Philosophiae Doctor - PhD / The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

La grande majorité des bactériophages connus ont un virion équipé d'une queue permettant la reconnaissance de l'hôte, la perforation de l'enveloppe bactérienne et l'éjection du matériel génétique viral directement dans le cytoplasme de la bactérie. La famille des Siphoviridae représente 60% des phages caudés et est caractérisée par une queue longue et non-contractile. Le tube de la queue est formé par un empilement de protéine majeur de tube (TTP) polymérisé autour de la protéine vernier (TMP). L'extrémité distale de la queue est équipé d'un complexe de protéine dans lequel se trouve les protéines de liaison au récepteur (RBP). La séquence d'évènement permettant l'éjection de l'ADN et l'infection est encore mal décrite.Au cours de cette thèse, la structure de pb6, la TTP du phage T5 qui s'assemble de façon non-canonique en trimères, a été résolue par cristallographie à une résolution de 2,2 Å. L'analyse de cette structure confirme cependant une homologie structurale de pb6 avec les autres TTPs et avec des protéines bactériennes du système de sécrétion de type VI et des pyocines R. Une étude RMN comparant pb6 dans ses états de monomère et de tube polymérisé est en cours et permettra à terme une description très fine de cet assemblage.De plus, les structures du complexe distal de queue et du tube de la queue (tube de pb6) ont été résolue des résolutions intermédiaires avant et après interaction avec le récepteur bactérien. Ces structures obtenues par cryo-microscopie électronique révèle une absence de changements structuraux au niveau du tube, en contradiction avec le modèle jusque là proposé que la TTP transmettait l'information de fixation du récepteur à la capside.Bien qu'à un stade préliminaire, les reconstructions du complexe distal sont très informatives sur les rôles de protéines pb2 et pb4.L'ensemble de ses données ainsi que des expériences biochimique et la comparaison avec d'autres systèmes bien décrits dans la littérature permet de proposer un nouveau modèle pour les premières étapes de l'infection des Siphoviridae. Ce modèle a également un intérêt pour l'étude du mécanisme d'autres familles de virus (Myoviridae). Les différences, similarité et parenté d'éléments de la queue du phage T5 avec d'autres systèmes de perforation de membrane sont discutés. / The vast majority (96%) of bacteriophages possess a tail that allows host cell recognition, cell wall perforation and safe viral DNA channelling from the capsid to the cytoplasm of the bacterium. Siphoviridae is a familly representing 60% of all tailed phages characterized by a long flexible tail. The tail tube is formed by stacks of hexamers of the tail tube protein (TTP) polymerised around the tape measure protein (TMP). At the distal end of the tail, the tail tip complex harbours the receptor binding proteins (RBP). For these phages, little is known on the mechanism that triggers DNA ejection after binding to the host.We report the crystal structure at 2.2 Å resolution of pb6, an unusual trimeric TTP, of siphophage T5. Structure analysis however confirms the homology of pb6 with all TTPs, related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin) and procapsid proteases. We fit this structure into the cryo-electron microscopy map of the tail tube determined at 6 Å resolution. Comparing the structure of the tail tube before and after interaction with the host receptor, we show that unlike previously proposed, the host binding information is not propagated to the capsid by the tail tube, as the two structures, at that resolution, are identical. An ambitious NMR comparative study of the TTP in its monomeric and tube form is underway to further describe this assembly.Moreover, the structures of the tail tip complex prior and after interaction with the bacterial receptor were solved at intermediate resolution. These structures reveal interesting conformationnal changes triggered by the RBP binding to the bacterial receptor. Those rearrangements are the first to occur after phage irreversible binding to its host and they induce the TMP ejection, the capsid opening, the enveloppe perforation and ultimately DNA channeling to the host cytoplasm.Together with biochemical data and comparison with other known system in the litterature we are able to propose a model for Siphoviridae very first steps of infection. These findings might be of interest for the mechanism of other viral familly (notably Myoviridae) and similarity with other membrane perforating systems is discussed.

Photoemission electron microscopy (PEEM) uses photoelectrons excited from material surfaces by incident photons to probe the interaction of light with surfaces with nanometer-scale resolution. The point resolution of PEEM images is strongly limited by spherical and chromatic aberration. Image aberrations primarily originate from the acceleration of photoelectrons and imaging with the objective lens and vary strongly in magnitude with specimen emission characteristics. Spherical and chromatic aberration can be corrected with an electrostatic mirror, and here I develop a triode mirror with hyperbolic geometry that has two adjacent, field-adjustable regions. I present analytic and numerical models of the mirror and show that the optical properties agree to within a few percent. When this mirror is coupled with an electron lens, it can provide a large dynamic range of correction and the coefficients of spherical and chromatic aberration can be varied independently. I report on efforts to realize a triode mirror corrector, including design, characterization, and alignment in our microscope at Portland State University (PSU). PEEM may be used to investigate optically active nanostructures, and we show that photoelectron emission yields can be identified with diffraction, surface plasmons, and dielectric waveguiding. Furthermore, we find that photoelectron micrographs of nanostructured metal and dielectric structures correlate with electromagnetic field calculations. We conclude that photoemission is highly spatially sensitive to the electromagnetic field intensity, allowing the direct visualization of the interaction of light with material surfaces at nanometer scales and over a wide range of incident light frequencies.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19180号 / 人健博第28号 / 新制||人健||3(附属図書館) / 32172 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 市橋 則明, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright ©American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jacs.5b10162. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19611号 / 医博第4118号 / 新制||医||1015(附属図書館) / 32647 / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 渡邊 直樹, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Previous findings showed that Listeria monocytogenes exhibits higher heat tolerance to thermal exposure at 60°C when pre-exposed to sublethal heat stress at 48°C for 30/60 min. The objective of this study was to investigate the morphological changes that occurred in L. monocytogenes serotype 1/2a cells as visualized by transmission electron microscopy (TEM) after exposure to sublethal heat stress at 48°C for 60 min and in combination with lethal concentration of carvacrol for 30 min. The TEM micrographs revealed thickening of cell wall and cell membrane, and clumping of cytoplasm when subjected to sublethal heat stress followed by carvacrol treatment. These studies indicate that L. monocytogenes cells when adapted to heat stress may alter its morphology to protect themselves against carvacrol. Further studies will investigate the effect of increased contact time with carvacrol and with other essential oils on the ultrastructural changes in L. monocytogenes cells adapted to heat stress.

The Wilcox Group includes the most abundant lignite bearing strata in Mississippi. Currently, the only lignite mine in Mississippi is located in Choctaw County with a proposed mine in Kemper County. Six lignite seams are currently mined in Choctaw County. One of those seams is believed to be continuous, from SE Choctaw County through central Winston County, NE Neshoba County into SW Kemper County (roughly 50 miles). Establishing a cross section from Choctaw to Kemper County verifying a continuous lignite seam would provide a stratigraphic marker horizon in correlation along strike, which is uncommon in the Wilcox Group sediments. Through observation of over 60 geophysical logs from test wells along strike, SEM and petrographic microscopy, the lignite seam was verified as being continuous from the mine in Choctaw County to the new proposed site in Kemper County and should be used as stratigraphic marker horizon for correlation along strike.