Francisella tularensis is the etiological agent of tularemia, a severe and potentially fatal disease in humans. It is extremely infectious by the aerosol route, being thought to cause disease in humans with an infectious dose as small as one to ten organisms, which led to its weaponization by several nations and classification as a category A select agent by the Centers for Disease Control and Prevention. An intracellular pathogen, relatively little is known about the mechanisms by which Francisella is capable of successfully modulating host cell processes to escape its phagosome and replicate within the cytosol and what genes beyond the Francisella pathogenicity island are required. Furthermore, in the context of aerosol exposure, it is unknown what cells F. tularensis initially interacts with and the overall contribution of those interactions to inhalational tularemia. I initiated this study by generating an in vitro model system to study interactions of F. tularensis with epithelial cell lines in tissue culture. Utilizing this system, I determined that F. tularensis LVS was capable of adherence to human epithelial cell lines of alveolar (A549), bronchial airway (HBE), and cervical carcinoma (HEp-2) origin. Furthermore, LVS was capable of invading these cell lines and growing productively within them. In order to detect genes important for virulence in this system, I generated a ~15,000 member transposon library in virulent strain Schu S4 that was could be screened in a high-throughput manner by transposon site hybridization. As uptake in the in vitro epithelial cell line system was relatively inefficient, I screened this library through human primary macrophages. Results of the screen implicated 207 genes as negatively selected in the human macrophage model. Of these, I generated mutants in genes residing in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238, to determine their virulence phenotypes. Mutants in these genes demonstrated significant vulnerability to complement-mediated lysis as compared with wild type Schu S4. Analysis of purified LPS and capsule from these mutants further showed that they had marked defects in O-antigen and capsular polysaccharide biosynthesis. Complementation of these mutants restored surface polysaccharide biosynthesis and further determined that FTT1236 and FTT1237 compose an operon, as a mutation in FTT1236 is polar onto FTT1237. Characterization of the intracellular defect of these mutants in the absence of active complement demonstrated that they were taken up more efficiently by primary human macrophages than wild type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic analysis of macrophages infected with mutant bacteria revealed that, as early as 16 hpi, these macrophages exhibited signs of cell death. In contrast, cells infected with Schu S4 exhibited a healthy, spread morphology as late as 32 h, despite significantly more extensive F. tularensis cytosolic replication. Quantitation of cell death by the release of lactate dehydrogenase, signifying membrane permeability, confirmed that mutants in FTT1236, FTT1237, and FTT1238 induced early cell death in infected macrophages as compared with wild type Schu S4. Together, this work contributes to our understanding of the factors, such as O-antigen and capsule, required for and genes involved in Francisella's lifecycle as an intracellular pathogen.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration.

Human colonic epithelial cell lines (T84, Caco2 and HT-29) were used to address the question of whether intestinal epithelial cells can detect and respond to activated complement via the anaphylatoxin receptors, considering the gut is host to large numbers of bacteria. All cell lines possess C3aR, C5aR and C5L2. Confocal microscopy confirmed that cells express apical C5aR and C5L2. C3a and C5a up-regulated CXCL8 and CXCL10 mRNA but not secreted protein levels within 48 hours. Protein levels were not increased using simultaneous treatment with subthreshold concentrations of LPS or TNF plus anaphylatoxin. C3a and C5a also increased the permeability of polarized monolayers. Anaphylatoxins also promoted the proliferation of T84 and HT-29. Inhibition of ERK signaling abolished these effects of anaphylatoxins. Our findings that multiple human cell lines possess functional anaphylatoxin receptors indicates that the colonic epithelium likely responds to the activation of complement in the lumen with an inflammatory outcome.

Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. At present, there are an estimated one million people in Canada with some form of AMD and this number is expected to double to two million by 2031. These estimates are sobering, and it is predicted that costs for treatment and care of individuals who suffer vision loss from AMD will have significant impact on the social and public health systems in Canada in the next two decades. There are treatments to slow the progression of vision loss, but unfortunately, there are currently no cures available for AMD. In order to develop effective second generation therapies and cures, further insights into how and why AMD develops are greatly needed. Recent studies have provided novel insights into the role of inflammation in the pathogenesis of AMD. Inflammation, or swelling of the retinal tissues, causes harmful processes that promote macular degeneration. The proposed studies will focus on the triggers of inflammation in the retina. It is hypothesized that macular degeneration may be slowed or stopped by eliminating the molecules that cause inflammation in the retina. This study will focus on amyloid beta (Aβ), a toxic molecule that has been implicated in retinal inflammation, and the role that it may play in gene expression of the retinal pigment epithelial cell. Amyloid beta is a well studied peptide in another age related disorder, Alzheimer’s disease. It is the major extracellular deposit in Alzheimer’s disease plaques, and has recently been discovered as a component of drusen, the hallmark extracellular deposits in the retina of patients with the ‘dry’ form of AMD. These studies will allow the development of new treatment regimens that target retinal inflammation and thus minimize the processes that ‘trigger’ the onset of macular degeneration. / Medicine, Faculty of / Graduate

Aspergillus fumigatus is a filamentous fungus and the main pathogen responsible for the often fatal respiratory condition, aspergillosis. Airway epithelial cells (AECs) are likely to be the first line of host defence that come into contact with the inhaled conidia of A. fumigatus. Recent evidence strongly suggests that the response of the airway epithelium to inhaled pathogens is pivotal in orchestrating immune responses by inducing phagocytic-like reactions and the secretion of inflammatory cytokines and antimicrobial peptides. However, the majority of previous work investigating A. fumigatus-host interactions has been performed using macrophages and neutrophils, thereby neglecting the epithelium. AECs have been shown to secrete inflammatory cytokines in response to A. fumigatus although these studies predominantly used transformed AEC lines that lack tight junctions and do not fully differentiate. Furthermore, most studies used culture filtrate or extract of A. fumigatus rather than live, whole organism and as a result, the direct interaction of the germinating fungus and the airway epithelium has been overlooked. During the early germination and growth period, the cell wall composition of A. fumigatus is dynamic, with various antigens exposed at different morphological stages. The aim of this thesis was to determine whether AECsare able to alter the germination and growth rate of A. fumigatus, and, conversely, if A. fumigatus affects AECs in terms of the secretion of inflammatory mediators. These studies used live, germinating A. fumigatus, and human primary differentiated AECs to obtain a more realistic in vitro model than those used in previous studies. Data showed that AECs are able to significantly inhibit the germination and growth of A. fumigatus, although this effect was less pronounced in differentiated primary AEC than in transformed AEC lines. A. fumigatus also significantly inducedthe expression and secretion of the inflammatory cytokines, IL-6 and IL-8, probably via the interaction of fungal cell wall β-glucans, and as of yet unidentified AEC receptor. The A1160pyrG+ strain of A. fumigatus secreted factors capable of inducing cytokine secretion whereas Af293 strain did not, highlighting diverse mechanisms of action for different strains. Upregulation of both cytokines was dependent on the stage of A. fumigatus growth with induction synchronous with germination. Despite being associated with fungal sensitisation in asthmatics, AEC-derived cytokines associated with this disease, namely TSLP, IL33 and IL25,did not appear to be upregulated by transformed AECs in response to A. fumigatus. Similarly, A. fumigatus did not seem to induce synthesis and secretion of the acute phase response protein, fibrinogen above baseline levels. The data presented in this thesis confirms the importance of the airway epithelium in directing anti-A. fumigatus immunity and the involvement of complex ligand-receptor interactions.

The choroid plexuses are largely responsible for cerebrospinal fluid (CSF) secretion and therefore play a fundamental role in brain homeostasis. The membrane proteins involved in CSF secretion are not fully known. Several electroneutral transporters have been identified by molecular methods in choroid plexus epithelial cells but there is a lack of functional data to support their expression making it impossible to elucidate their role in CSF secretion fully. The activity of many of these transporters can be observed in cell volume regulation. Thus, the main aim of the present study was to determine the ability of mammalian choroid plexus epithelial cells to regulate their volume in response to anisosmotic challenge and to investigate the transporters involved.Experiments were performed on cells isolated from the mouse fourth ventricle choroid plexus. Cells were isolated using a combination of manual perturbation, the enzyme dispase and a Ca2+ free incubation to disrupt tight junctions. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pL.Cells exhibited a HCO3- dependent regulatory volume increase (RVI) in response to hypertonic challenge. Strong evidence is presented that the Na+/H+ exchanger (NHE1) and the Cl-/HCO3- exchanger (AE2) contribute to the RVI but the Na+K+2Cl- cotransporter (NKCC1) and the epithelial Na+ channel (ENaC) do not. Choroid plexus cells exhibit a HCO3- dependent regulatory volume decrease (RVD) in response to hypotonic challenge. The RVD was unaffected by DIOA (an inhibitor of KCC activity), the K+ channel inhibitors TEA+, Ba2+ or 4AP or the Cl- channel inhibitors DIDS or NPPB. However removal of extracellular Ca2+ completely abolished cell swelling in response to hypotonic challenge. This sensitivity of volume change to Ca2+ was specific to cell swelling as cell shrinkage in hypertonic artificial CSF was unaffected by removal of extracellular Ca2+.Thus functional evidence is presented to further elucidate the role of several proteins in the choroid plexus cell volume regulatory response to anisosmotic challenge.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22004号 / 医博第4518号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 中川 一路, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM