 / Chapter 3.2 --- Cord Blood Sample Collection --- p.28 / Chapter 3.3 --- Cord Blood Neutrophil Isolation --- p.30 / Chapter 3.3.1 --- Isolation of Neutrophils --- p.30 / Chapter 3.3.2 --- Analysis of Neutrophil Purity by Flow Cytometry --- p.31 / Chapter 3.3.3 --- Cell Viability Test by Trypan Blue Exclusion Assay --- p.31 / Chapter 3.4 --- In Vitro Stimulation of Neutrophils by LPS or PGN --- p.33 / Chapter 3.5 --- Total RNA and Protein Isolation --- p.34 / Chapter 3.5.1 --- Total RNA Isolation --- p.34 / Chapter 3.5.2 --- Protein Isolation --- p.35 / Chapter 3.6 --- Preparation of Total RNA Samples for Expression Profiling and Quantitative Real Time Polymerase Chain Reaction (qPCR) --- p.37 / Chapter 3.6.1 --- DNase Treatment --- p.37 / Chapter 3.6.2 --- Total RNA Cleanup --- p.37 / Chapter 3.6.3 --- Purity Assessment of the Purified Total RNA Sample --- p.38 / Chapter 3.6.4 --- Integrity Assessment of the Purified Total RNA Sample --- p.39 / Chapter 3.6.5 --- Assessment of Tumor Necrosis Factor Alpha (TNF-α) mRNA Expression Level in Neutrophils --- p.42 / Chapter 3.7 --- Determination of the PGN Concentration for Neutrophil Activation --- p.43 / Chapter 3.8 --- "Expression Profiling of the LPS, PGN Stimulated or Unstimulated CB Neutrophils" --- p.44 / Chapter 3.8.1 --- cRNA Preparation and Array Hybridization --- p.44 / Chapter 3.8.2 --- Expression Profiling Data Analysis --- p.46 / Chapter 3.9 --- Validation of Candidate Genes Using qPCR --- p.48 / Chapter 3.10 --- Gram-Negative Bacterial Endotoxin Assay --- p.50 / Chapter CHAPTER FOUR --- LPS Stimulation Induced Transcriptional Changes in Cord Blood Neutrophils --- p.61 / Chapter 4.1 --- Result --- p.61 / Chapter 4.1.1 --- Gene Expression Profile of CB Neutrophils in Response to LPS Stimulation --- p.61 / Chapter 4.1.1.1 --- Up-regulated Genes in LPS-stimulated CB Neutrophils --- p.61 / Chapter 4.1.1.2 --- Down-regulated Genes in LPS-stimulated CB Neutrophils --- p.62 / Chapter 4.1.1.3 --- Network Analysis of Genes Induced by LPS Stimulation --- p.63 / Chapter 4.2 --- Discussion --- p.64 / Chapter 4.2.1 --- Robust Transcriptional Response in CB Neutrophils --- p.64 / Chapter 4.2.2 --- LPS Modulated Transcriptional Responses --- p.64 / Chapter 4.2.2.1 --- LPS-induced NF-kB Pathway --- p.64 / Chapter 4.2.2.2 --- LPS-induced Expression of Various Transcription Factors --- p.66 / Chapter 4.2.2.3 --- LPS-induced Regulation of Apoptosis --- p.67 / Chapter CHAPTER FIVE --- PGN Stimulation Induced Transcriptional Changes in Cord Blood Neutrophils --- p.83 / Chapter 5.1 --- Result --- p.83 / Chapter 5.1.1 --- Gene Expression Profile of PGN-stimulated CB Neutrophils --- p.83 / Chapter 5.1.2 --- Up-regulated Genes in PGN-stimulated CB Neutrophils --- p.83 / Chapter 5.1.3 --- Down-regulated Genes in PGN-stimulated CB Neutrophils --- p.84 / Chapter 5.1.4 --- Network Analysis of Genes Induced by PGN Stimulation --- p.84 / Chapter 5.2 --- Discussion --- p.86 / Chapter 5.2.1 --- Robust Transcriptional Response in CB Neutrophils --- p.86 / Chapter 5.2.2 --- PGN Modulated Transcriptional Responses --- p.86 / Chapter 5.2.2.1 --- PGN-induced NF-kB Pathway --- p.86 / Chapter 5.2.2.2 --- Possible Role of STAT3 in PGN-stimulated CB Neutrophil --- p.89 / Chapter 5.2.2.3 --- Possible Role of c-Jun in PGN-stimulated CB Neutrophil --- p.90 / Chapter CHAPTER SIX --- Comparison and Validation of LPS- and PGN-activated Transcriptomes in Cord Blood Neutrophils --- p.106 / Chapter 6.1 --- Result --- p.106 / Chapter 6.1.1 --- Comparison of the Transcriptional Changes of LPS- and PGN- stimulated CB Neutrophils --- p.106 / Chapter 6.1.2 --- Common Transcriptional Changes of LPS- and PGN-Stimulated CB Neutrophils --- p.106 / Chapter 6.1.2.1 --- Commonly Up-regulated Genes in LPS- and PGN- Stimulated Neutrophils --- p.107 / Chapter 6.1.2.2 --- Commonly Down-regulated Genes in LPS- and PGN- Stimulated Neutrophils --- p.107 / Chapter 6.1.2.3 --- Network Analysis of Genes Commonly Regulated by LPS and PGN --- p.108 / Chapter 6.1.3 --- Differential Transcriptional Changes of LPS- and PGN- Stimulated CB Neutrophils --- p.108 / Chapter 6.1.4 --- Real Time qPCR Validation of the Expression Levels of Selected Genes --- p.109 / Chapter 6.1.5 --- Expression Changes of the Confirmed Target Genes in Response to High-dose LPS Stimulation --- p.110 / Chapter 6.2 --- Discussion --- p.111 / Chapter 6.2.1 --- Activation of NF-kB and Related Genes by Both LPS- and PGN-stimulation in CB Neutrophils --- p.111 / Chapter 6.2.2 --- Commonly Expressed Genes - Transcription Factor MAFF --- p.112 / Chapter 6.2.3 --- Commonly Expressed Genes - Novel Gene G0S2 --- p.113 / Chapter 6.2.4 --- Suspected Commonly Expressed Genes - Transcription Factor NR4A3 --- p.114 / Chapter 6.2.5 --- Differentially Expressed Genes - Heat Shock Proteins --- p.115 / Chapter 6.2.6 --- Differentially Expressed Genes 226}0ؤ AP-1 Transcription Factor Complex --- p.118 / Chapter 6.2.7 --- Other Differentially Expressed Genes --- p.121 / Chapter CHAPTER SEVEN --- General Discussion and Conclusion --- p.164 / Bibliography --- p.168

A deeper understanding of the diversity of carbohydrates and the many applications of oligo- and polysaccharides found in nature are of high interest. Many of the processes involving carbohydrates affect our everyday life. This thesis is based on six papers all contributing to an extended perspective of carbohydrate property and functionality. An introduction to carbohydrate chemistry together with a presentation of selected carbohydrate synthesis and analysis methods introduces the reader to the research field. The first paper is an NMR spectroscopy reinvestigation of the structures of the O-antigens from the lipopolysaccharides (LPS) of Shigella dysenteriae type 3 and Escherichia coli O124. The repeating units were concluded to be built of identical branched pentasaccharides now with the correct anomeric configurations. Paper II is a structural investigation of the O-antigen from the LPS of E. coli O74 which is built of branched tetrasaccharide repeating units including the uncommon monosaccharide d-Fuc3NAc. Paper III is a conformational study of a rhamnose derivative, using NMR spectroscopy and X-ray crystallography. The benzoyl ester group positioned at C4 prefers an “eclipsed” conformation in the crystal as well as in solution. The use of site-specifically 13C-labeled compounds in conformational studies is discussed in Papers IV and V. The disaccharide α-L-Rhap-(1→2)-α-L-Rhap-OMe was synthesized together with two 13C-isotopologues and studied with NMR spectroscopy to give seven J-couplings related to torsion angles φ and ψ. The trisaccharide α-L-Rhap-(1→2)[α-L-Rhap-(1→3)]-α-L-Rhap-OMe was synthesized with 13C-labeling at two positions which presented a solution to a problem of overlapping signals in the 1H NMR spectrum. The site-specific labeling also facilitated the measurement of two 3JCC and two 2JCH coupling constants. Finally, chapter 6 gives a short introduction to glycosynthase chemistry and discusses the synthesis of α-glycosyl fluorides. A novel cyclic heptasaccharide was synthesized from α-laminariheptaosyl fluoride using a mutant of the enzyme laminarase 16A and subsequently analyzed by NMR spectroscopy. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Nous nous sommes intéressés à la compréhension des mécanismes régissant la symbiose Rhizobium-Acacia dans les conditions de stress salin. Les lipopolysaccharides jouent un rôle important dans les étapes de cette symbiose. Le but était de mettre en évidence les modifications pariétales de la bactérie en réponse au stress salin par l'étude de la structure des lipopolysaccharides des souches isolées du désert marocain tolérant NaCl 7%. Ainsi, une nouvelle méthode d'hydrolyse des lipopolysaccharides sensible, non destructive et compatible avec la spectrométrie de masse a été développée. En présence de stress salin, nous avons montré que la membrane externe devenait plus hydrophobe en augmentant l'acylation de la région lipidique ainsi qu'en réduisant la présence des molécules de LPSs à longues chaînes de sucres.Des essais d'évaluation de l'efficience et de l'infectivité des Rhizobia étudiés ont été mis en œuvre pour déterminer l'impact de ces modifications des LPSs sur la symbiose sous stress salin.

Pferde als Pflanzenfresser benötigen für die Verdauungsvorgänge im Magen-Darm-Kanal eine Vielzahl von Mikroorganismen. In der Pathogenese der equinen Kolikerkrankungen spielen die aus einem Teil dieser Bakterien stammenden Endotoxine (Lipopolysaccharide, LPS) eine wichtige Rolle. Das Caecum und das Colon ascendens scheinen der Ort einer pathologischen Endotoxinabsorption beim Pferd zu sein. Mit Hilfe von Limulus-Amoebozyten-Lysat-Tests (chromogenes Substrat, Endpunkt Methode) wurden die Endotoxinkonzentrationen bei 52 gesunden Pferden und 105 an Magen-Darm-Kolik erkrankten Pferde bestimmt. Durch wiederholte Messungen wurde die Entwicklung der Endotoxinkonzentration bei Kolikpferden im Krankheitsverlauf untersucht. Im Plasma aller gesunden Pferde wurden Endotoxine nachgewiesen, mit einem Mittelwert von = 5,90 pg/ml ± 2,78 pg/ml. Bei 90,5% der Pferden mit Kolik lag die Endotoxinkonzentration in der ersten Probe nach Einlieferung in die Klinik über 10 pg/ml. Kolikformen mit grundsätzlich hohen Endotoxinkonzentrationen konnten herausgefunden werden. In dieser Untersuchung waren das die Hernia foraminis omentalis mit einem LPS-Mittelwert von 91,57 pg/ml, die Dünndarmstrangulation durch Lipoma pendulans mit einem LPS-Mittelwert von 89,32 pg/ml und die Torsio coli totalis 360° mit einem LPS-Mittelwert von 88,21 pg/ml. / Endotoxaemia in colic illnesses in horses; Quantitative analysis and clinical relevance Horses as herbivores require a multitude of micro-organisms for the digestive processes in the gastrointestinal tract. The endotoxins (lipopolysaccharides, LPS) originating from a part of the bacteria play an important role in the pathogenesis of equine colic illnesses. The caecum and the colon ascendens appear to be the site of a pathological absorption of endotoxins in horses. With the aid of limulus-amoebocyte-lysate tests (chromogeneous substrate, end-point method) the endotoxin concentrations were analysed in 52 healthy horses and 105 horses suffering from gastrointestinal colic. The development of the endotoxin concentration in the case of horses suffering from colic was investigated through repeated measurements throughout the course of the illness. Endotoxins were identified in the plasma of all healthy horses at a mean value of = 5.90 pg/ml ± 2.78 pg/ml. In 90.5% of the horses with colic, the concentration of endotoxins in the first sample subsequent to admission to the clinic was over 10 pg/ml. It was possible to determine specific forms of colic accompanied by fundamentally high concentrations of endotoxins. In this investigation these were omental foramen hernia with a mean LPS value of 91.57 pg/ml, small intestinal strangulation by lipoma pendulans with a mean LPS value of 89.32 pg/ml and colon torsion 360° with a mean LPS value of 88.21 pg/ml.

Dissertation (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 72-96.

Thesis (Ph.D.)--McMaster University (Canada), 1992. / Source: Dissertation Abstracts International, Volume: 54-08, Section: B, page: 4124. Adviser: D. B. MacLean.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.