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Entwicklung und Einsatz eines In-vitro-Ischämiemodels zur Untersuchung zellulärer Pathomechanismen der Klauenrehe des Rindes: Development and experimental application of an in vitro ischemia model for investigating the cellular pathomechanism of laminitis in cattleLübbe, Katharina 05 May 2015 (has links)
Die subklinische Klauenrehe oder claw horn disruption (CHD) ist von großer wirtschaftlicher Bedeutung für die Rinderhaltung, da sie zu Lahmheiten, Beeinträchtigungen des Allgemeinbefindens sowie einer eingeschränkten Leistungsfähigkeit der Tiere führt. Trotz zahlreicher Untersuchungen sind die pathophysiologischen Grundlagen der CHD noch immer nicht vollständig geklärt. Die derzeitigen Hypothesen weisen auf eine Ischämie in den noch lebensfähigen Epidermisschichten infolge einer veränderten dermalen Mikrozirkulation. Diese hat pathophysiologische Veränderungen zur Folge, die eine Störung der epidermalen Zellproliferation, eine Schädigung der dermo-epidermalen Verbindung sowie eine veränderte Keratinisierung und Hornproduktion umfassen. Von Bedeutung sind daher In-vitro-Ischämiemodelle, um die epidermale Reaktionsmechanismen auf die pathologischen Veränderungen der Dermis zu untersuchen.
Ziel in der vorliegenden Arbeit war die Etablierung eines In-vitro-Ischämiemodells auf der Grundlage boviner Keratinozyten aus der Klauenepidermis. Mithilfe dieses Modells sollten die zellulären Pathomechanismen infolge einer Ischämie, einer Hypoxie sowie eines Glukoseentzugs untersucht werden. Des Weiteren stand die Analyse des Differenzierungsverhaltens der Keratinozyten infolge ischämischer, hypoxischer und hypoglykämischer Konditionen im Mittelpunkt.
Für die Etablierung des In-vitro-Ischämiemodells diente als Grundlage das oxygen glucose deprivation (OGD)-Modell, das die Untersuchung eines gleichzeitigen Sauerstoff- und Glukosemangels sowie lediglich einer Hypoxie und eines Glukoseentzugs bei bovinen Keratinozyten ermöglichte. Die Versuche wurden in eine Kurzzeitanalyse über 96 Stunden sowie eine Langzeitanalyse über drei Wochen geteilt. Nach erfolgter Exposition wurde die Zellviabilität mittels LDH(Lactatdehydrogenase)- und MTT(3-(4,5-Dimethylhiazol-2-yl)-2,5-diphenyltetrazoliumbromid)-Assay untersucht. Des Weiteren wurde das veränderte Differenzierungsverhalten der Keratinozyten infolge der veränderten Kultivierungsbedingungen mittels Western Blot-Analyse anhand der Involukrin- und Lorikrin-Expression untersucht.
Die Keratinozyten zeigten infolge einer OGD nach kurzer Expositionsdauer die höchsten zytotoxischen Effekte, die von einer zeitabhängigen Abnahme der Zellviabilität sowie massiven morphologischen Veränderungen gefolgt wurde. Hypoxische Bedingungen bewirkten eine zeitabhängige Abnahme der Zellviabilität, die erst nach zweiwöchiger Inkubation die größte Zytotoxizität aufwies, sowie eine geringgradig veränderte Zellmorphologie bei Erhaltung des Zellverbands. Der Glukoseentzug bewirkte eine stark verminderte Zellviabilität sowie starke morphologische Zellveränderungen. In der Western Blot-Analyse konnte eine gesteigerte Involukrin- und Lorikrin-Expression infolge einer OGD, einer Hypoxie und eines Glukoseentzugs nachgewiesen werden.
In der vorliegenden Arbeit konnte erstmalig ein auf bovinen Keratinozyten basierendes In-vitro-Ischämiemodell etabliert werden, das die Untersuchung zellulärer Mechanismen der Epidermis ermöglichte. Die OGD zeigte den stärksten Einfluss auf die Zellviabilität sowie eine veränderte Zelldifferenzierung der Keratinozyten, was die pathophysiologischen Veränderungen im Rahmen der CHD reflektiert. Die ebenfalls starken Zellveränderungen infolge eines Glukoseentzugs verdeutlichen die Rolle der Glukose im Zellmetabolismus der Keratinozyten. Solch ein epidermaler Glukosemangel ist in Verbindung mit der negativen Energiebilanz der Rinder im peripartalen Zeitraum denkbar. Die Ergebnisse infolge einer Hypoxie verweisen auf vielfältige Adaptationsmechanismen der Keratinozyten an hypoxische Bedingungen, denen sie in der Epidermis in vivo während der Zelldifferenzierung ausgesetzt werden. Damit besitzt das In-vitro-Ischämiemodell ein großes Potenzial für den Einsatz in der Klauenreheforschung, um einerseits die mit einer Ischämie einhergehenden pathologischen Veränderungen der CHD untersuchen zu können. Andererseits liefert das Modell wertvolle Informationen zu den physiologischen Grundlagenmechanismen der Epidermis, die mit der Zelldifferenzierung einhergehen. / The subclinical laminitis or claw horn disruption (CHD) is of great economic importance in the dairy industry as it causes lameness, poor general condition and reduced performance. Despite extensive research efforts, the pathomechanism of CHD remains widely unclear. The current hypotheses on CHD pathogenesis include ischemic alterations of the epidermal keratinocytes resulting from an impaired blood supply. This causes an alteration of cell proliferation, a dermo-epidermal separation and an impaired keratinization and horn production. Therefore, in vitro ischemia models are of critical importance in clarification of the epidermal responses to an altered microcirculation.
The aim of this study was the establishment of an in vitro ischemia model based on bovine claw keratinocytes. This in vitro model should enable the investigation of cellular pathomechanisms following exposure to ischemia, hypoxia and glucose deprivation. An additional aim was the analysis of the differentiation pattern of keratinocytes under ischemic, hypoxic and hypoglycaemic conditions.
To establish the in vitro ischemia model, the keratinocytes were exposed to oxygen-glucose deprivation (OGD). In addition, this model allowed the parallel examination of hypoxic and hypoglycaemic conditions on bovine claw keratinocytes. The experiments were divided into a short-term analysis over 96h and a long-term analysis over three weeks. Measurement of cell viability was performed by LDH(lactatedehydrogenase) and MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide) assays. Furthermore, the differentiation pattern of the keratinocytes after exposure to ischemia, hypoxia and glucose deprivation was detected by western blot analysis of the focus on expression of involucrin and loricrin.
The highest cytotoxic effect was measured after short exposure to OGD followed by a time-dependent decrease of cell viability and extensive morphological changes of the keratinocytes. Hypoxic conditions lead to a time-dependent decrease of cell viability with the highest cytotoxicity after two weeks. The keratinocytes showed slight changes in cell morphology while maintaining a confluent cell layer. Exposure of keratinocytes to glucose deprivation showed a high decrease of cell viability and strong morphological changes. Furthermore, western blot analysis showed an altered expression pattern with increased involucrin and loricrin levels after exposure to OGD, hypoxia and glucose deprivation.
The present study established for the first time an in vitro ischemia model based on bovine claw keratinocytes to study the cellular mechanisms of the epidermis. After exposure to OGD, keratinocytes showed the highest loss in cell viability and an altered cell differentiation. This reflects the pathophysiological changes following epidermal ischemia occurring during the pathogenesis of CHD. The massive cellular alterations after glucose deprivation provide good evidence for the importance of glucose in the cellular metabolism of keratinocytes. An epidermal glucose deficiency may occur in combination with a negative energy balance during peripartal period in cattle. The results of hypoxia show the different adaptive mechanisms of keratinocytes to hypoxic conditions which are present in the epidermis during cell differentiation. Thus, the in vitro ischemia model has a great potential for use in research into CHD pathogenesis and pathomechanisms associated with ischemia. On one side, it is possible to investigate the pathological changes following ischemia during CHD. On the other side, the model offers useful information on physiological response mechanisms of the epidermis that correlate with cell differentiation.
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Implication de la lysyl oxydase au cours de la différenciation épidermique en modèles in vitro / Determination of lysyl oxidase implication during epidermal differentiation using in vitro modelsLe Provost, Gabrielle 05 July 2010 (has links)
La lysyl oxydase (LOX) est une enzyme extracellulaire dont le rôle canonique est de catalyser la réticulation des fibres de collagènes et de l’élastine, assurant ainsi l’intégrité des tissus conjonctifs. La LOX agit également dans plusieurs types cellulaires comme régulateur de différents processus biologiques, soulignant des fonctions à la fois extra et intracellulaires. Ces travaux de thèse ont contribué à améliorer la compréhension du rôle de LOX dans l’épiderme, et plus précisément au cours de la différenciation des kératinocytes. Le développement d’un modèle de culture en monocouche à confluence, et d’un modèle tridimensionnel d’épiderme reconstruit ont permis d’aborder l’étude de LOX au cours de l’induction du programme de différenciation des kératinocytes et du processus de différenciation terminale conduisant à la formation d’un épiderme pluristratifié, cornifié et fonctionnel. L’expression de LOX est induite au cours des premières étapes de la différenciation de kératinocytes primaires ainsi que d’une lignée de kératinocytes immortalisés. Grâce à l’établissement de lignées de kératinocytes éteignant l’expression de LOX de façon stable, nous avons mis en évidence l’implication de la protéine LOX, indépendamment de son activité enzymatique, dans la régulation des premières étapes de différenciation des kératinocytes. En absence de LOX, l’initiation du programme de différenciation est perturbée, affectant la différenciation terminale et fonctionnelle des épidermes reconstruits. Ainsi, une régulation fine de l’expression de LOX est nécessaire au déroulement normal du processus de différenciation des kératinocytes, et donc au maintien de l’homéostasie épidermique / Lysyl oxidase (LOX) is an extracellular enzyme that catalyzes the cross-linking of fibrillar collagens or elastin, thereby regulating the structural integrity of connective tissues. Moreover, LOX displays multiple roles in different cell types, acting as a regulator of various biological processes at both extra and intracellular levels. The aim of the present work was to shed light on LOX functions in the epidermis, especially during keratinocyte differentiation. The development of culture models, consisting of confluent monolayers or reconstructed-epidermis allowed us to study LOX functions during the induction of the differentiation program, and furthermore during the terminal differentiation process leading to the formation of a pluristratified, cornified and functional epidermis. LOX expression is induced at the onset of the commitment to differentiation, both in primary and immortalized keratinocytes. Stable silencing of LOX expression affects the induction of the differentiation program and strongly impairs terminal and functional epidermal differentiation in reconstructed-epidermis. Therefore, LOX protein acts during the first steps of keratinocyte differentiation, independently of its enzymatic activity, and is implied for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is required for normal keratinocyte differentiation, and thus for maintenance of epidermal
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A study of an epithelial-mesenchymal transition-inducing transcriptional factor Snail in prostate cancer using a newly-developed three-dimensional culture model. / CUHK electronic theses & dissertations collectionJanuary 2008 (has links)
In recent years, three dimensional (3D)-culture technique has emerged as a very popular approach to reconstruct tissue architectures and develop experimental models for studying epithelial cancers. However, 3D culture models of prostate epithelial cells to mimic prostate cancer development are relatively rare, making it highly desirable to develop and characterize novel 3D culture models suitable for studying prostate cancer. Recently, epithelial-mesenchymal transition (EMT) has emerged as an important mechanism for cancer cell invasion. The zinc finger transcriptional factor Snail as a key regulator of EMT has been found to contribute to aggressive progression in many types of neoplasms. Even though several studies corroborated that EMT is implicated in prostate cancer, the expression patterns of Snail in normal prostate and prostate cancer, and the functional role of Snail in prostate cancer as well as its relation with EMT are still unknown. Based on this background, my major efforts were to establish a 3D culture model of human prostatic epithelial cells with structural and functional relevance to prostate gland and to employ this model to study the functional role of Snail in the prostate cancer. / When embedded in Matrigel for 3D culture, BPH-1 cells developed into growth-arrested acinar structures with a hollow lumen. Ultrastructural examination of BPH-1 spheroids by electricon microscopy indicated that BPH-1 spheroids displayed a polarized differentiation phenotype. Immunoflurescence analysis of polarized epithelial markers further confirmed that BPH-1 spheroids were polarized. In contrast, tumorigenic BPH-1CAFTD cells exhibited disorganized and continuously proliferating structures in Matrigel, with polarized epithelial markers randomly diffused or completely lost. In addition, BPH-1 CAFTD cells displayed significantly higher invasive capacity in comparison to BPH-1 cells by transwell invasion assay. Moreover, LY294002 treatment of BPH-1CAFTD1 and BPH-1CAFTD3 cells in 3D cultures resulted in impaired cell proliferation as evidenced by reduced colony size and decreased Ki-67 index, and western blot analysis showed that cyclin D1 protein levels were significantly decreased, while p21 protein levels were slightly up-regulated in LY294002-treated 3D cultures. Additionally, LY94002 significantly decreased the invasive capacity of BPH-1CAFTD1 and BPH-1CAFTD3 cells. Interestingly, LY294002 treatment completely reverted the disorganized non-polar 3D structures of BPH-1CAFTD1 cells to well-organized polarized spheroid structures in Matrigel, but failed to restore the polarized differentiation in 3D cultures of BPH-1CAFTD3 cells, which still formed compact aggregates as shown by confocal immunofluorescence analysis. Snail protein was barely detected in the epithelial cells of human benign prostatic tissue but significantly elevated as nuclear protein in primary prostate cancer and bone metastatic specimens by immunohistochemical analysis. Snail transcript levels were weakly expressed in a majority of nonmalignant prostatic epithelial cell lines, while markedly increased in almost all tested cancer cell lines. Snail expression induced a morphological switch to more scattered and spindle-shaped appearance in BPH-1 and BPH-1CAFTD1 cells in 2D culture, and immunofluorescence analysis of several EMT specific markers indicated that Snail-expressing cells underwent EMT. In 3D contexts, Snail-expressing cells developed into more disorganized structures with many cords or protrusions, with a concurrent EMT change as evidenced by reduced E-cadherin and increased vimentin expression. In addition, Snail expression augmented the invasive capacities in both BPH-1 cells and BPH-lCAFTD1 cells, but did not significantly affect the migratory capacities. Snail expression enhanced the MMP2 activity in BPH-1 cells and promoted both MMP-2 and MMP-9 activities in BPH-1CAFTD1 cells. Moreover, Snail expression enhanced anchorage-independent growth capability in BPH-1 cells, but failed to initiate tumor formation in nude-mice. Lastly, Snail expression induced a dramatic increase in FoxC2 and SPARC transcripts but a marked decrease in claudin-1 and p63 transcripts. / Chu, Jianhong. / Adviser: Franky Chan Leung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3448. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 143-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
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Transcriptional regulation of mouse epidermal permeability barrier development and homeostasis by Ctip2Wang, Zhixing 05 June 2012 (has links)
Skin is the largest organ in the body that protects the organism from environmental, chemical and physical traumas of each passing day. The protective skin epidermal permeability barrier (EPB) is formed within the exterior layers of the epidermis, which are regularly sloughed off and repopulated by movement of inner cells. The epidermal permeability barrier is established during in utero development and maintained through lifetime. Impaired epidermal barrier formation is one of the major features of several dermatoses such as psoriasis and atopic dermatitis.
Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (Ctip2), also known as Bcl11b, is a C���H��� zinc finger protein expressed in many organs and tissues. It has been shown to regulate the development of thymocyte, tooth and corticospinal motor neurons. Ctip2 is highly expressed in mouse epidermis during skin organogenesis and in adulthood. It is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Germline (Ctip2- null mice) and selective ablation of Ctip2 in mouse epidermis (Ctip2[superscript ep-/-] mice) leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation and terminal differentiation as well as altered lipid distribution during embryogenesis. Sphingolipids account for ~50% of total skin lipids by weight and are crucial components of epidermal barrier. We have recently identified Ctip2 as a key regulator of skin lipid metabolism. Germline deletion of Ctip2 in mouse embryos leads to altered lipid composition in the developing mouse epidermis by modulating the expression levels of key enzymes involved in lipid metabolism (bio-synthesis and catabolism). We also demonstrated that Ctip2 is recruited to the promoter regions of several genes involved in the ceramide and sphingomyelin biosynthesis pathways and could directly regulate their expression. Thus, we have identified Ctip2 as a key regulator of several lipid metabolizing genes and hence epidermal sphingolipid biosynthesis during skin development.
To study the role of Ctip2 in adult skin homeostasis, we have utilized Ctip2[superscript ep-/-] mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. We showed that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as high infiltration of T lymphocytes and immune cells. We have also observed increased expression of Th2-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression is significantly upregulated in the mutant epidermis as early as postnatal day 1 and Ctip2 was recruited to the promoter region of the TSLP gene in mouse epidermal keratinocytes. The results suggest that upregulation of TSLP expression in the Ctip2[superscript ep-/-] epidermis could be due to a derepression of gene transcription in absence of Ctip2. Thus, our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult skin, as well as a non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines in adult mouse skin. Present results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism mediated by Ctip2 in mouse epidermal keratinocytes.
Altogether, our study indicates that Ctip2 could be involved in a diverse range of biological events in skin including barrier formation, maintenance and epidermal homeostasis. Ctip2 appears to be a master regulator in skin barrier functions by directly regulating the transcription of a subset of genes involved in lipid metabolism and inflammatory responses. / Graduation date: 2013
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Infections ostéo-articulaires à Staphylococcus aureus et Staphylococcus epidermidis : épidémiologie moléculaire et corrélation entre expression clinique et interactions hôte – bactérie / Pas de titre anglaisValour, Florent 15 December 2014 (has links)
Le genre Staphylococcus, première étiologie des infections ostéo-articulaires (IOA), est associé à des formes particulièrement difficiles à traiter. Trois mécanismes phénotypiques ont été rattachés à ce fort taux de chronicité et de rechutes, permettant l'adaptation bactérienne à la vie au sein du tissu osseux et un échappement au système immunitaire de l'hôte et à l'action des antibiotiques : la formation de biofilm, la persistance des staphylocoques dans les ostéoblastes, et l'évolution vers le morphotype de small colony variant (SCV). Longtemps considéré comme simple commensal cutanéo-muqueux, S. epidermidis est désormais reconnu comme un agent étiologique majeur des IOA sur matériel. Or, si le portage est universel, l'infection est un phénomène rare. A ce jour, aucun facteur génotypique n'a pu être associé au pouvoir invasif de certaines souches de portage. Notre travail a permis de montrer l'absence de pouvoir discriminant des capacités d'internalisation des ostéoblastes et de formation de biofilm entre souches commensales et invasives. Par ailleurs, un très faible taux d'internalisation de S. epidermidis dans les ostéoblastes a été mis en évidence, suggérant une importance moindre de ce mécanisme dans la physiopathologie des IOA à S. epidermidis par rapport aux IOA à S. aureus. Les principales études ayant porté sur les capacités d'interaction de S. aureus avec les ostéoblastes et de formation de biofilm ont cherché à en explorer les mécanismes à partir de souches de laboratoire ou de souches représentatives de quelques clones de S. aureus résistants à la méticilline (SARM). Dans notre cas, nous avons souhaité étudier une large collection de souches cliniques de S. aureus (n=95) sensible à la méticilline (SASM) responsables d'IOA aiguës ou chroniques. La caractérisation des fonds génétiques de cette collection, puis en élargissant notre étude à des collections de différents villes françaises, a d'abord permis de décrire une forte prévalence du clone émergent de SASM CC398 dans les IOA en France / Pas de résumé en anglais
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Engineered infected epidermis model for in vitro study of the skin proinflammatory responseJahanshahi, Maryam 24 January 2020 (has links)
Wound infection is a major clinical burden that can significantly impede the healing process and cause severe pain. Prolonged wound infection can lead to long-term hospitalization or death. Pre-clinical research to evaluate new drugs or treatment strategies relies on animal studies. However, animal studies have several challenges including interspecies variations, cost, and, ethics question the success of these models. Recent advances in tissue engineering have enabled the development of in vitro human skin models for wound infection modeling and drug testing. The existing skin models are mostly representative of the healthy human skin and its normal functions. However, to study the wound healing process and the response of skin to the infection, there is still a need to develop a skin model mimicking the wound infection. This work presents a simplified functional infected epidermis model, fabricated with enzymatically crosslinked gelatin hydrogel. The immortalized human keratinocytes, HaCaT cells, was successfully cultured and differentiated to a multilayer epidermis structure at the air-liquid interface, and expressed terminal differentiation marker, filaggrin, in the outer layer. The barrier function of the epidermis model was studied by measuring the electrical resistance and tissue permeability across the layer. The results showed that the developed epidermis model offered a higher electrical resistance and a lower drug permeability compared to the cell monolayer on gelatin and cell-free gelatin. To show the capability of the developed epidermis model in wound modeling and drug, the model was infected with Escherichia coli and the inflammatory response of keratinocytes was studied by measuring the level of proinflammatory cytokines, including IL-1β and TNF-α. The results demonstrated the proinflammatory response of the epidermis model to infection by producing a higher level of TNF-α and IL-1β compared to the control group. While treating with antibiotic ciprofloxacin terminated the proinflammatory response and reduced the level of TNF-α and IL-1β. The robust fabrication procedure and functionality of this model suggest that this model has great potential for wound modeling and high throughput drug testing. / Graduate
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Chemical Composition of Soybean Root Epidermal Cell WallsFang, Xingxiao January 2006 (has links)
The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (<em>Glycine max</em>) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to <em>Phytophthora sojae</em> and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF<sub>3</sub>-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, ω-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,ω-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.
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The Role of Spindle Orientation in Epidermal Development and HomeostasisSeldin, Lindsey January 2015 (has links)
<p>Robust regulation of spindle orientation is essential for driving asymmetric cell divisions (ACDs), which generate cellular diversity within a tissue. During the development of the multilayered mammalian epidermis, mitotic spindle orientation in the proliferative basal cells is crucial not only for dictating daughter cell fate but also for initiating stratification of the entire tissue. A conserved protein complex, including LGN, Nuclear mitotic apparatus (NuMA) and dynein/dynactin, plays a key role in establishing proper spindle orientation during ACDs. Two of these proteins, NuMA and dynein, interact directly with astral microtubules (MTs) that emanate from the mitotic spindle. While the contribution of these MT-binding interactions to spindle orientation remains unclear, these implicate apical NuMA and dynein as strong candidates for the machinery required to transduce pulling forces onto the spindle to drive perpendicular spindle orientation. </p><p> In my work, I first investigated the requirements for the cortical recruitment of NuMA and dynein, which had never been thoroughly addressed. I revealed that NuMA is required to recruit the dynein/dynactin complex to the cell cortex of cultured epidermal cells. In addition, I found that interaction with LGN is necessary but not sufficient for cortical NuMA recruitment. This led me to examine the role of additional NuMA-interacting proteins in spindle orientation. Notably, I identified a role for the 4.1 protein family in stabilizing NuMA's association with the cell cortex using a FRAP (fluorescence recovery after photobleaching)-based approach. I also showed that NuMA's spindle orientation activity is perturbed in the absence of 4.1 interactions. This effect was demonstrated in culture using both a cortical NuMA/spindle alignment assay as well as a cell stretch assay. Interestingly, I also noted a significant increase in cortical NuMA localization as cells enter anaphase. I found that inhibition of Cdk1 or mutation of a single residue on NuMA mimics this effect. I also revealed that this anaphase localization is independent of LGN and 4.1 interactions, thus revealing two independent mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. </p><p> After gaining a deeper understanding of how NuMA is recruited and stabilized at the cell cortex, I then sought to investigate how cortical NuMA functions during spindle orientation. NuMA contains binding domains in its N- and C-termini that facilitate its interactions with the molecular motor dynein and MTs, respectively. In addition to its known role in recruiting dynein, I was interested in determining whether NuMA's ability to interact directly with MTs was critical for its function in spindle orientation. Surprisingly, I revealed that direct interactions between NuMA and MTs are required for spindle orientation in cultured keratinocytes. I also discovered that NuMA can specifically interact with MT ends and remain attached to depolymerizing MTs. To test the role of NuMA/MT interactions in vivo, I generated mice with an epidermal-specific in-frame deletion of the NuMA MT-binding domain. I determined that this deletion causes randomization of spindle orientation in vivo, resulting in defective epidermal differentiation and barrier formation, as well as neonatal lethality. In addition, conditional deletion of the NuMA MT-binding domain in adult mice results in severe hair growth defects. I found that NuMA is required for proper spindle positioning in hair follicle matrix cells and that differentiation of matrix-derived progeny is disrupted when NuMA is mutated, thus revealing an essential role for spindle orientation in hair morphogenesis. Finally, I discovered hyperproliferative regions in the interfollicular epidermis of these adult mutant mice, which is consistent with a loss of ACDs and perturbed differentiation. Based on these data, I propose a novel mechanism for force generation during spindle positioning whereby cortically-tethered NuMA plays a critical dynein-independent role in coupling MT depolymerization energy with cortical tethering to promote robust spindle orientation accuracy. </p><p> Taken together, my work highlights the complexity of NuMA localization and demonstrates the importance of NuMA cortical stability for productive force generation during spindle orientation. In addition, my findings validate the direct role of NuMA in spindle positioning and reveal that spindle orientation is used reiteratively in multiple distinct cell populations during epidermal morphogenesis and homeostasis.</p> / Dissertation
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Analyse einer chromosomalen Deletion und Entdeckung einer neuartigen nicht-translatierten RNA in Staphylococcus epidermidis / Analysis of a chromosomal deletion and discovery of a novel non-translated RNA in Staphylococcus epidermidisEckart, Martin January 2006 (has links) (PDF)
Staphylococcus epidermidis ist ein wichtiger Bestandteil der gesunden Hautflora des Menschen, gleichzeitig aber auch der häufigste Erreger nosokomialer Infektionen bei immunsupprimierten Patienten. Die Forschungsarbeiten haben sich in den vergangenen Jahren besonders auf Faktoren und Mechanismen konzentriert, welche zur Etablierung der Spezies als Pathogen beigetragen haben. Eine typische Eigenschaft klinischer Isolate ist die Fähigkeit, auf künstlichen Oberflächen Biofilme zu bilden. Gegenstand der vorliegenden Arbeit war die Untersuchung der IS-vermittelten Genomflexibilität und der Vergleich der Genomstruktur nosokomialer und kommensaler Isolate von S. epidermidis. Dazu wurde eine 260 kb große spontane Deletion im Chromosom des Biofilm-bildenden Stammes S. epidermidis 307 sequenziert und annotiert, von der bekannt war, dass sie durch eine homologe Rekombination zweier IS256-Kopien ausgelöst wurde. Auf dem deletierten Fragment fanden sich neben dem ica-Operon zahlreiche potentielle Virulenz-assoziierte Gene. Das überraschende Ergebnis dieser Analyse war jedoch die Identifizierung eines neuartigen SCC-Elementes, das den rechten Rand der Deletion begrenzt. Dies ist die erste Beschreibung eines SSC-Elementes mit einer CcrC-Rekombinase, dem das Methicillin-Resistenzgen mecA fehlt. In der Nähe des Rekombinasegens fand sich S.-haemolyticus-spezifische DNA. Weitere Untersuchungen zeigten, dass das SCC-Element durch die IS256/Tn4001 -vermittelte Deletion in der Mutante verkürzt wurde. Genomvergleiche ica-positiver und ica-negativer Stämme von S. epidermidis mittels Microarrays, sowie in-silico-Analysen zweier vollständiger S.-epidermidis-Genome ergaben, dass sich kommensale und pathogene Stämme in nur wenigen Faktoren unterscheiden. Diese befinden sich jedoch fast alle in einer Region um den oriC, in dessen Nähe auch die Insertionsstelle für die SCCmec-Inseln lokalisiert ist. Das deletierte DNA-Fragment von S. epidermidis 307 konnte ebenfalls dieser Region zugeordnet werden, die offenbar eine Zone hoher genomischer Flexibilität darstellt, in der fremde DNA integriert werden kann. Im Rahmen dieser Arbeit konnte außerdem gezeigt werden, dass das ica-Operon diesen variablen Genomabschnitt begrenzt. Die exakte Grenze zwischen ica-positiven und ica-negativen Stämmen bildet eine Thr-tRNA, die in einer 1,4 kb großen intergenischen Region stromabwärts des icaR-Regulatorgens lokalisiert ist. In unmittelbarer Nachbarschaft konnte ein Transkript nachgewiesen werden, welches nur in ica-positiven S. epidermidis vorkommt. Gestützt auf bioinformatische Analysen wurden zunächst die Polarität und Länge des Transkriptes, sowie der korrespondierende Promotor experimentell bestimmt. Demnach handelt es sich um eine 487 nt lange RNA, die entgegengesetzt zur Thr-tRNA orientiert ist und mit dieser teilweise überlappt. Da die Nukelotid-Sequenz weder eine Ribosomen- Bindungsstelle noch einen größeren Leserahmen aufweist, ist es sehr wahrscheinlich, dass es sich um eine nicht-translatierte RNA handelt. Qualitative RT-PCR-Experimente zeigten, dass die IGRica-RNA kostitutiv exprimiert wird. Spontane IS256 –Insertionsmutanten in der Promotorregion der IGRica-RNA wiesen phänotypisch eine deutlich verminderte Biofilmbildung auf. Daher ist zu vermuten, dass die IGRica-RNA in die Regulation dieses wichtigen Virulenzfaktors involviert ist. In der vorliegenden Arbeit konnte weiterhin gezeigt werden, dass S. epidermidis das in vielen Spezies konservierte Hfq-Protein exprimiert, welches ein Bindungspartner und Chaperon für zahlreiche regulatorische RNAs darstellt. gel-shift-Experimente mit rekombinantem Hfq-Protein aus S. epidermidis ergaben jedoch keine nachweisbare Wechselwirkung der IGRica-RNA mit diesem Faktor in vitro. Insgesamt hat die Studie gezeigt, dass S. epidermidis eine Spezies mit einer außerordentlich hohen Genomflexibilität ist, die sich hauptsächlich in einer bestimmten Genomregion abspielt und für die IS-Elemente von besonderer Bedeutung sind. Die Identifizierung von DNA aus anderen Staphylokokken-Arten in dieser Region zeigt, dass S. epidermidis zu horizontalem Gentransfer über Speziesgrenzen hinweg in der Lage ist. Dies, sowie die Daten zur neuen SCC-Kassette, unterstreichen die Bedeutung von S. epidermidis als Reservoir für die Entwicklung neuartiger Resistenz- und Virulenzdeterminanten, die gerade im Krankenhausmilieu auf Spezies mit höherem Virulenzpotential übertragen werden können und so einen wichtigen Faktor für die anhaltende Problematik nosokomialer Infektionen mit S. aureus darstellen. Die Entdeckung einer RNA mit möglicherweise regulatorischer Funktion ist der erste Faktor dieser Art, der – abgesehen von einigen phylogenetisch hoch konservierten RNAs – bisher in S. epidermidis nachgewiesen wurde. Die funktionale Analyse der IGRica-RNA und die Suche nach weiteren regulatorischen RNAs in Staphylokokken eröffnen ein Forschungsfeld, das zum besseren Verständnis der Lebensweise dieser bedeutenden Erreger beitragen kann. / Staphylococcus epidermidis is an important part of the human skin flora, but also the most frequent cause of nosocomial infections in immune-suppressed patients. Research in the last years focused on the factors and mechanisms leading to the establishment of the species as a pathogen. A typical feature of clinical isolates of S. epidermidis is the ability to form biofilms on artificial surfaces. Topic of this work was the IS-mediated genome flexibility and the comparison of genome structure from nosocomial and commensal S. epidermidis isolates. A 260 kb large spontaneous deletion in the chromosome of the biofilm-forming strain S. epidermidis 307 was sequenced and annotated. The deletion was caused by homologous recombination of two IS256 copies. It contained many putative virulence-associated genes besides the ica operon. However, the surprising result of this analysis was the identification of a new SCC element that limits the right border of the deletion. This is the first report of a SCC element containing a CcrC recombinase but lacking the mecA gene. DNA of S. haemolyticus is located near the recombinase gene ccrC. Further studies showed that the SCC element is truncated due to IS256/Tn4001 mediated deletion in the mutant. Genome comparison of ica-positive and ica-negative S. epidermidis strains using microarrays revealed that commensal and pathogenic strains differ only in very few factors. This was confirmed by in silico comparison of two complete genomes of S. epidermidis. Most of the virulence factors are harbored within a region around the oriC. The insertion sites of the SCCmec islands and the deleted fragment of S. epidermidis are also located in this part of the genome, that obviously represents a region of high genomic flexibility and in which foreign DNA can be integrated. Furthermore, this work showed that the ica operon delimits this variable segment of the genome. The exact border between ica-positive and ica-negative strains represents a Thr-tRNA within the 1.4 kb intergenic region downstream of the icaR regulatory gene. In the direct vicinity a transcript could be detected that is only present in ica-positive S. epidermidis. Based on bioinformatical analysis the polarity, length, and the corresponding promoter were experimentally determined. The RNA ranges 487 nt in length and overlaps partly with the Thr-tRNA on the opposite strand. Neither a ribosomal binding site nor an appropriate open reading frame can be found; so, it is likely that the RNA is not translated. Qualitative RT-PCR experiments showed constitutive expression of the IGRica-RNA. Spontaneous IS256 insertion mutants within the promoter region of the IGRica-RNA exhibited a clearly diminished biofilm formation. Therefore, it is tempting to speculate that the IGRica-RNA is involved in the regulation of this important virulence factor. The experiments show that S. epidermidis also expresses the highly conserved Hfq protein that functions as a binding partner and a chaperon for many regulatory RNAs. However, gel shift experiments with recombinant Hfq from S. epidermidis revealed no detectable interaction of the IGRica-RNA with this factor in vitro. In summary, the study shows that S. epidermidis is a species with extraordinary high genome flexibility in a certain region of the genome where IS elements have a special influence. Identification of DNA from other staphylococcal species demonstrates that S. epidermidis is capable of horizontal gene transfer beyond the species barrier. This and the newly discovered SCC element emphasize the importance of S. epidermidis as a reservoir for the development of new resistance and virulence determinants. These factors can be transferred easily within the hospital environment to species with a higher virulence potential. This represents an important factor for the long-standing problem of nosocomial infections with S. aureus. The discovered RNA with a putative regulatory function is the first factor of this kind that has been identified in S. epidermidis so far, besides some phylogenetically conserved RNAs. Functional analysis of the IGRica-RNA and investigation for other regulatory RNAs in staphylococci open a wide field of research that can contribute considerably to the understanding of these important pathogens.
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Proteínas do Tegumento de Abelhas Apis mellifera em Metamorfose: Identificação por Espectrometria de Massa / Integument Protein of Honeybee Apis mellifera under Metamorphosis: Identification by Mass SpectrometryMicas, André Fernando Ditondo 19 December 2012 (has links)
Como qualquer inseto holometábolo, a abelha Apis mellifera sofre metamorfose completa, apresentando grandes mudanças na forma e fisiologia quando passa do estágio larval para o estágio de pupa (muda metamórfica). Após esta muda, com o prosseguimento do desenvolvimento, o tegumento pupal (cutícula e a epiderme subjacente), extensivamente remodelado, é substituído pelo tegumento adulto, definitivo, que passa por intensa melanização e esclerotização. Eletroforese bidimensional e espectrometria de massas foram utilizadas neste trabalho para caracterizar as mudanças do padrão proteico no tegumento em desenvolvimento de operárias e zangões. No total foram identificadas 51 proteínas diferentes no tegumento torácico extraído de larvas, pupas e adultos (adultos-faratos). Quatorze proteínas foram identificadas como genuinamente cuticulares: Apidermina-3,1-like, Apidermina-2, Cuticular proteins analogous to peritrophins-3C e 3D, AmelCPR3, 12, 16 e 27, Glicoproteína SgAbd-2-like, e cinco outras proteínas homólogas à proteínas cuticulares de outras espécies de insetos contendo um domínio de ligação à quitina. As proteínas diferiram principalmente quantitativamente entre as fases de desenvolvimento e sexo, e poucas diferenças qualitativas foram observadas. Por exemplo, Apidermina-2 é típica de tegumentos fortemente esclerotizados e pigmentados. As diferenças quantitativas foram destacadas pela comparação da abundância de algumas proteínas e seus respectivos RNA mensageiros (utilizando RT-PCR em tempo real) entre as fases de desenvolvimento e entre os sexos. Várias proteínas cuticulares mostraram mais de uma forma molecular, aparentemente derivadas de modificações pós-traducionais. Além de conferir suporte experimental para a validação de genes de A. mellifera preditos, ou não-anotados, nossos dados forneceram novas informações sobre as proteínas que atuam no tegumento em desenvolvimento. / As a holometabolous insect, the honey bee undergoes complete metamorphosis, displaying a marked change in shape and physiology when passing from the larval to the pupal stage (metamorphic molt). As development progresses, the extensively remodeled pupal integument (cuticle and subjacent epidermis) is replaced by the adult integument, which undergoes intense sclerotization and melanization. Two-dimensional electrophoresis and mass spectrometry were here used to characterize the changing protein patterns in the developing integument of workers and drones. Overall, we identified 51 different proteins in the thoracic integument extracted from larvae, pupae and adults (pharate adults). Fourteen proteins were identified as genuine cuticular proteins: Apidermin-3,1-like protein, Apidermin-2, Cuticular Proteins Analogous to Peritrophins-3C and 3D, AmelCPR3, 12, 16 and 27, Glycoprotein SgAbd-2-like, and 5 other proteins homologous to cuticular proteins from other insect species, and containing the chitin-binding domain. Integument proteins mainly differed quantitatively among the developmental stages and sexes, although few qualitative differences have also been detected. For example, Apidermin-2 is typical of the heavily pigmented and sclerotized integument. The quantitative differences were highlighted by comparing the levels of some of these proteins and their respective mRNAs (using RT-qPCR) among the developmental phases and between sexes. It is noteworthy that several cuticle proteins showed more than one molecular form, apparently derived from post-translational modifications. In addition to give experimental support for validation of predicted, or unannotated, honey bee genes, our data provided new information on proteins acting in the metamorphosing integument.
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