The dairy industry today is beginning to dedicate more focus on the growth of the calf from birth to first breeding to better improve the milk production as well as the overall performance of the individual cows. While the development of the rumen is one of the most vital contributors to the performance of the calf, it remains unknown what molecular mechanisms are responsible for the development of the rumen, and more specifically the proliferation of rumen epithelial cells. The objectives of this study were to investigate the existing data on rumen development through meta-analysis and to explore the effects of sodium butyrate and lipopolysaccharide (LPS) on rumen development in calves through experiment. In the first study a meta-analysis was performed to summarize the literature on calf performance and derive equations that relate rumen (e.g., rumen pH, reticulorumen weight, papillae area) and non-rumen factors (e.g., feed composition, form of feed, housing) to animal performance (e.g., intake of milk replacer (MR), starter, and forage; average daily gain (ADG); and feed efficiency). We looked at four different relationships to further investigate the connections between rumen, non-rumen, and performance factors. In the first and second relationships of interest, the effect of dietary and environmental variables on rumen variables and performance variables were examined, respectively. The third relationship of interest was how rumen variables influenced performance variables. The final relationship of interest was investigating the additive effects of the rumen, dietary, and environmental variables on the performance variables. Forward selection, multiple regression was used to derive equations to select variables that explained variation in the response variable in each model. Results showed that the variation in calf ADG was explained by daily forage intake, calves that were weaned, total starter intake, and total MR intake (concordance correlation coefficient (CCC) = 0.976). The variation in feed to gain ratio was explained by the weight of the ruminal contents, daily forage, MR, and starter intakes, percent of starter in the diet, and total starter intake (CCC = 0.992). The variation in daily forage intake was explained by the percent of the diet that was starter or MR (CCC = 0.998). The variation in daily starter intake was explained by the percent of acid detergent fiber in the starter, a pelleted starter (versus a texturized), diets including starter and forage (versus a milk replacer only diet), and the percent of the diet that was MR (CCC = 0.998). The variation in daily MR intake was explained by the percent of the diet that was starter, final body weight, ruminal propionate concentration, and daily starter intake (CCC = 0.918). Based on these analyses, although dietary and environmental factors are closely associated with calf performance, ruminal factors such as volatile fatty acid (VFA) concentration and ruminal contents appear to have additional, additive influences on calf performance. In the second study, 24 Holstein bull calves were challenged with oral doses of LPS and sodium butyrate. The hypothesis here was that LPS and sodium butyrate would instigate rumen cell proliferation independently and additively. Calves were assigned to one of four treatments: control (CON; n=5), butyrate (BUTY; n=5), LPS only (LPS-O) (n=6), or LPS plus butyrate (LPSB; n=6). All treatments were administered orally twice daily consisting of either: 0.9% saline (CON); 11 mM sodium butyrate (BUTY); LPS ranging from 2.5 to 40 µg/kg metabolic body weight (BW0.75, LPS), or both butyrate and LPS (LPSB). Calves were fed milk replacer (22% CP, 20% fat, as-fed) and starter (20% CP, 3% fat, as-fed) based on metabolic BW, or about 12% BW of MR and 3% BW of starter. Feed intake, fecal and respiratory scores, and rectal temperature were recorded daily. Calf BW, hip height, jugular blood samples, and rumen content samples (via oroesophageal tube) were collected weekly. Calves were weaned at 6 wk of age and euthanized at 8 wk of age, whereupon ruminal weights and ruminal samples for papillae area and epithelial thickness were collected. Blood and rumen samples were analyzed for concentrations of beta-hydroxybutyrate, glucose, LPS-binding protein, and VFA. Data were analyzed as a 2x2 factorial with the repeated effect of week. Three non-orthogonal contrasts (CON versus the average of all other treatments; LPS-O versus LPSB, and LPSB versus BUTY) were investigated. Feed intake, health measures, and blood metabolites did not differ by treatment. Calf BW increased by week (P < 0.0001). Irrespective of week, LPS calves weighed more and had higher ADG than BUTY calves (P = 0.020). Irrespective of week, withers height was greater in LPS compared to CON (P = 0.006). Rumen pH and rumen VFA concentrations did not differ by treatment but did decrease and increase, respectively, with week in conjunction with increased starter intake. Total empty forestomach (P = 0.014) and reticulorumen weights (P = 0.012) were greater in LPSB compared to BUTY. Overall, LPS and sodium butyrate appeared to have synergistically affected some, but not all rumen measurements without affecting calf growth, intake, or health. Results from the meta-analysis emphasize the importance of continuing to focus on the solid feed intake of the calf from birth through weaning. Implications from the LPS study are imperative to other dairy scientists who will attempt to further study the effects of LPS on the rumen. / Master of Science in Life Sciences / Dairy calves are born with an under-developed stomach. The stomach has four compartments: the rumen, reticulum, omasum, and abomasum. The rumen is the largest component where finger-like projections called papillae grow to absorb nutrients for the calf. It is vital to the calf that the rumen develops not only the papillae to absorb nutrients but also to foster a microbe-rich environment so the microbes can act as a defense mechanism for the calf to aid in fighting disease. While it is known that things like solid feed support the development of the rumen, the mechanism behind how that is happening still remains unclear in the literature. The objective of this study was first to better understand the relationships that exist in the literature between dietary, environmental, and ruminal factors, and second to investigate the claim that certain components of the bacteria in the rumen are stimulating rumen development independently and additively with sodium butyrate. In order to investigate the relationships amongst the dietary, environmental, and ruminal parameters, a computer program called R Studio was used to analyze over 30 different models that extracted data from a database that included a collection of 36 studies from the literature. This is also known as a meta-analysis. The associations of interest that we found were: average daily gain (ADG) of the calf was associated with daily forage intake, calves that were weaned, total starter intake, and total MR intake. Feed efficiency of the calf was associated with the weight of the ruminal contents, daily forage, milk replacer (MR), and starter intakes, percent of the diet composed of starter, and total starter intake. Daily forage intake was associated with the percent of the diet that was starter or MR. Daily starter intake was associated with acid detergent fiber in the starter, a pelleted starter (versus a texturized starter), diets including starter and forage (versus a MR only diet), and the percent of the diet that was MR. Daily MR intake was associated with the percentage of the diet that was starter, final body weight (BW), ruminal propionate concentration, and daily starter intake. These relationships emphasized that although dietary and environmental factors are more closely associated with calf performance, ruminal factors such as rumen contents and volatile fatty acid concentrations appear to have additional, additive influences on calf performance. The second part of the study objective was to explore an idea that, to our knowledge, has not been published in the literature. In the second study, 24 dairy calves were challenged with oral doses of a gram-negative bacteria lipopolysaccharide (LPS), and a short-chain fatty acid sodium butyrate. The hypothesis in this study was that the LPS and sodium butyrate would trigger metabolic pathways on the rumen cell membranes to a greater extent together, versus independently, to increase the amount of cells growing. Calves were assigned to one of four treatments: control (CON), butyrate (BUTY), LPS only (LPS-O), or LPS plus butyrate (LPSB). To study this effect, each treatment group was administered their respective treatment orally as a liquid twice daily. To measure the results, the following data was collected: feed intake, fecal and respiratory scores, rectal temperature BW, hip and withers height, blood samples, rumen content and pH samples, papillae area, epithelial thickness, and organ weights. Blood and rumen samples were analyzed for blood metabolites and volatile fatty acids concentrations respectively. Data were analyzed and results showed no difference amongst feed intake, health measures, rumen pH, rumen VFA concentration, and blood metabolites by treatment. Calves on the LPS treatment weighed more and had higher ADG than BUTY treatment calves. Withers height was higher in the LPS group when compared to CON. Stomach weights were higher in the LPSB group when compared to the BUTY group.

Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences. / Ph. D.

Thesis advisor: Christina Faherty / Shigella, a Gram-negative bacterial pathogen, induces inflammation and diarrhea by invading the colonic epithelium. Annually, millions of Shigella infections occur globally, mainly in malnourished children. Despite extensive research, no effective vaccine exists. This work explores the mechanisms of Shigella proliferation before colonic infection, where an adverse environment is encountered, including bile salts exposure. One means of bile salts evasion is possibly lipopolysaccharide (LPS) modification. LPS—O-antigen, the polysaccharide core, and the lipid A—is a crucial outer membrane component for virulence. Transposon mutant analysis suggested a role of LPS in bile salts resistance; thus, the goal of this study was to define Shigella LPS modifications following bile salts exposure. LPS mutants were investigated to distinguish crucial components of the LPS structure for bile salts resistance. Mutants were analyzed relative to wild type for growth in bile salts and biofilm formation. The LPS from all strains was purified and analyzed by polyacrylamide gel electrophoresis. Stained gels show modifications in the Oag, lipid A, and core components. Key bands were sent for mass spectrophotometry sequencing. Results indicate that the O-antigen regulates Shigella bile salts resistance, as the complete O-antigen deletion mutant and partial deletion mutants exhibited slow growth in bile salts and failed to form a biofilm in the presence of bile salts. This work highlights the importance of bile salts exposure for Shigella in future targeted antibodies against the pathogen. / Thesis (BS) — Boston College, 2019. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Introduction: L'injection ip de lipopolysaccharide (LPS) d'E.coli à la rate gestante aboutit à un phénotype cognitivo-comportemental de pathologies neuropsychiatriques chez la progéniture mâle. L'objectif principal était de vérifier l'hypothèse d'une atteinte structurelle et d'un déséquilibre entre excitation et inhibition dans l'hippocampe. L'objectif secondaire était de dégager des stratégies thérapeutiques ciblées.Méthodes: 500 μg/kg de LPS d'E.coli de sérotype O55:B5 ou 2 ml/kg de sérum physiologique étaient injectés ip à la rate au 19e jour de gestation. La progéniture mâle était étudiée à différents stades du développement. L'étude structurelle reposait sur de l'immunohistochimie, l'étude fonctionnelle sur des enregistrements électro-physiologiques de l'activité des cellules pyramidales de l'aire CA1. L'effet protecteur de la N-acétylcystéine (NAC) donnée po à la rate gestante après l'injection de LPS était testé. Résultats: Les animaux soumis à un stress prénatal par le LPS présentaient une désorganisation durable de la couche pyramidale de l'aire CA3, un déficit transitoire de neurones exprimant la reeline, une altération de la dépression à long terme des synapses glutamatergiques (LTDe) liée à un déficit des récepteurs NMDA et du système GABAergique. Un inhibiteur de la recapture du GABA parvenait à corriger les anomalies de la LTDe. La NAC prévenait les anomalies cyto-architecturales.Conclusion: Cette thèse confirme l'impact d'un stress immuno-inflammatoire maternel sur la structure et la fonction hippocampique. Elle démontre l'intérêt d'un traitement prénatal par la NAC et de la modulation du tonus GABAergique pour corriger les troubles cognitifs associés. / Introduction: A late gestational exposure to lipopolysaccharide (LPS) leads to a behavioral and cognitive phenotype of neuropsychiatric disorders in male offspring. The main goal was to test the hypothesis of structural damage and imbalance between excitation and inhibition in the hippocampus. The secondary goal was to identify targeted therapeutic strategies.Methods: Pregnant rats were ip injected with either 500 μg/kg LPS from E.coli O55:B5 or 2 ml/kg saline vehicle on gestational day 19. Male offspring were studied at different developmental stages. The structural study was based on immunohistochemistry, the functional study on electrophysiological recordings of the activity of pyramidal cells in the CA1 area. The protective effect of N-acetylcysteine (NAC) given to pregnant rats after LPS injection was tested.Results: In male offspring, LPS induced late gestational immune challenge led to sustainable disarray of the pyramidal layer in the CA3 area, transient deficit of reelin expressing neurons, impaired long term depression of glutamatergic synapses (LTDe), due to NMDA receptor and GABAergic system dysfunction. An inhibitor of GABA reuptake completely restored plasticity lost after prenatal stress. NAC prevented cyto-architectural abnormalities.Conclusion: This thesis confirms the impact of a late prenatal immune challenge on hippocampal structure and function. It demonstrates that prenatal treatment with NAC and GABAergic tone modulation are valuable therapeutic strategies for the cognitive impairment associated with prenatal immune challenge.

Legionella pneumophila (Lp) est une bactérie ubiquitaire dans les environnements aqueux et responsable d’une pneumopathie potentiellement sévère : la légionellose. La majorité des souches impliquées appartiennent au sérogroupe 1 (Lp1) et à un sous- groupe spécifique de souches portant un épitope particulier dites mAb3/1+. Cependant, la différence de distribution entre les souches retrouvées dans l’environnement et celles impliquées en clinique n’est pas clairement élucidée. Notre travail a porté sur la détection de deux facteurs de virulence de Lp. Nous avons voulu mettre en évidence l’existence de Phenols Soluble Modulines (PSMs), peptides uniquement décrit chez Staphylocoques et avons ainsi pu démontrer l’activité de peptides prédits par analyse in silico chez Lp capables d’activer la réponse inflammatoire par la voie du NF-?B et sont dotés d’une action cytotoxique. Notre deuxième axe d’étude a porté sur le lipopolysaccharide (LPS) de Lp. Afin de vérifier si la prédominance de certaines souches était liée à un biais diagnostique, nous avons voulu tout d’abord vérifier la sensibilité de 3 tests urinaires diagnostiques envers le LPS extrait de souches de différents sous- groupes de Lp1 et sérogroupes de Lp et avons ainsi pu montrer que ces tests sont capables de détecter tous les LPS de Lp1. La sensibilité envers le LPS des autres sérogroupes est très variable mais reste insuffisante pour permettre leur détection. Nous avons ensuite utilisé ces LPS extraits pour vérifier la réponse immunitaire innée en fonction des souches de Lp1. Ainsi les souches mAb3/1+ activent moins le système immunitaire que les souches mAb3/1-, ce qui pourrait expliquer alors une moins bonne clairance de ces souches permettant leur multiplication à l’origine d’une infection. Au final, notre travail a permis d’étudier deux facteurs de virulence potentiels au sein de Lp, pouvant expliquer partiellement la prédominance de certaines souches en pathologie humaine / Legionella pneumophila (Lp) is a ubiquitous intracellular bacterium found widely in the environment and is the cause of an opportunistic infection named legionellosis. The majority of the strains involved belong to serogroup 1 (Lp1) and to a specific subgroup named mAb3/1+, linked to a specific epitope expressed at the cell membrane. However the distribution difference between the strains found in the environment and the ones involved in pathology is not fully understood. We here studied two virulence factors of Lp. We first demonstrated the existence of Phenols Soluble Modulines (PSMs), smalls peptides that only have been described for Staphylococcus and found that the peptides that were predicted for Lp by in silico analysis were able to activate the innate immune response by NF-?B pathway and were able to have a cytotoxic activity. We also studied the lipopolysaccharide (LPS) of Lp. To found out if the predominance of some strains was linked to a diagnosis biais, we first evaluated the sensitivity of 3 urinary antigens tests against extracted LPS of strains belonging to all the sous-groups of Lp1 and serogroups of Lp. We then demonstrated that those tests are able to detect all LPS of Lp1, independently of mAb3/1 character. The sensitivities of the 3 tests were very variable for the other serogroups of Lp, but were too low to be able to detect those LPS in practice. We then used these extracted LPS to evaluate the innate immune response for different strains of Lp1. We demonstrated that mAb3/1- strains needed lower dose of LPS to activate the innate immune response than mAb3/1+ strains, which could be linked to a better clearance of the bacteria from the host, which doesn’t develop an infection. This work has studied two potentially virulent factors of Lp, which could partially explain the predominance of some strains of Lp in human pathology

Avec 800 000 morts par année, les maladies diarrhéiques sont la seconde cause de mortalité chez les enfants de moins de cinq ans. La shigellose, causée par des bactéries Gram négatif appelées Shigella, est l’une des quatre grandes maladies entériques touchant cette population. L’infection naturelle protège contre la réinfection et la composante polysaccharidique du lipopolysaccharide bactérien est la principale cible de l’immunité humorale. Chez S. sonnei, espèce prévalente dans les pays en développement et développés, ce polysaccharide spécifique, à caractère zwitterionique, a pour unité répétitive un disaccharide composé de deux hexosamines rares : l’acide 2-acétamido-2-désoxy-L-altruronique (A) et le 2-acétamido-4-amino-2,4,6-tridésoxy-D-galactose (B, aussi appelé AAT) associés par des liens glycosidiques 1,2-trans (I). ->4-a-L-AltpNAcA-(1->3)-b-D-FucpNAc4N-(1-> (I). Ces travaux s’intègrent dans un programme visant le développement d’un vaccin issu de sucres de synthèse à couverture large contre les infections par Shigella. Le premier objectif de la stratégie développée contre les infections par S. sonnei est l’identification des épitopes saccharidiques, cibles des anticorps protecteurs. Dans ce but, nous avons entrepris la synthèse d’une diversité de fragments du polysaccharide d’intérêt. Des synthèses multi-grammes de précurseurs orthogonalement protégés des monosaccharides A et B ont été mises au point afin d’accéder aux intermédiaires donneurs et accepteurs impliqués dans les étapes de glycosylation. En particulier, deux voies originales d’accès au précurseur B ont été développées. D’autre part, l’optimisation des conditions de glycosylation et d’oxydation a conduit à un bloc disaccharidique AB compatible avec la synthèse d’oligosaccharides d’ordres supérieurs. Les synthons mono- et disaccharidiques identifiés ont été validés à travers l’obtention de quatre disaccharides portant ou non des modifications de la répartition des charges, de deux trisaccharides ainsi que d’un tétrasaccharide. / 800,000 children die each year of diarrhoeal diseases, making it the second cause of death among children under five. Shigellosis, caused by a Gram negative bacterium, Shigella, is one of the four major forms of diarrhoeal diseases in this population. Natural infection protects against reinfection and the humoral response is primarily directed against the specific polysaccharide moiety of the bacterial lipopolysaccharide. S. sonnei, the prevalent species in developed and transitional countries, displays a zwitterionic polysaccharide, whose disaccharide repeating unit is made of two rare aminosugars: a 2-acetamido-2-deoxy-L-altruronic acid (A) and a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (B, AAT) 1,2-trans linked to one another (I). ->4-a-L-AltpNAcA-(1->3)-b-D-FucpNAc4N-(1-> (I). This work is part of the program aimed at the development of a synthetic carbohydrate-based broad coverage vaccine against Shigella infections. In order to define the protective epitopes located on the O-specific polysaccharide of S. sonnei, we tackled the synthesis of fragments thereof. First, multigram-scale syntheses of orthogonally protected precursors to residues A and B were undertaken to access donor and acceptor intermediates in the glycosylation reactions. In particular, two original routes to precursors of residue B were developed. Careful optimisation of the glycosylation and oxidation reaction conditions gave the disaccharide building block AB equipped for the synthesis of chain extension at both ends. Selected mono- and disaccharide building blocks were validated by the synthesis of four disaccharides, bearing modification of the charge pattern or not, two trisaccharides and a tetrasaccharide.

No description available.

Hospital acquired pneumonia is a major problem in the nosocomial environment worldwide. The rise in the number and level of antibiotic resistant strains of bacteria means that conventional therapies are no longer as effective as they once were. Many of the main causative organisms are Gram-negative rods, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae and one that has become a greater problem in the last twenty years Acinetobacter genospecies 13 TU. Lipopolysaccharide (LPS) is a molecule that is found on the cell surface of all Gram-negative organisms. LPS is a vital part of the outer membrane of Gram-negative bacteria and is a major factor in these organisms’ ability to cause serious infection and disease. While many Gram-negative organisms, such as E. coli and Klebsiella pneumoniae, are well characterised, other species that have become potential nosocomial pathogens more recently, such as Acinetobacter genospecies 13 TU, are much less well characterised. It is unknown as to how widespread exposure to Acinetobacter genospecies 13 TU is in a healthy population. Also, little is also about pathogenesis of Acinetobacter genospecies 13 TU such as the capacity for induction of cytokines by the Acinetobacter genospecies 13 TU LPS LPS was extracted with the aqueous phenol method and re-purified by Voegel’s method from eight strains of Acinetobacter genospecies 13 TU, four strains of Haemophilus influenzae, two strains of Pseudomonas aeruginosa, two strains of Klebsiella pneumoniae and two strains of E. coli. These LPSs were used in enzyme linked immunosorbant assays (ELISAs) with serum taken from 475 blood donors from the Southeast Scotland Blood Transfusion Service. The results from the ELISAs were averaged for each individual blood donor across all the species tested. These averaged results were compared across the species. LPS from two strains of each species, ten in all, were used to challenge the THP-1 human monocytic cell line and the mRNA was extracted and used in quantitative polymerase chain reactions to measure cytokine induction. It was seen that exposure to Acinetobacter genospecies 13 TU LPS is about as widespread in a healthy population from Southeast Scotland as exposure to Pseudomonas aeruginosa LPS and somewhat similar to Klebsiella pneumoniae LPS. Antibodies to E. coli LPS and Haemophilus influenzae LPS were similarly widespread in a healthy population from Southeast Scotland. These last two were much more widely spread than the other organisms tested. Some individuals seem to produce antibodies at high levels to all of the LPSs tested. It may be possible to use serum from these individuals to make a hyper-immune immunoglobulin preparation to be used in the immunotherapy of hospital associated pneumonia. The LPS from one of the strains of Acinetobacter genospecies 13 TU was able to induce similar levels of cytokine production as Klebsiella pneumoniae and Pseudomonas aeruginosa. It was able to induce higher levels of cytokine production over a greater number of cytokines than both Haemophilus influenzae and E. coli LPS. LPS from the other strain of Acinetobacter genospecies 13 TU tested induced lower levels of cytokines compared to the other strain. These levels were lower than those developed by Haemophilus influenzae and E. coli LPS as well as those induced by the LPSs from Klebsiella pneumoniae and Pseudomonas aeruginosa. It seems that there is a range of different levels of cytokine production induced by Acinetobacter genospecies 13 TU LPS with some strains inducing high levels and others inducing low levels of cytokines.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of Gram-negative bacteria and plays a fundamental role in protecting the bacteria from harsh environments and toxic compounds. The LPS transport system is responsible for transporting LPS from the periplasmic side of the inner membrane (IM) to the OM, in a process involving seven LptA-LptG proteins. The current model for lipopolysaccharide transport (Lpt) suggests that LPS is initially extracted by a four-protein complex, LptBCFG, from the inner membrane to the periplasm, where LptA mediates further transport to the OM. Another two protein complex, LptD/E, catalyses the assembly of LPS at the OM cell surface. However, the details of this transport mechanism have remained unknown, mainly due to a lack of structural information. In chapter 1 and 2 of this thesis, I report materials and methods for all LptD/E, and Schmallenberg virus (SBV) nucleoprotein (NP) experiments and the theories and software that were used in determining structures of LptD/E, SBV NP and the SBV NP/RNA complex. In chapter 3 of this thesis, I report the first crystal structure of the outer membrane protein LptD/E complex. LptD forms a 26-strand ß-barrel in a closed form and LptE is a roll-like structure located inside LptD to form “barrel and plug” architecture. Through structural analysis, function assay and molecular dynamics simulation, we proposed a mechanism in which the hydrophilic head of LPS molecule, including the oligosaccharide core and the O antigen, directly penetrates through the hydrophilic ß- barrel whilst the hydrophobic lipid A tail is inserted into an intramembrane hole, with a lateral opening between strand ß1 and ß26 of the LptD. LptE may assist this process. In chapter 4, I report the crystal structure of the SBV NP in two conformations: tetrameric when the protein was purified under native conditions, and trimeric when denatured and refolded during purification. The SBV NP has a novel fold and we have also identified that the N-terminal arm is crucial for RNA binding, and the N- and the C-terminal arm is essential for RNA multimerisation with adjacent protomers and for viral RNA encapsidation. Chapter 5 describes the crystal structure of SBV NP in complex with a 42 nucleotide long RNA (polyU). This ribonucleoprotein (RNP) complex was crystallized as a ring-like tetramer with each protomer bound to 11 ribonucleotides. Eight of these nucleotides are bound in a positively charged cleft between N- and C- terminal domains and three are bound in the N-terminal arm. I also compared the structure to that of other NPs from negative-sense RNA viruses, and found that SBV NP sequesters RNA using a different mechanism. Furthermore, the structure suggests that when RNA binds the protein, there are conformational changes in the RNA-binding cleft, and in the N- and C-terminal arms. Thus our results reveal a novel mechanism of RNA encapsidation by orthobunyaviruses NP.

Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.