ABSTRACT This thesis research involved investigations into the effects of inflammation on maturation of L-carnitine homeostasis in developing rat neonates. The overall hypothesis was an inflammatory stimulus will alter the ontogeny of L-carnitine homeostasis pathways and this depends upon when the inflammatory stimulus occurs in postnatal development. The objective was to investigate the potential effect of inflammation on carnitine transporter expression in different age groups of neonates and evaluation of effect of inflammation on ontogeny and activity of enzymes involved in carnitine biosynthesis and whether this differs depending upon when in postnatal development the inflammatory stimulus occurs. Rat pups at postnatal day 3, 7, and 14 received an intraperitoneal injection of lipopolysaccharide (LPS) at a dose known to cause a febrile reaction in rat neonates. L-Carnitine homeostasis pathways underwent significant ontogenesis during postnatal development in the rat. LPS administration caused a significant decrease in free L-carnitine levels in serum and heart tissue and a decrease in mRNA expression levels of the high affinity carnitine transporter, Octn2, in kidney, heart and intestine at all postnatal ages. Furthermore, significant decreases in mRNA expression levels of key enzymes involved in carnitine biosynthesis was observed, while an increase in carnitine palmitoyltransferase mRNA levels were observed at all postnatal ages. Reductions in butyrobetaine hydroxylase mRNA expression were paralleled by reductions in enzyme activity only at postnatal day 3 and 7. Heart creatine phosphate levels were deceased significantly in LPS treated groups in all postnatal ages; however, ADP and ATP levels were unaffected. Collectively, this research provided experimental evidence for a significant effect of inflammation on changes in L-carnitine homeostasis maturation in early neonatal stages. The maturation of physiological processes may be altered by external factors in early postnatal life.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Increasing evidence highlights that term labour is an inflammatory event associated with increased production of pro-­‐inflammatory mediators and leukocyte influx into the intrauterine tissues. Preterm labour (PTL), defined as labour before 37 weeks gestation, is a major clinical problem, and preterm birth is the leading cause of neonatal mortality and morbidity worldwide. The causes of PTL are poorly understood, but intrauterine infection and inflammation have been shown to be important factors. Therefore, there is growing interest in the hypothesis that preterm labour may occur as a result of the premature activation of the inflammatory pathways normally initiated with labour at term, either idiopathically, or in response to a pathological intrauterine infection. The aim of this thesis was to use a mouse model of infection-­induced PTL to: characterise the local inflammatory and immune response to an intrauterine infection; investigate the potential of anti‐inflammatory agents to delay delivery of pups and to improve their survival; and to investigate the role of specific immune cell populations in infection-­induced preterm labour. To characterise the inflammatory and immune response to intrauterine infection, CD1 mice received an intrauterine injection of PBS vehicle or increasing doses of bacterial-­derived lipopolysaccharide (LPS) on day 17 of gestation. Time to delivery, and the number of live born pups were determined. Intrauterine administration of increasing doses of LPS dose-­dependently induced preterm labour and reduced the proportion of live born pups. Analysis of tissues harvested six hours post-­surgery demonstrated that in response to intrauterine LPS administration, there was increased expression of inflammatory cytokines and chemokines within the utero-­placental tissues, amniotic fluid and maternal serum; and an influx of neutrophils into the decidua, compared to mice receiving PBS. Given these results, the potential of anti‐inflammatory agents to delay LPS-­induced preterm delivery and improve pup survival was then investigated using the same mouse model. Prior to intrauterine LPS administration, mice were pre-­‐treated with epi-­lipoxin, BML-­111 (a stable lipoxin analogue), or IL-­10. Time to delivery was unaffected by pre-­treatment with the anti-­inflammatory agents, however epi-­lipoxin significantly increased the proportion of live born pups in mice delivering preterm, compared to mice receiving only LPS. To further investigate the role of immune cells in infection-­induced PTL, antibody-­based depletion strategies were used to selectively deplete specific immune cell populations to determine whether they played a causative role in LPS­‐induced preterm delivery. Despite successful depletion of macrophages or neutrophils, it was not found to significantly affect LPS-­induced preterm delivery, suggesting these immune cells are not required for the induction of preterm labour in response to intrauterine infection. However, it is likely that they contribute to the intrauterine inflammatory response as depletion resulted in altered inflammatory signalling within the intrauterine tissues. Collectively, this work has demonstrated that the presence of intrauterine bacterial LPS, as a surrogate model of infection, induces a robust inflammatory and immune response within the utero‐placental tissues that involves the increased production of inflammatory mediators and the influx of immune cells into the decidua, which ultimately leads to PTL. Whilst the anti-­inflammatory treatments tested here did not delay LPS-­induced PTL, epi-­lipoxin attenuated LPS-­induced mortality in pups born preterm, suggesting this anti‐inflammatory agent may be useful in protecting the fetus from the adverse effects of infection-­induced preterm birth. Using models such as the one described here, are vital to improving our understanding of the events regulating the induction of PTL and will ultimately aid the search for novel therapeutic options for the treatment of PTL.

Thesis advisor: Thomas N. Seyfried / Two major metabolic phenomena observed in cancer cells include the Warburg effect and Crabtree effect. The Crabtree effect is the in vitro inhibition of respiration by glucose. The influence of glucose on the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of tumorigenic RAW264.7 and VM-M3 macrophage cells, as well as non-tumorigenic BV-2 microglia cells, was studied using the Seahorse XF96 extracellular flux analyzer. RAW264.7, VM-M3, and BV-2 cells incubated in glucose medium displayed a significantly lower OCR and higher ECAR compared to cells incubated in no glucose medium. Furthermore, when glucose medium was added to the RAW264.7 and BV-2 cells in real-time using the Seahorse XF96 injection ports, a rapid decrease in OCR and increase and ECAR was observed. Therefore, RAW264.7, VM-M3, and BV-2 cells display a robust Crabtree effect in vitro, as assessed by OCR and ECAR. Additionally, it is important to consider the Crabtree effect when studying in vitro energy metabolism of all cell and tissue types. It was also found that the elimination of the Crabtree effect through glucose deprivation resulted in dynamic cardiolipin (CL) fatty acid changes in VM-M3 cells. VM-M3 cells incubated in 10 mM glucose medium for four hours displayed a short-chain, saturated (immature) CL fatty acid composition, while VM-M3 cells incubated in no glucose media for four hours displayed long-chain, unsaturated (mature) CL fatty acid composition. Cardiolipin (CL) is a phospholipid highly enriched in the inner mitochondrial membrane. Mature, long-chain, unsaturated CL molecular species are involved in maintaining mitochondrial function and membrane integrity. Overall, these data suggest that CL fatty acid composition may function as a structural component of the Crabtree effect in vitro. The Warburg effect, or aerobic glycolysis, is the observation that tumor cells consume less oxygen and more glucose than normal, untransformed cells in the presence of oxygen. It has been shown that immune cells display a Warburg effect upon activation by changing their core metabolism from oxidative phosphorylation to glycolysis. In this study, it was observed that both RAW264.7 macrophage cells and BV-2 microglia cells display a significantly lower OCR and higher ECAR following LPS-activation. However, this observation is dependent on the concentration of LPS. Therefore, these data suggest that both RAW264.7 and BV-2 cells display a LPS concentration-dependent change in metabolism from oxidative phosphorylation to glycolysis upon LPS-activation in vitro. The in vitro lipid profiles that resulted from the Crabtree effect and the LPS-activated Warburg effect were also studied in the RAW264.7 cell line. The lipids phosphatidylserine (PS) and cardiolipin (CL) displayed the most robust changes in the RAW264.7 cells. Both PS and CL have been shown to be associated with cellular respiration. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Macrophages are present in virtually all tissues and account for approximately 10% of all body mass. Although classically credited as the scavenger cells of innate immune system, ridding a host of pathogenic material and cellular debris though their phagocytic function, macrophages also play a crucial role in embryogenesis, homeostasis, and inflammation. De-regulation of macrophage function is therefore implicated in the progression of many disease states including cancer, arthritis, and atherosclerosis to name just a few. The diverse range of activities of this cell can be attributed to its exceptional phenotypic plasticity i.e. it is capable of adapting its physiology depending on its environment; for instance in response to different types of pathogens, or specific cocktail of cytokines detected. This plasticity is exemplified by the macrophages capacity to adjust rapidly its transcriptional profile in response to a given stimulus. This includes interferons which are a group of cytokines capable of activating the macrophage by interacting with their cognate receptors on the cell. The different classes of interferons activate downstream signalling cascades, eventually leading to the expression (as well as repression) of hundreds of genes. To begin to fully understand the properties of a dynamic cell such as the macrophage arguably requires a holistic appreciation of its constituents and their interactions. Systems biology investigations aim to escape from a gene-centric view of biological systems. As such this necessitates the development of better ways to order, display, mine and analyse biological information, from our knowledge of protein interactions and the systems they form, to the output of high throughput technologies. The primary objectives of this research were to further characterise the signalling mechanisms driving macrophages activation, especially in response to type-I and type- II interferons, as well as lipopolysaccharide (LPS), using a ‘systems-level’ approach to data analysis and modelling. In order to achieve this end I have explored and developed methods for the executing a ‘systems-level’ analysis. Specifically the questions addressed included: (a) How does one begin to formalise and model the existing knowledge of signalling pathways in the macrophage? (b) What are the similarities and differences between the macrophage response to different types of interferon (namely interferon-β (IFN-β) and interferon-γ (IFN-γ))? (c) How is the macrophage transcriptome affected by siRNA targeting of key regulators of the interferon pathway? (d) To what extent does a model of macrophage signalling aid interpretation of the data generated from functional genomics screens? There is general agreement amongst biologists about the need for high-quality pathway diagrams and a method to formalize the way biological pathways are depicted. In an effort to better understand the molecular networks that underpin macrophage activation an in-silico model or ‘map’ of relevant pathways was constructed by extracting information from published literature describing the interactions of individual constituents of this cell and the processes they modulate (Chapter-2). During its construction process many challenges of converting pathway knowledge into computationally-tractable yet ‘understandable’ diagrams, were to be addressed. The final model comprised 2,170 components connected by 2,553 edges, and is to date the most comprehensive formalised model of macrophage signalling. Nevertheless this still represents just a modest body of knowledge on the cell. Related to the pathway modelling efforts was the need for standardising the graphical depiction of biology in order to achieve these ends. The methods for implementing this and agreeing a ‘standard’ has been the subject of some debate. Described herein (in Chapter-3) is the development of one graphical notation system for biology the modified Edinburgh Pathway Notation (mEPN). By constructing the model of macrophage signalling it has been possible to test and extensively refine the original notation into an intuitive, yet flexible scheme capable of describing a range of biological concepts. The hope is that the mEPN development work will contribute to the on-going community effort to develop and agree a standard for depicting pathways and the published version will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest. With a desire to better understand the transcriptional response of primary mouse macrophages to interferon stimulation, genome wide expression profiling was performed and an explorative-network based method applied for analysing the data generated (Chapter-4). Although transcriptomics data pertaining to interferon stimulation of macrophages is not entirely novel, the network based analysis of it provided an alternative approach to visualise, mine and interpret the output. The analysis revealed overlap in the transcriptional targets of the two classes of interferon, as well as processes preferentially induced by either cytokine; for example MHC-Class II antigen processing and presentation by IFN-γ, and an anti-proliferative signature by IFN-β. To further investigate the contribution of individual proteins towards generating the type-I (IFN-β) response, short interfering RNA (siRNA) were employed to repress the expression of selected target genes. However in macrophages and other cells equipped with pathogen detection systems the act of siRNA trasfection can itself induce a type-I interferon response. It was therefore necessary to contend with this autocrine production of IFN-β and optimise an in vitro assay for studying the contribution of siRNA induced gene-knock downs to the interferon response (described in Chapter-5). The final assay design incorporated LPS stimulation of the macrophages, as a means of inducing IFN-β autonomously of the transfection induced type-I response. However genome-wide expression analysis indicated the targeted gene knock-downs did not perturb the LPS response in macrophages on this occasion. The optimisation process underscored the complexities of performing siRNA gene knockdown studies in primary macrophages. Furthermore a more thorough understanding of the transcriptional response of macrophages to stimulation by interferon or by LPS was required. Therefore the final investigations of this thesis (Chapter-6) explore the transcriptional changes over a 24 hour time-course of macrophage activation by IFN-β, IFN-γ, or LPS and the contribution of the macrophage pathway model in interpreting the response to the three stimuli. Taken together the work described in this thesis highlight the advances to be made from a systems-based approach to visualisation, modelling and analysis of macrophage signalling.

Orientador: Erick José Ramo da Silva / Resumo: O epidídimo é responsável por transformar os espermatozoides imaturos em células funcionalmente competentes, capazes de adquirir motilidade progressiva e de reconhecer e fertilizar o oócito. Além disso, o epidídimo é primordial para proteger e armazenar o gameta masculino antes da ejaculação. Alterações da fisiologia epididimária podem afetar a sua função, causando infertilidade masculina. A inflamação do epidídimo, conhecida como epididimite, é uma das doenças mais prevalentes do trato urogenital masculino, sendo um fator relevante de infertilidade masculina. Sua principal etiologia envolve a invasão de bactérias, como Escherichia coli, via ascensão retrógrada pela uretra. O epidídimo expressa constitutivamente diversos componentes do sistema imunológico inato, incluindo os receptores do tipo Toll (TLRs), como o TLR4, que é ativado pelo lipopolissacarídeo (LPS) de bactérias Gram-negativas, além de proteínas com atividade antimicrobiana, como os inibidores da protease do tipo WFDC (Whey-acidic protein four disufide core), indicando que este órgão está em constante estado de alerta para combater infecções bacterianas. Além da ação antimicrobiana e inibidora de protease, as proteínas WFDC apresentam ação anti-inflamatória, imunomoduladora e sobre a função espermática, sendo proteínas multifuncionais no trato reprodutor masculino. Neste estudo, testamos a hipótese de que a expressão de genes Wfdc no epidídimo é modificada por estímulos inflamatórios, como parte da resposta tecid... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The epididymis is responsible for transforming immature sperm into functionally competent cells capable of acquiring progressive motility and recognizing and fertilizing the oocyte. In addition, the epididymis is paramount for protecting and storing the male gamete before ejaculation. Changes in epididymal physiology may affect sperm maturation, transport or storage, causing male infertility. Epididymal inflammation, known as epididymitis, is one of the most prevalent diseases of the male urogenital tract, being a relevant factor of male infertility. Its main etiology involves the invasion of bacteria, such as E. coli, through retrograde ascension through the urethra. The epididymis constitutively expresses several elements of the innate immune system, including Toll-like receptors (TLRs), like the TLR4, which is activated by the lipopolysaccharide (LPS) of Gram-negative bacteria, as well as proteins with antimicrobial activity, like the WFDC (Whey-acidic difulfide core)-type protease inhibitors, indicating that this organ is constantly alert to fight bacterial infections. In addition to antimicrobial and protease inhibitory activities, WFDC proteins show anti-inflammatory, immunomodulatory and reproductive functions, being multifunctional proteins in the male reproductive tract. In this study, we tested the hypothesis that Wfdc gene expression in the epididymis is modified by inflammatory stimuli as part of tissue defense mechanisms to microbial aggression. For that, we char... (Complete abstract click electronic access below) / Mestre

Endotoxin, a kind of lipopolysaccharide (LPS), is the glycolipid composition of cell walls of all Gram negative bacteria. The main biological reaction triggered by LPS is inflammation reaction. The surface epithelium of intestinal mucosa contains a large number of goblet cells which function as secreting mucus to soak chime and form a protection for digestive tract. The purpose of this experiment was to study the variations of the mucus secretion of intestinal gland goblet cells and whether dimethylthiourea (DMTU), a hydroxyl radical scavenger, could exert an inhibitory effect at different time points after LPS application. Sprague-Dawley rats received an intravenous injection of LPS (15 mg/kg, over 5 seconds) through the femoral vein. India ink was used to label the leaky microvessels where the extravasated colloidal particles of dye were trapped between the endothelium and basement membrane. 5 and 30 minutes after LPS injection, strips of intestinal tissues were dehydrated and embedded with glycol methacrylate. Tissue sections 2 micrometers in thickness were histochemically stained with Alcian blue-PAS reagent. The number of goblet cells in intestinal glands of duodenum and ileum were counted. Mucus area and epithelial area of sampled glands were recorded with SimplePCI and statistically analyzed. 5 or 30 minutes after LPS, the percent (%) of the number of discharging goblet cells in intestinal glands significantly increased to 7-8 times as the saline control in duodenum. In the ileum, the number of discharging goblet cells in intestinal glands significantly increased to 5-18 times as the saline control. 30 min after LPS, the percent (%) of goblet cell mucus in epithelial area of intestinal glands decreased to half the value of control. DMTU prior to LPS, the mucus-discharging ability of goblet cells was inhibited. In duodenum, DMTU pretreatment 30 min earlier than LPS, the percent (%) of goblet cell mucus in epithelial area of intestinal glands increased to 1.4 times and the percent (%) of the number of discharging goblet cells in intestinal glands significantly was reduced by 90%. In ileum, DMTU pretreatment 5 min earlier than LPS, the percent (%)of the number of discharging goblet cells in intestinal glands decreased to one half as the control. From the experiment results, we could come to a conclusion that intravenous injection of a high dose of LPS induced an compound exocytotic release of mucus granules resulting in cavitation of goblet cell supranuclear cytoplasm and the released mucus temporarily accumulated in the lumen of glands. It is suggested that hydroxyl radicals were involved in LPS-induced mucus release from goblet cells.

As phosphorylation represents the pivotal mechanism for regulation of biological processes, kinases belong to one of the most biologically significant enzyme classes. The development of analytical techniques for characterization of kinase activity, in particular at a global scale, is a central priority for proteomic and cell biology researchers. In order to facilitate global analysis of cellular phosphorylation, a new paradigm of microarray technology which focuses on analysis of total cellular kinase activity, kinome, has emerged in the past few years. As the specificity of many kinases is dictated primarily by recognition of residues immediately surrounding the site of phosphorylation a logical methodology is to employ peptides representing these immediate sequences as experimental substrates. Microarray chips carrying hundreds of such substrate targets have been developed for human kinome analysis, however, lack of similar tools for species outside research mainstream has limited kinome analysis in these species.<p> Based on sequence alignment of orthologous phosphoproteins from mammalian species, conservation of amino acid identity is reported to be 80 %. Accordingly, the potential exists to utilize phosphorylation sequence databases to extrapolate phosphorylation sites in other species based on their genomic sequence information. Peptides representing these proposed phosphorylation sites can then be utilized as substrates to quantify the activity of the corresponding kinase. Based on these principles, a bovine microarray of 300 unique peptide targets was constructed. The bovine phosphorylation targets were selected to represent a spectrum of cellular events but with focus on processes related to innate immunity. Initial application and validation of the bovine peptide arrays was carried out for kinome analysis of bovine blood monocytes stimulated with either lipopolysaccharide (LPS) or CpG-ODNs; ligands for Toll-like receptors (TLR) 4 and 9, respectively. The arrays confirmed activation of the known TLR signaling pathway as well as identifying receptor-specific phosphorylation events. Phosphorylation events not previously attributed to TLR activation were also identified and validated by independent bioassays. This investigation offers insight into the complexity of TLR signaling and more importantly verifies the potential to use bioinformatics approaches to create tools for species-specific kinome analysis based on genomic information.

As phosphorylation represents the pivotal mechanism for regulation of biological processes, kinases belong to one of the most biologically significant enzyme classes. The development of analytical techniques for characterization of kinase activity, in particular at a global scale, is a central priority for proteomic and cell biology researchers. In order to facilitate global analysis of cellular phosphorylation, a new paradigm of microarray technology which focuses on analysis of total cellular kinase activity, kinome, has emerged in the past few years. As the specificity of many kinases is dictated primarily by recognition of residues immediately surrounding the site of phosphorylation a logical methodology is to employ peptides representing these immediate sequences as experimental substrates. Microarray chips carrying hundreds of such substrate targets have been developed for human kinome analysis, however, lack of similar tools for species outside research mainstream has limited kinome analysis in these species.<p> Based on sequence alignment of orthologous phosphoproteins from mammalian species, conservation of amino acid identity is reported to be 80 %. Accordingly, the potential exists to utilize phosphorylation sequence databases to extrapolate phosphorylation sites in other species based on their genomic sequence information. Peptides representing these proposed phosphorylation sites can then be utilized as substrates to quantify the activity of the corresponding kinase. Based on these principles, a bovine microarray of 300 unique peptide targets was constructed. The bovine phosphorylation targets were selected to represent a spectrum of cellular events but with focus on processes related to innate immunity. Initial application and validation of the bovine peptide arrays was carried out for kinome analysis of bovine blood monocytes stimulated with either lipopolysaccharide (LPS) or CpG-ODNs; ligands for Toll-like receptors (TLR) 4 and 9, respectively. The arrays confirmed activation of the known TLR signaling pathway as well as identifying receptor-specific phosphorylation events. Phosphorylation events not previously attributed to TLR activation were also identified and validated by independent bioassays. This investigation offers insight into the complexity of TLR signaling and more importantly verifies the potential to use bioinformatics approaches to create tools for species-specific kinome analysis based on genomic information.

Infectious agents such as bacteria or viruses can grow rapidly. If a microorganism invades a host, it must be recognized rapidly and destroyed before it overwhelms the immune system. Limiting infection to a minimum in the early stage is critical for the outcome and the recovery from infection. The innate immune system has evolved to recognize a few highly conserved, constitutive structures present only in microorganisms, such as bacterial lipopolysaccharide (LPS), called pathogen-associated molecular patterns (PAMP). Toll-like receptors are the host receptors that recognize PAMP, ultimately activating a variety of transcription factors to induce expression of a wide spectrum of immune related genes, e.g. defensins. Defensins are antimicrobial peptides that play an important role in innate defense against microorganisms in plants and animals. Beta-defensins are the largest family of antimicrobial peptides, which can directly kill microorganisms and have regulatory effects on the immune system. Thirteen beta-defensins have been identified; however, the regulation of these genes has not been well-investigated in the chicken. The objective of this research was to understand constitutive and inducible gene expression of beta-defensins in chicken white blood cells. Real-time RT-PCR was used to quantify gene expression level before and after LPS stimulation. Transcription factor binding sites in the genes were identified to understand the gene expression regulation. From the expression profile results, most chicken beta-defensins had induced gene expression by LPS stimulation in the early phase (0- to 3-hour) and reduced gene expression in the late phase (3- to 8-hour). As for the level of gene expression, the results show that the induced gene expression in the early phase corresponded to the higher levels of expression at 3-hours after LPS stimulation, and the reduced gene expression in the late phase corresponded to the lower levels of gene expression at 8-hours after LPS stimulation.