Purpose. In the genetically defective Royal College of Surgeons (RCS) rat model for retinal degeneration, a breakdown occurs in the retinal pigment epithelial (RPE) cell tight junctions just as the photoreceptors begin to degenerate. These experiments sought to determine the impact of the RPE genetic defect on this alteration in the RPE cell tight junctions. Methods. Retinal pigment epithelial cell cultures prepared from RCS and control rats were treated with hormonally defined medium (HDM), base medium conditioned by RCS or control retinas, or unconditioned base medium. The tight junctions formed by these cultures were assayed functionally by measuring transepithelial electrical resistance and permeability. Junction structure was evaluated by immunolocalization of the tight junction protein zonula occludens I and of the junction-associated actin microfilaments. Results. Retinal pigment epithelial cultures from dystrophic rats formed structurally and functionally normal tight junctions when maintained in hormonally defined medium. The junctions remained stable when the medium bathing the apical surface was switched to base medium preconditioned by normal retinas. In contrast, cultures treated with medium preconditioned by degenerating dystrophic retinas or with unconditioned medium exhibited a breakdown in their tight junctions. Conclusions. Retinal pigment epithelial cells isolated from dystrophic RCS rats can form tight junctions normally in vitro. Normal, but not dystrophic, retinas release factors that support RPE tight junctions. Therefore, the junctional abnormality seen in dystrophic rat RPE cells in vivo is probably caused by the loss of trophic factors normally provided by the healthy neural retina rather than by a direct effect of the genetic defect on the tight junctions.

<p>In sum, these studies described in this dissertation showed that both mycotoxin deoxynivalenol exposure and nutrient starvation can increase endocytosis and degradation of tight junction proteins in the lysosome. Therefore, manipulation of endocytic pathway as well as the signaling pathway involved in this process can be used to prevent intestinal barrier dysfunction in animals.</p> <p><br></p>

No description available.

No description available.

No description available.

The small intestine and yolk sac (YS) are important organs for nutrient absorption and innate immunity in chickens during the post-hatch or prehatch periods. These organs share a similar structure of epithelial cell-lined villi with tight junctions between adjacent cells. Probiotics have been reported to improve chicken growth performance and gut health including promotion of intestinal morphology. However, there are few studies that show the effect of probiotics on ontogeny of intestinal epithelial cells and antimicrobial peptides, or intestinal integrity in young healthy chicks. Heat stress during incubation was shown to increase mortality and decrease hatchability of chicks, while no studies have investigated the effect of heat stress on the integrity of the YS, which might be related to hatching performance. There were four studies conducted in this research: 1) a comparison of the effect of two probiotics on the ontogeny of small intestinal epithelial cells in young chicks; 2) the effect of two probiotics on mRNA abundance of tight junction proteins in the small intestine of young chicks; 3) the effect of high incubation temperature on mRNA abundance of tight junction proteins in the YS of broiler embryos; and 4) comparison of avian defense peptide mRNA abundance in the YS of broilers and layers. In study 1, Probiotics transiently decreased body weight gain (BWG) from day 2 to day 4, but did not affect body weight (BW) from day 2 to day 8, and small intestinal weight and intestinal morphology from day 2 to day 6. Probiotics did not affect marker gene expression of intestinal stem cells (Olfm4) and goblet cells (Muc2) in all small intestinal segments, but did increase expression of a marker gene of proliferating cells (Ki67), and decreased an antimicrobial peptide (liver-enriched antimicrobial peptide 2, LEAP2) in the jejunum at day 4. Probiotic 1 decreased PepT1, a marker of enterocytes in the duodenum at day 4. These results suggest that probiotics did not improve growth performance and intestinal morphology in young healthy chicks, but temporarily promoted intestinal epithelial cell proliferation and decreased LEAP2 antimicrobial peptide expression in the jejunum. In situ hybridization (ISH) showed that Ki67+ proliferating cells were mainly located in the crypt region and the blood vessels of villi. In study 2, Probiotic supplementation to newly hatched chicks for less than one week did not affect mRNA abundance of the tight junction proteins in the small intestine. Occludin (OCLN) mRNA, which was detected by ISH to be expressed in intestinal epithelial cells in both the villus and crypt regions, was greater in the duodenum of female chicks than males. In study 3, high incubation temperature starting from embryonic day 12 (E12) affected mRNA abundance of the tight junction proteins in the YS, including increased zonula occluden 1 (ZO1) at E13, increased junctional adhesion molecule A (JAMA) and heat shock protein 90 (HSP90) at E17, but decreased tight junction protein JAMA at E19 and OCLN at day of hatch (DOH). These results showed that the YS tight junction proteins were increased by short term heat exposure but decreased by long term heat exposure. In study 4, the expression of avian β defensin 10 (AvBD10), CATHs and toll-like receptors in the YS was examined. Toll-like receptors were highly expressed in the YS at early incubation stages (E7), while CATHs showed a peak expression from E9 to E13, which was similar to the expression pattern of AvBD10. CATHs and AvBD10 mRNA temporal expression patterns were similar in broilers and layers, while their expression levels were different. Layers, especially brown layers, had greater mRNA abundance for antimicrobial peptides such as AvBD10, CATH1, and CATH2 in the YS. These results demonstrate that the antimicrobial peptide temporal expression patterns in the YS are not affected by breed, but their expression levels are affected by breed. In summary, the small intestine and the YS are essential for nutrient uptake, innate immunity, and maintenance of integrity. The ontogeny of intestinal epithelial cells, such as proliferating cells can be modulated by probiotic supplementation. Similar to the small intestine, the YS can also express tight junction proteins, which can be affected by high incubation temperature. Antimicrobial peptide expression in the intestine of healthy young chicks is also transiently decreased by probiotic supplements. Avian defensin and cathelicidin expression patterns in the YS were not affected by breed. / Doctor of Philosophy / The small intestine and yolk sac are important organs for nutrient absorption in hatched chicks or embryonic chicks. These organs also serve as a barrier to prevent pathogens from entering the blood circulation. Intestinal epithelial cells along the villi renew rapidly by proliferation and differentiation. In this research, probiotics which are also known as direct fed microbials temporarily increased expression of the proliferating cell marker Ki67 in the jejunum of healthy young chicks, which suggests that probiotics promote intestinal epithelial cell proliferation. However, probiotics transiently decreased expression of an antimicrobial peptide, which may reduce immune protection in the gut. The yolk sac can also express tight junction proteins. The expression of tight junction proteins was affected by elevated incubation temperature in broiler embryos, which might be related to low hatchability of eggs exposed to heat stress. Avian defense peptides and pathogen recognition receptors were expressed in the YS, which implied that the yolk sac contained an innate immune function. The expression pattern of avian defense peptides was affected by breed (broilers and layers), while the expression level of avian defense peptides was greater in layers than broilers. In summary, the small intestine and the yolk sac are multifunctional organs. Their cell composition, structural integrity, and secretion of antimicrobial peptides can be affected by environmental factors, such as probiotic supplementation or high incubation temperature.

Curdlan, a (1,3)-linked linear homopolysaccharide composed of beta-D-glucan, is produced by the bacterium Alcaligenes faecalis var. myxogenes. Several strategies to synthesize chemically modified curdlan derivatives have been reported, but there have been few reports of regioselective functionalization at specific positions of the curdlan backbone, especially of aminated curdlan derivatives which have remarkable potential in biomedical and pharmaceutical applications. We demonstrate herein the design, synthesis and characterization of a family of regioselectively aminated curdlan derivatives including 6-deoxy-6-(bromo/azido/amino/amido/ammonium) curdlans starting from 6-bromo/azido-6-deoxycurdlan. A key reaction that enabled the whole synthesis of new curdlan derivatives at C-6 described in this dissertation was the highly selective bromination of curdlan. The resultant 6-bromo-6-deoxycurdlan, prepared with high regioselectivity, was treated with trialkylamines or heterocyclic amines to produce a range of water-soluble curdlan ammonium salts. The bromide was then nucleophilically displaced by sodium azide to produce the versatile precursor 6-azido-6-deoxycurdlan. Its water solubility was enhanced either by the incorporation of hydrophilic trioxadecanoate esters into O-2/4 positions or by the borohydride reduction to afford 6-amino-6-deoxycurdlan. The iminophosphorane intermediate generated during Staudinger reactions was further investigated for subsequent syntheses: i) 6-amino or 6-amido-6-deoxycurdlan by in situ reaction with water or excess carboxylic anhydride, ii) 6-monoalkylamino curdlan by reductive amination using aldehydes and sodium cyanoborohydride, and iii) 6-dialkylamino-/tri-alkylammoniocurdlans by reacting with methyl iodide. Such derivatives could have properties useful for a range of biomedical applications, including interactions with proteins, encapsulation of drugs, and formulation with genes or other biological compounds. / Ph. D.

Die Tight Junction (TJ) reguliert den parazellulären Transport von Ionen, Wasser und Soluten an Epithelien und Endothelien und ist von entscheidender Bedeutung für die Aufrechterhaltung der Funktion von Organen und Geweben. Obwohl Claudin-3 zu den zuerst identifizierten und ubiquitär exprimierten Komponenten der TJ gehört, konnte seine spezifische Funktion bislang nicht geklärt werden. Für die funktionelle Charakterisierung des humanen Claudin-3-Proteins wurden stabile Überexpressionsklone der lecken Nierenepithel-Zelllinie MDCK II generiert. Die Überexpression von Claudin-3 führte zu einer deutlichen Änderung des TJ-Strangmusters sowie zu einer starken Zunahme des transepithelialen Widerstandes und einer verminderten Permeabilität für Ionen und Moleküle der Größe 332 Da und 4000 Da. Der parazelluläre Durchtritt von Wasser war unverändert. Claudin-3 konnte eindeutig als abdichtende Komponente der TJ identifiziert werden. Anhand des endothelialen Zellkulturmodells HUVEC wurden Expression und Regulation von Claudin-3 und anderen TJ-Proteinen unter dem Einfluss mechanischer Strömungsverhältnisse und des Sauerstoffpartialdrucks analysiert. Die Behandlung mit fehlender Wandschubspannung führte zur Hochregulation der abdichtenden TJ-Proteine Occludin, Claudin-3, Claudin-5 und Claudin-11, nicht aber Claudin-23. Die Regulation der einzelnen TJ-Komponenten wurde durch unterschiedliche Signalwege vermittelt, wobei der verstärkten Proteinexpression jeweils eine Hochregulation auf mRNA-Ebene zugrunde lag. Die kombinierte Behandlung mit fehlender Wandschubspannung und Hypoxie resultierte in einer sehr stark erhöhten Expression von Claudin-3. Durch die Hochregulation abdichtender TJ-Komponenten unter Bedingungen fehlender Wandschubspannung und Hypoxie, wie sie in verschiedenen physiologischen und pathologischen Situationen auftreten, könnte einem unerwünschten Durchtritt von Substanzen aus dem Blut in das umliegende Gewebe vorgebeugt werden. / The tight junction (TJ) regulates the paracellular transport of ions, water and solutes in epithelia and endothelia and is of particular importance for a correct function of organs and tissues. Although claudin-3 is one of the first identified and ubiquitously expressed TJ components, its specific function was unsolved as yet. For functional characterization, human claudin-3 was stably overexpressed in the leaky epithelial cell line MDCK II. Overexpression of claudin-3 led to a marked alteration of TJ meshwork pattern, a strong increase in transepithelial resistance and a decrease in permeability for ions and paracellular tracers (332 or 4000 Da). Paracellular water transport was not affected. It was proved that claudin-3 acts as a „tightening“ TJ component. The endothelial cell culture model HUVEC was used for analysis of expression and regulation of claudin-3 and several other TJ proteins under different conditions of wall shear stress and oxygen saturation. Treatment with lacking wall shear stress led to an upregulation of the “tightening” TJ proteins occludin, claudin-3, claudin-5, and claudin-11, but not claudin-23. Upregulation of all proteins was due to increased mRNA levels. Apparently, different signaling pathways were involved in regulation of particular TJ components. Combined treatment with lacking shear stress and hypoxia resulted in drastically increased claudin-3 expression. Upregulation of tightening TJ components under lacking shear stress and hypoxic conditions as occuring in different physiological or pathological situations would limit the passage of solutes from the blood into the surrounding tissue.

Die tight junction schränken die Diffusion durch den parazellulären Raum in Epithel- und Endothelzellschichten für viele Moleküle stark ein. Dadurch behindern sie die Aufnahme von wasserlöslichen Medikamenten in das dahinterliegende Gewebe. Zwei Proteine, die am tight junction Aufbau mitwirken, sind Zonula Occludens Protein 1 (ZO-1) und Occludin. Eine Öffnung der tight junctions stellt eine Möglichkeit für die Verabreichung von Medikamenten dar. Deshalb wurden die tight junction Proteine ZO-1 und Occludin auf ihre Funktion, Struktur und Regulation untersucht. Für die Interaktion beider Proteine gab es ein Modell, welches eine Oligomerisierung der Bindungspartner als Voraussetzung ihrer Interaktion über helikale Wechselwirkungen vorhersagte. Die Annahmen aus dem Modell der Interaktion von ZO-1 und Occludin konnten experimentell bestätigt werden. Für den C-Terminus von Occludin wurde darüber hinaus eine Interaktion über Disulfidbrücken nachgewiesen. Diese Interaktion könnte in der Zelle von pathologischer Bedeutung bei Schlaganfall und Ischchämie sein. Beide Erkrankungen verursachen eine Öffnung der tight junction im Zusammenhang mit oxidativem Stress. ZO-1 bindet über PDZ Domänen eine Vielzahl von tight junction Proteinen, die an der Abdichtung des parazellulären Raums beteiligt sind. Deshalb wurde die Interaktion und Regulation der PDZ-Domänen aus ZO-1 untersucht. Eine Phosphorylierung der PDZ durch die Proteinkinase C alpha sowie eine Interaktion mit den Phosphatasen 2A und 4 konnte nachgewiesen werden. In vitro konnte gezeigt werden, dass die Phosphorylierung der PDZ-Domänen die Bindung an Membranproteine der tight junction beeinflusst. Diese Arbeit leistet einen Beitrag, die Mechanismen, die zum Verschluss des parazellulären Spaltes führen, aufzuklären. Damit zeigt sie Ansatzpunkte für eine pharmakologische Beeinflussung der Permeabilität der tight junction auf. / Tight junctions restrict diffusion through the paracellular gap in endothelia and epithelia. Thereby they constrain the uptake of water soluble drugs to the tissue. Zonula occludens protein 1 (ZO-1) and occludin are some of proteins involved in tight junction assembly. The opening of tight junctions is a possibility to apply drugs. Therefore the structure, function and regulation of ZO-1 and occludin is characterised. In previous studies, a model predicted the interaction of occludin and ZO-1 through helices. It was proposed that the interaction is mediated by oligomers of ZO-1 and Occludin. This author´s experimental research supports these hypotheses. Furthermore, occludin is shown to self assemble via disulfide bridges. This interaction could be of importance during stroke and ischemia. Both diseases cause the opening of tight junctions in combination with oxidative stress. In addition, this author investigated the interaction and regulation of the PDZ domains of ZO-1. It was shown that the PDZ domains are phosphorylated by protein kinase C alpha and interact with protein phosphatases 2A and 4. Phosphorylation led to a reduction in affinity of PDZ to membrane proteins in vitro. This thesis contributes to the understanding of the mechanisms which are involved in the sealing of the paracellular gap.

<p>Our knowledge of the fine structure of the Human Spiral Ganglion (HSG) is still inadequate and new treatment techniques for deafness using electric stimulation, call for further information and studies on the neuronal elements of the human cochlea. This thesis presents results of analyses of human cochlear tissue and specimens obtained during neurosurgical transpetrosal removal of life-threatening meningeomas. The use of surgical biopsies produced a well-preserved material suitable for ultrastructural and immunohistochemical studies on the human inner ear. The SG was studied with respect to fine structure, using TEM technique and the immunostaining pattern of synaptophysin, which is an integral membrane protein of neuronal synaptic vesicles. The immunostaining patterns of the tight junctional protein ZO-1 and the gap junctional proteins Cx26 and Cx43 in the human cochlea were also studied. The ultrastructural morphology revealed an absence of myelination pattern in the HSG, thus differing from that in other species. Furthermore, formation of structural units as well as signs of neural interaction between the type 1 neurons could be observed. Type 1 cells were tightly packed in clusters, sharing the ensheathment of Schwann cells. The cells frequently made direct physical contact in Schwann cell gaps in which membrane specializations appeared. These specializations consisted of symmetrically or asymmetrically distributed filamentous densities along the apposed cell membranes. The same structures were also present between individual unmyelinated nerve fibres and the type 1 cells. Synapses were observed on the type 2 neurons, with nerve fibres originating from the intraganglionic spiral bundle. Such synapses, though rare, were also observed on the type 1 cells. The ultrastructural findings were confirmed by the synaptophysin study. A 3-D model of a Schwann cell gap between two type 1 cells was constructed, describing the distribution pattern of membrane specializations. In the immunohistochemical studies on the human cochlea, ZO-1 was expressed in tissues lining scala media, thus contributing to the formation of a closed compartment system, important for the maintenance of the specific ionic composition of the endolymph. Protein Cx26 could be identified in non-sensory epithelial cells of the organ of Corti, in connective tissue cells of the spiral ligament and spiral limbus, as well as in the basal and intermediate cell layers of stria vascularis. Cx26 in this region may be involved in the recycling of potassium. Protein Cx43 stained weakly in the spiral ligament, but intense staining in the SG may indicate that gap junctions exist between these neurons. A different functional role for the HSG can be assumed from the morphological characteristics and the signs of a neural interaction between the SG cells. This might be relevant for neural processing mechanisms in speech coding and could have implications for cochlear implant techniques.</p>