In Anlehnung an die Definition beim Pferd (SCHOON et al. 1992, 1997) wird die bovine Endometrose als endometriale periglanduläre und/oder stromale Fibrose mit Alteration der betroffenen Drüsen definiert (RODENBUSCH 2011). Eine eingehende histomorphologische und immunhistologische Charakterisierung der Endometrose existiert bisher bei der Stute (KENNEY u. DOIG 1986, SCHOON et al. 1992, HOFFMANN et al. 2009), jedoch nicht beim Rind. Das Ziel dieser Arbeit ist daher die histomorphologische und immunhistologische Charakterisierung der verschiedenen Endometroseformen beim Rind. Die Auswertung der Proben erfolgt am Institut für Veterinär-Pathologie der Universität Leipzig. In Abhängigkeit von den klinisch-gynäkologischen Befunden werden diese Proben in drei Gruppen unterteilt. Gruppe A1: Endometriumbioptate (n=12) von vier klinisch-gynäkologisch gesunden fertilen Rindern in definierten Zyklusphasen; Gruppe A2: Endometriumbioptate (n=36) von 36 klinisch genitalgesunden Rindern mit mindestens einer Abkalbung und Gruppe B: Uterusquerschnitte (n=69) von 69 sub-/ infertilen Rindern. Die Proben werden anhand der von HOFFMANN (2006) definierten Kriterien auf histopathologische Veränderungen hin untersucht und charakterisiert. Das histomorphologische Erscheinungsbild der involvierten periglandulären Stromazellen erlaubt die Einteilung der Endometrose in eine aktive, inaktive und gemischte Fibrose, die, je nach der Integrität des Drüsenepithels, einen destruierenden oder nicht destruierenden Charakter aufweist. Zur Charakterisierung der Endometroseformen werden neben histomorphologischen Kriterien die Intermediärfilamente Desmin, Vimentin und Zytokeratin sowie die Expression von α-Aktin und Laminin berücksichtigt. Die deskriptive statistische sowie die Inferenzstatistik-Auswertung erfolgen unter Zuhilfenahme der Software SPSS 18. Eine aktive Fibrose tritt bei 96,2 % der Rinder auf; 1,9 % weisen eine inaktive und 1,9 % eine gemischte Endometrose auf. Ein nicht destruierendes Erscheinungsbild der Endometrose kann bei 88,6 % der untersuchten Rinder nachgewiesen werden. Bei 11,4 % der untersuchten Rinder liegt eine Endometrose mit destruierendem Charakter vor. Die Endometrose betrifft bei 81 % der Rinder Einzeldrüsen und bei 19 % Drüsennester. Drüsennester sind häufiger bei mittel- und hochgradigen Endometrosen nachweisbar. 20 % der Proben mit nicht destruierender Endometrose und 58 % der Proben mit destruierender Endometrose weisen eine entzündliche Infiltration der Drüsen auf, wobei ein Zusammenhang zwischen der entzündlichen Infiltration der endometrotischen Drüsen und dem destruierenden Charakter der Endometrose festgestellt werden kann. Zusätzlich zur Endometrose zeigen 61 % der Rinder eine Endometritis, 12,4 % eine Perivaskulitis und 66 % eine interkarunkuläre und/oder intrakarunkuläre Angiosklerose. Insgesamt findet sich nur bei 24 % der Fälle ausschließlich eine Endometrose, bei 10,5 % eine Endometrose und zugleich eine Endometritis, bei 16 % liegt eine Endometrose zusammen mit einer Angiosklerose vor. Eine Kombination von Endometrose, Angiosklerose und Endometritis ist bei 49,5 % der Proben nachweisbar. Insgesamt bestehen jedoch keine erkennbaren statistischen Zusammenhänge zwischen den einzelnen Befundkombinationen. Aufgrund der immunhistologischen Untersuchung kann konstatiert werden, dass die periglandulären Stromazellen innerhalb der Endometrose eine stromale Koexpression von Desmin, Vimentin und α-Aktin aufweisen, welche ein für Myofibroblasten charakteristisches Merkmal ist. Ein kleiner Prozentsatz der Drüsenepithelzellen in der destruierenden Endometrose reagiert multifokal positiv mit dem Vimentinantikörper. Dies ist möglicherweise Ausdruck einer Fehldifferenzierung zur Stabilisierung der Zelle oder Anzeichen einer intensivierten (pathologischen) Proliferation. Die Lamininexpression der Basallamina der endometrotisch veränderten Drüsen ist, insbesondere bei der destruierenden Endometrose, diskontinuierlich und geht mit einer Auffaserung der Basallamina einher. Vermutlich lassen sich die umfangreichen Basallaminaalterationen auf von Myofibroblasten sezernierte Enzyme zurückführen. Bei Rindern kann kein Zusammenhang zwischen der Endometrose und dem Alter der Rinder oder der Anzahl der Kalbungen festgestellt werden. In der vorliegenden Arbeit dominiert die geringgradig aktive nicht destruierende Endometrose gegenüber den anderen bovinen Endometroseformen. Die sub-/ infertilen Kühe (Gruppe B) zeigen häufiger eine schwerere und destruierende Endometrose als die klinisch gesunden Rinder (Gruppe A1, A2). Die klinisch-gynäkologisch gesunden Rinder in definierten Zyklusphasen weisen variable Endometroseformen oder Endometrosegrade auf. Die sub-/ infertilen Rinder zeigen eine höhere Güstzeit (252,82 ± 163,83 Tage) als die klinisch-gynäkologisch gesunden Tiere mit mindestens einer Abkalbung (94 ± 28,4 Tage). Die längere Güstzeit bei den sub- und infertilen Rindern könnte somit eine Folge des insgesamt in Charakter und Grad stärker geschädigten Endometriums bei dieser Gruppe sein. Somit kann unter Berücksichtigung der vorliegenden Ergebnisse angenommen werden, dass die aufgeführten Alterationen die Fertilität des Rindes negativ beeinflussen. Die Ergebnisse ermöglichen eine histomorphologische und immunhistologische Charakterisierung der Endometrose beim Rind. Anhand der Ergebnisse der hier durchgeführten detaillierten Untersuchungen ist es möglich, eine präzisere Deskription degenerativer endometrialer Befunde vorzunehmen. In Hinblick auf die Fertilität bei Vorliegen einer bovinen Endometrose wird somit die Grundlage für zukünftige prognostische Bewertungen gelegt.

<p>Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Additionally, while cell delivery to the pathological IVD has significant therapeutic potential for enhancing NP regeneration, the development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the NP region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into biomaterial scaffolds for NP tissue engineering or cell delivery to the disc may be beneficial for promoting NP cell survival and phenotype. In this dissertation, laminin-111 (LM111) functionalized poly(ethylene glycol) (PEG) hydrogels were developed and evaluated as biomaterial scaffolds for cell-based NP regeneration. </p><p>Here, PEG-LM111 conjugates with functional acrylate groups for crosslinking were synthesized and characterized to allow for protein coupling to both photocrosslinkable and injectable PEG-based biomaterial scaffolds. PEG-LM111 conjugates synthesized using low ratios of PEG to LM111 were found support NP cell attachment and signaling in a manner similar to unmodified LM111. A single PEG-LM111 conjugate was conjugated to photocrosslinkable PEG-LM111 hydrogels, and studies were performed to evaluate the effects of hydrogel formulation on immature NP cell phenotype in vitro. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, softer LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels. </p><p>A novel, injectable PEG-LM111 hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. PEG-LM111 conjugates were crosslinked via a Michael-type addition reaction upon the addition of PEG-octoacrylate and PEG-dithiol. Injectable PEG-LM111 hydrogel gelation time, mechanical properties, and ability to retain delivered cells in the IVD space were evaluated. Gelation occurred in approximately 20 minutes without an initiator, with dynamic shear moduli in the range of 0.9 - 1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 hydrogel carrier, as compared to cells in liquid suspension. </p><p>The studies presented in this dissertation demonstrate that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers and cell-cell interactions characteristic of an immature NP cell phenotype. Furthermore, these findings suggest that this novel, injectable laminin-functionalized biomaterial may be an easy to use and biocompatible carrier for delivering cells to the IVD.</p> / Dissertation

層黏蛋白 (laminin) 是基底膜內主要的糖蛋白，屬於細胞外基質。研究指出層黏蛋白在學習與記憶扮演重要角色，且內生性laminin β1表現於海馬迴神經，但是目前對於層黏蛋白在中樞神經的瞭解仍不是很清楚。因此，本實驗室利用水迷津學習實驗探討laminin β1對大白鼠空間學習與記憶的影響，實驗結果發現，經過學習試驗後的大白鼠，其海馬迴CA1區域的laminin β1 mRNA和蛋白質表現量明顯地減少，laminin β2的表現則不受到影響。為瞭解laminin β1在空間學習與記憶力程中扮演的角色及其下游的訊息傳遞路徑，我們轉染laminin β1 siRNA至海馬迴CA1區域會促進大白鼠在水迷津空間學習與記憶的能力，由西方墨點法也觀察到抑制海馬迴中laminin β1的表現量會促進SGK Ser422及ERK1/2的磷酸化，但是AKT Ser473的磷酸化則無顯著差異。當轉染質體基因laminin β1至CA1區域，則會抑制大白鼠水迷津空間學習與記憶的能力，同時也抑制SGK Ser422及ERK1/2的磷酸化。由於過去證實SGK1於大白鼠空間學習記憶歷程中扮演重要角色，我們推測laminin β1與SGK Ser422的磷酸化有關，為確認laminin β1與SGK1之間的關係，我們同時轉染laminin β1 siRNA和SGK siRNA至海馬迴CA1區域，原先laminin β1促進大白鼠在水迷津空間學習與記憶的能力反而受到抑制。由於許多文獻指出mitogen-activated protein kinase /extracellular signal-regulated kinase (MAPK/ERK) 能整合不同訊息傳遞路徑進而調控基因的表現，對神經突觸的可塑性及記憶的形成是個重要分子，我們進一步探討laminin β1是否透過ERK1/2調控下游的SGK1進而影響大白鼠的空間記憶與學習。因此我們先轉染laminin β1 siRNA，48小時後再給予U0126 (MEK抑制劑) 以抑制MAPK/ERK的活性。結果顯示U0126會阻斷laminin β1所引起SGK ser422的磷酸化。本篇研究提出laminin β1會參與哺乳類動物學習記憶的形成，同時也證實laminin β1會經由抑制ERK1/2及SGK1的活化進而抑制大白鼠空間學習與記憶的形成。

GABAB receptors are G-protein coupled receptors for the major inhibitory neurotransmitter in the mammalian central nervous system, γ-aminobutyric acid (GABA). The receptor is linked to a variety of disorders including epilepsy, pain, spasticity, drug addiction and cognitive impairment and is, therefore of major importance for drug discovery. The most abundant receptor isoforms GABABR1a and R1b differ by the presence in R1a of a pair of Nterminal extracellular complement control protein modules (CCP1 and CCP2) which - in other proteins - are generally involved in mediating specific protein-protein recognition. The CCP1 module contains disulphides but is natively disordered. In the current work, the yeast two-hybrid system was used to confirm an interaction of CCP1 of GABABR1a with the extracellular protein fibulin-2. Further work with the yeast twohybrid system extablished the novel interaction of the abundant extracellular matrix protein laminin, with GABABR1a CCP1, via its laminin globular (LG) domains. The laminin interaction was further characterised by surface plasmon resonance, demonstrating that several different domains are involved in the binding to the GABAB receptor CCPs. The primary binding site is located on laminin α5 LG4-5, but the E10 domains of the β1 chain and LG1-3 on α1 may also be involved. The pharmacological properties of the GABABR1a and R1b isoforms were studied by transient expression in Xenopus laevis oocytes. It was demonstrated that the agonist baclofen, as well as the antagonist CGP55845, appear to be more potent at GABABR1b compared to GABABR1a. Intriguingly, when recorded in the precence of laminin, GABABR1b/R2 expressing oocytes exhibited an increased baclofen-evoked response while the response in GABABR1a/R2 was completely abolished. In conclusion, the work demonstrates that laminin is a binding partner for GABABR1a CCPs. Such an interaction between the metabotropic GABA receptor and the extracellular matrix may lie behind the recently reported roles of GABA in neuronal migration and the laying down of neuronal circuitry during the development of parts of the central nervous system.

<p>Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with numerous cell-cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with associated changes in NP cell phenotype, morphology and proteoglycan synthesis that may contribute to IVD degeneration. Studies demonstrate healthy, juvenile NP cells exhibit potential for preservation of multi-cell clusters and NP cell phenotype when cultured upon soft, laminin-containing substrates; however, the mechanisms that regulate metabolism and phenotype of these NP cells are not understood. N-cadherin is a cell adhesion molecule that is present in juvenile NP cells, but disappears with age. The goal of this dissertation was to reveal the role of N-cadherin for NP cells in multi-cell clusters that contribute to the maintenance of the juvenile NP cell morphology and phenotype in vitro, and to evaluate the potential for laminin- functionalized poly(ethylene glycol) (PEG-LM) hydrogels to promote human NP cells towards a juvenile NP cell phenotype. </p><p>In this dissertation, juvenile porcine IVD cells were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype on soft, laminin-rich hydrogels. In the first part of this dissertation, preservation of the porcine juvenile NP cell phenotype and presence of N-cadherin was analyzed by culturing porcine NP cells on soft, laminin-rich or PEG-LM hydrogels. Secondly, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Finally, human IVD cells were cultured on PEG-LM hydrogels to investigate the potential to revert degenerate, human NP cells toward a juvenile NP cell phenotype and morphology. </p><p>Findings reveal soft (<500 Pa), laminin-rich substrates promote NP cell clustering, a key feature of the juvenile NP cell that is associated with N-cadherin positive expression. Additionally, N-cadherin-mediated cell-clustering regulates NP cell matrix production and gene expression of NP-specific and NP-matrix related markers. Inhibition of N-cadherin-mediated contacts resulted in decreased expression of juvenile NP cell features. Finally, juvenile human NP cells are also able to form N-cadherin positive cell clusters on soft, PEG-LM hydrogels with higher expression of juvenile NP cell features compared to culturing on stiff PEG-LM hydrogels. Some degenerate, human NP cells are also able to form N-cadherin positive cell clusters with some features of the juvenile NP cell. </p><p>The studies presented in this dissertation support the proposed hypothesis and establish the importance of soft, laminin-rich substrates in promoting NP cell clustering behaviors with associated features of a juvenile cell phenotype and morphology. Additionally, these studies establish a regulatory role for N-cadherin in juvenile NP cells and suggest that preservation of N-cadherin-mediated cell-cell contacts is important for preserving the juvenile NP cell phenotype and morphology. Furthermore, findings from this dissertation reveal the ability to promote degenerate, mature human NP cells towards a juvenile NP cell phenotype, demonstrating the potential to use PEG-LM hydrogels as a means for autologous cell delivery for the restoration of healthy IVD.</p> / Dissertation

The extracellular matrix (ECM) offers a protective shelter for cells and provides signaling paths important for cell to cell communication. ECM consists of basement membranes (BM) and interstitial matrix. BMs provide mechanical support for parenchymal cells, influence cell proliferation, survival, migration and differentiation. They are also important for tissue integrity. Laminins (LM) are the major non-collagenous component of BMs. Cell-ECM interactions, mediated by receptors, are indispensable during embryonic development, wound healing, remodeling and homeostasis of tissues. The integrins are the major cell-adhesion receptors. The expression of alpha11 integrin chain in the cornea is of great interest, as it is part of the alpha11beta1 integrin receptor for collagen type I, the predominant component of the corneal stroma. The aims were to thoroughly characterize the ECM in the developing and adult human eye, with particular focus on the cornea, LM and alpha11 integrin chains, and to examine alpha11 integrin chain in an animal model of corneal wound healing and remodeling. Human fetal eyes, 9-20 weeks of gestation (wg), and adult human corneas with different diagnosis were treated for immunohistochemistry with specific antibodies against LM and alpha11 integrin chains. Normal and knockout (ko) mice were treated with laser surgery to create a deep wound in the corneal stroma. The wound healing process was followed at different time points. The cellular source of alpha11 integrin chain was studied in cell cultures. In the fetal eyes, the BM of the corneal epithelium, the Descemet’s membrane (DM) and the Bruch’s membrane each had their specific combinations of LM chains and time line of development, whereas the lens capsule and the internal limiting membrane showed constant LM chain patterns. The epithelial BMs of normal and diseased adult corneas contained similar LM chains. The normal morphology of the epithelial BM was altered in the different diseases, particularly when scarring was present. In the scarred keratoconus corneas there were excessive LM chains. The majority of keratoconus corneas also expressed extra LM chains in the DM. At 10-17 wg alpha11 integrin chain was present in the human corneal stroma, especially in the anterior portion, but it was scarce at 20 wg, in normal adult corneas and in Fuchs’ endothelial dystrophy. In contrast, it was increased in the anterior portion of the stroma in keratoconus corneas with scarring. Alpha11 integrin ko mice had a defective healing with subsequent thinner corneas. Alpha11 integrin expression correlated to the presence of alpha-smooth muscle actin in vivo as well as in vitro. The distinct spatial and temporal patterns of distribution for alpha11 integrin and each of the LM chains suggest that they play an important role in human ocular differentiation. The selectively affected LM composition and the novel expression of alpha11 integrin chain in scarred keratoconus corneas as well as the pathologic healing in ko mice, indicate that alpha11 integrin and LM chains also play an important role in the process of corneal healing, remodeling and scarring and might participate in the pathogenesis of corneal disease. This knowledge is of practical importance for future topical therapeutic agents capable of modulating the corneal wound healing processes.

Multizelluläre Gemeinschaften in Form bakterieller Biofilme stellen aus medizinischer Sicht ein großes klinisches Problem dar. Häufig lassen sich chronische oder rezidivierende Erkrankungen aber auch nosokomiale Infektionen auf die multizelluläre Lebensweise von humanpathogenen Erregern zurückführen. Sowohl fakultativ als auch obligat pathogene Escherichia coli-Stämme besitzen eine Vielzahl unterschiedlicher Faktoren, die die Biofilmbildung beeinflussen. Daran beteiligt sind unter anderem Flagellen, extrazelluläre polymere Substanzen, Adhäsine oder Oberflächen-assoziierte Proteine wie Autotransporter. Gegenstand der vorliegenden Arbeit ist die funktionale Charakterisierung des Proteins Antigen 43 (Ag43). Aufgrund seiner Autoaggregation-vermittelnden Eigenschaft trägt Ag43 ebenfalls zur Mikrokoloniebildung und Biofilmreifung bei. Antigen 43 ist ein Autotransporterprotein, welches innerhalb der Bakterienspezies Escherichia coli weit verbreitet ist. Interessanterweise besitzen viele E. coli-Isolate gleich mehrere identische oder ähnliche Kopien von agn43, die in der Regel von variablen Genombereichen (genomische Inseln, Plasmide) kodiert werden. Am Beispiel der Antigen 43-Varianten des uropathogenen Escherichia coli (UPEC)-Stammes 536 (O6:K15:H31), des kommensalen E. coli Isolats Nissle 1917 (O6:K5:H1) sowie des E. coli K-12-Laborstammes MG1655 (OR:H48:K-) ist die Bedeutung des Autotransporterproteins im Rahmen dieser Arbeit näher untersucht worden. Hierfür wurden die verschiedenen agn43-Allele in ein geeignetes Vektorsystem kloniert und im Adhäsin-freien Escherichia coli K-12-Stamm MG1655 &#916;fim&#916;flu exprimiert. Da Antigen 43 in Wildtypstämmen posttranslational glykosyliert vorliegt, sind die Experimente zusätzlich unter Einfluß der heterologen, AIDA I-spezifischen Heptosyltransferase Aah (‘Autotransporter Adhesin Heptosyltransferase’) durchgeführt worden. Anhand von Bindungsstudien (intermolekulare Autoaggregation, Zelladhäsionstests) wurde gezeigt, daß sich einzelne Ag43-Varianten teilweise in ihren Eigenschaften unterscheiden. Im direkten Vergleich mit AIDA-I (‘Adhesin Involved in Diffuse Adherence’) enteropathogener Escherichia coli (EPEC) konnte für Ag43 nur eine Funktion als schwaches Adhäsin nachgewiesen werden. Die heterologe O-Glykosylierung beeinflußte die Funktionalität des Antigen 43 in unterschiedlichem Ausmaß. Je nach Autotransporter-Variante führte die Heptosylierung entweder zu signifikant reduzierten Affinitäten, oder sie hatte keinen Effekt auf die Bindungskapazität des Ag43. Antigen 43 weist zudem strukturelle Homologien zu vergleichbaren Domänen anderer Autotransporter auf. Für viele dieser Proteine konnte bereits eine adhäsive oder invasive Funktion nachgewiesen werden. Eine mögliche Interaktion von Ag43 mit eukaryontischen Rezeptoren ist hingegen noch nicht bzw. nur unvollständig untersucht worden. In Overlay assays und ELISAs wurde für Antigen 43 hier erstmals die spezifische Bindung an die extrazellulären Matrixkomponenten Kollagen und Laminin gezeigt. Zusammenfassend deuten die Ergebnisse dieser Arbeit darauf hin, daß das Escherichia coli spezifische Autotransporterprotein Antigen 43 nicht nur an der bakteriellen Biofilmbildung, sondern auch an der Besiedlung epithelialer Gewebe beteiligt sein kann. Seine Expression verschafft Bakterien einen Kolonisationsvorteil, der mit erhöhter Fitneß einhergeht. Die Aah-vermittelte O-Glykosylierung scheint für die Funktionalität von Ag43 nicht zwingend erforderlich zu sein. Des weiteren ist im Rahmen der vorliegenden Arbeit ein Testsystem entwickelt worden, das auf der Basis von Fluoreszenz-in-situ-Hybridisierungen (FISH) die Differenzierung von verschiedenen (uro-)pathogenen Mikroorganismen ermöglicht. Das etablierte Protokoll eignet sich nicht nur für die diagnostische Erregeridentifizierung, sondern auch in Abhängigkeit des Probenmaterials zur Untersuchung von (Multispezies-)Biofilmen. / Bacteria living in multicellular communities, i.e. biofilms, have evolved into a major health problem. Most chronic or recurrent diseases including nosocomial infections can be traced back to the multicellular lifestyle of pathogenic agents. Both facultative and obligate pathogenic strains of Escherichia coli express a multitude of diverse factors contributing to biofilm formation like flagella, extracellular polymeric substances, adhesins, or surface-exposed proteins, e.g. autotransporters. This work presents the functional characterization of the surface-associated protein antigen 43 (Ag43). Due to its autoaggregating phenotype, Ag43 is also involved in the formation of microcolonies and biofilms. Antigen 43 represents an autotransporter protein frequently expressed by all Escherichia coli pathotypes. Interestingly, many E. coli isolates encode multiple identical or orthologous copies of agn43 which are usually associated with mobile genetic elements (genomic islands, plasmids). Here, the antigen 43 variants of uropathogenic Escherichia coli (UPEC) strain 536 (O6:K15:H31), commensal isolate E. coli Nissle 1917 (O6:K5:H1), and E. coli K-12 strain MG1655 (OR:H48:K-) have been analyzed. For this purpose, the different agn43-alleles were cloned into a capable vector system and subsequently expressed in Escherichia coli K-12 strain MG1655 &#916;fim&#916;flu lacking the genes encoding type 1-fimbrial adhesins (fim) and antigen 43 (flu). Due to the occurrence of posttranslational glycosylation of Ag43 in wild type strains, experiments were additionally carried out upon co-expression of the heterologous AIDA-I-specific heptosyl transferase Aah (Autotransporter Adhesin Heptosyltransferase). On the basis of binding studies (intermolecular autoaggregation, adhesion to eukaryotic cells) individual properties could be detected for each Ag43-variant. However, compared to AIDA-I (Adhesin Involved in Diffuse Adherence) of enteropathogenic Escherichia coli (EPEC) strains, antigen 43 was shown to act as a weak adhesin. Additionally, O-glycosylation partly influenced protein functions. Depending on the variant tested, posttranslational modification by Aah either resulted in significantly reduced affinities or had no effect on the binding capacity of Ag43. Furthermore, antigen 43 exhibits structural homologies compared to certain domains of related autotransporters. For many of these proteins, an adhesion- or invasion-mediating function could be demonstrated. To date, the interaction of Ag43 with a putative eukaryotic receptor has not been determined, yet. Here, it is shown for the first time that antigen 43 specifically binds to components of the extracellular matrix. Overlay assays and ELISAs identified collagen and laminin as epithelial receptors for Ag43. Taken together, the results obtained in this study confirm the functional role of antigen 43 in biofilm formation as well as its effect during bacterial colonization of epithelial tissues. Expression of the Escherichia coli-specific autotransporter protein is directly correlated to the improved fitness of bacteria infecting the human host. Aah-mediated O-glycosylation seems not to be strictly required for the function of Ag43. The second aim of this work was the design and development of a test system which can be used for the differentiation of (uro-)pathogenic microorganisms in biofilms. For this purpose, the FISH (Fluorescence-in-situ-Hybridization)-technique has been optimized for diagnostic applications. Depending on the specimen, the protocol can also be adapted to the analysis of (multispecies) biofilms.

Trypanosoma cruzi expressa um grupo de glicoproteínas de superfície, as Tc85, que pertencem à superfamília gênica das gp85/trans-sialidases. Neste trabalho mostramos um modelo de estrutura para um membro desta família, a proteína Tc85-11, a qual tem propriedades adesivas para laminina e para a superfície da célula. Esta estrutura consiste em um domínio N-terminal com pregamento ß-propeller e um domínio C-terminal ß-sandwich conectados por uma um &#945;-hélice longa. A proteína recombinante que corresponde ao domínio amino terminal (Tc85-N), mas não ao domínio carboxi-terminal (Tc85-C), liga-se a laminina de uma maneira específica. Cinco peptídeos sintéticos, contidos no domínio N-terminal, aderem na superfície das células LLC-MK2 e inibem a infecção pelo T. cruzi. Dois destes peptídeos também podem inibir a interação Tc85-N - laminina de modo específico e poderiam representar o sítio de ligação a laminina. Estes resultados reforçam a hipótese que a Tc85-11 é uma proteína multi-adesiva, já que o domínio C-terminal, liga-se a citoqueratina-18. Por outro lado, o tratamento de células em cultura com Tc85-N aumenta a expressão de laminina, efeito já observado in vivo e quando culturas de células foram incubadas com antígenos liberados pelo parasita. A Tc85-11 pode ter, por tanto, um papel relevante na interação do parasita com o hospedeiro modulando ainda a expressão de componentes da matriz extracelular. / Trypanosoma cruzi expresses the Tc85 proteins, a set of surface glycoproteins belonging to the gp85/trans-sialidase supergene family. In this report we show a structure model for Tc85-11 a member of this family, which has adhesive properties to laminin and to the host cell surfaces. That structure consists in an N-terminus &#946;-propeller and a C-terminus &#946;-sandwich domains connected by a long &#945;-helix. The recombinant protein corresponding to the N-domain (Tc85-N), but not to the C-domain (Tc85-C), was able to bind laminin in a specific manner. Five synthetic 20-mer peptides from the N-domain adhere onto LLC-MK2 cell surface and inhibit the T. cruzi infection. Two of these peptides can also inhibit specifically Tc85-N - laminin interaction and may represent the laminin-binding site. These results reinforce the hypothesis that the Tc85-11 protein is a multi-adhesive protein, since it also binds to citokeratin-18 by C-terminus domain. On the other hand, the treatment of host cells with Tc85-N increases the expression of laminin in the cell culture, as was previously reported for treatment with T. cruzi released antigens. In summary, Tc85-11 protein may play an important role in the host-parasite interaction, including the modulation of ECM expression.

A disseminação metastática de células tumorais malignas envolve múltiplas etapas e é uma das principais causas de mortalidade. O microambiente tumoral apresenta um importante papel no processo de tumorigênese. A laminina é um componente do microambiente que pode ser clivado em peptídeos bioativos, como o peptídeo AG73 (domínio globular da cadeia 1) que aumenta a formação/atividade de invadopódios. Invadopódios são protrusões de membrana ricas em actina com atividade proteolítica da matriz pericelular. Além da actina, os invadopódios exibem outras proteínas importantes, como a cortactina e o MT1-MMP. Recentemente, descobriu-se que a podoplanina pode desempenhar um papel importante na atividade dos invadopódios. A podoplanina é uma glicoproteína transmembrana do tipo I, intimamente associada à progressão maligna do câncer. A podoplanina e o peptídeo AG73 influenciam a biologia do câncer. Tal particularidade levou-nos a investigar o papel do peptídeo AG73 na regulação da da podoplanina e invadopódios em linhagem celular derivada de carcinoma de células escamosas oral (Cal 27). As células foram cultivadas em DMEM com SFB e tratadas com o peptídeo AG73. As células tratadas por peptídeo de sequência embaralhada serviram como controles. Imunofluorescência foi realizada para analisar os níveis de proteína de podoplanina, cortactina e MT1-MMP. Os resultados mostraram que o peptídeo AG73 diminuiu os níveis de podoplanina nas células Cal27 em comparação aos controles. Nenhuma alteração foi observada em relação à cortactina e MT1-MMP. Estes resultados foram confirmados por imunofluorescência. As células tratadas com AG73 diminuíram a distribuição da podoplanina em todo o citoplasma em comparação com os controles. Nossos resultados sugerem que o peptídeo derivado da laminina AG73 inibe a podoplanina, uma molécula reguladora do câncer, em células malignas humanas. / The metastatic spread of malignant tumor cells involves multiple steps, and is one of the major causes of mortality. The tumor microenvironment has an important role in tumorigenesis process. Laminin is a component of the microenvironment that can be cleaved into bioactive peptides, such as peptide AG73 (globular domain of 1 chain) that increase invadopodia formation/activity. Invadopodia are actin-rich membrane protrusions with proteolytic activity of peri-cellular matrix. In addition to actin invadopodia exhibit other key proteins such as cortactin and MT1-MMP. Recently it has been discovered that podoplanin may play an important role in the invadopodia activity. Podoplanin is a type I transmembrane glycoprotein, closely associated with the malignant progression of cancer. Podoplanin and the peptide AG73 influence cancer biology. This prompted us to investigate the role of peptide AG73 in the regulation of podoplanin and invadopodia formation in a cell line derived from oral squamous cell carcinoma (Cal 27). Cells were cultured in DMEM with FBS and treated with peptide AG73. Cells treated by scrambled peptide served as controls. Immunoblot was carried out to analyze protein levels of podoplanin, cortactin and MT1-MMP. Results showed that the peptide AG73 decreased podoplanin levels in Cal27 cells compared to controls. No alterations were observed with regard to cortactin and MT1-MMP. These results were confirmed by immunofluorescence. Cells treated by AG73 decreased podoplanin distribution throughout the cytoplasm compared to controls. Our results suggest that the laminin-derived peptide AG73 inhibits podoplanin, a cancer regulator molecule, in human malignant cells.

Made available in DSpace on 2015-03-04T18:50:49Z (GMT). No. of bitstreams: 1 dissertacao_aline.pdf: 3429789 bytes, checksum: 82cf452f7244c55bd99e241aadcec89f (MD5) Previous issue date: 2007-05-07 / Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior / The extracelullar matrix (ECM) is formed by an assembly of proteins and glycoproteins which surrounds the cells, in various tissues. The laminin is a glycoprotein localized in the ECM that consists of three polypeptidic chains cross- shaped. It functions by anchoring epithelial cells to basal lamin, through associations with integrins, collagen, elastin and fibronectin. Integrins are adhesion receptors localized on cellular surface, that mediate interactions between cells and ECM. The interactions between proteins of ECM and cellular proteins are crucial for many normal biological processes such as cell differentiation, signal transduction, immune responses, wound healing and metastasis formation. The nature of interactions between laminin and integrin has not been fully identified yet. The present work aims to analyze, in an molecular scale, the interaction between human α6β1 integrin and laminin 111. In this work we conducted many studies, including: i) structural and sequential alignments of β-propeller and βA regions in lacking I-domain integrins; ii) model building of β-propeller e βA regions of α6β1 integrin complexed with small ECD or RGD peptide antagonists; iii) sequential alignment of various laminin LG domains; iv) model building of laminin 111 LG1 domain; and v) model building of the first described complex between the N-terminal portion of α6β1 integrin and laminin 111 LG1 domain. In order to do this, homology modeling and molecular dynamics techniques were applied, together with alignments between integrins α and β chains, and laminin LG domains. Our initial results show that the loop between blades 3 and 4 of α6 integrin subunit discriminates ligands by electrostatic interactions. Therefore we assumed that α6β1 integrin preferentially interacts with ECDF based peptides. It was demonstrated that the laminin 111 LG1 domain interacts with α6β1 integrin by the contact of this H β strand and α6 β-propeller. Further, by its electrostatic function and proximity to H β strand, LG1 residue Asp82 adheres to Mg+2 containing MIDAS in α6β1 integrin. This interaction appears to be indispensable for α6β1 and laminin 111 binding. / A matriz extracelular (ECM) é definida como um complexo de proteínas e glicoproteínas que envolve as células nos mais diversos tecidos. A laminina é uma glicoproteína que é formada por três cadeias polipeptídicas dispostas em cruz. Sua função é a de ancorar as células epiteliais à ECM, da qual faz parte, através de associações a outras proteínas como as integrinas, o colágeno, a elastina e a fibronectina. As integrinas são receptores de adesão localizadas na superfície celular, que medeiam a interação célula-ECM. Estas interações são cruciais para diversos processos biológicos, tais como a diferenciação celular, a transdução de sinais, a resposta imunológica, a cicatrização de ferimentos e a formação de metástases. Porém a forma estrutural com que a interação entre a integrina e a laminina ocorre ainda não foi esclarecida. Neste contexto este trabalho visa analisar, em escala molecular, a forma com que a integrina α6β1 e a laminina 111 humanas interagem. Assim, foram conduzidos vários estudos, entre eles: i) alinhamentos estruturais e seqüenciais das regiões β-propeller e βA de integrinas não-possuidoras de domínio I; ii) construção do modelo das regiões β-propeller e βA da integrina α6β1 em complexo com pequenos inibidores peptídicos do tipo ECD ou RGD; iii) alinhamento entre os domínios LG de lamininas; iv) construção do modelo do domínio LG1 da laminina 111; e v) construção do primeiro modelo descrito do complexo formado pela porção N- terminal da integrina α6β1 e o domínio LG1 da laminina 111. Para tanto, foram aplicadas as técnicas de modelagem por homologia e dinâmica molecular, além de alinhamentos entre as cadeias α e β de integrinas, e dos domínios LG de lamininas. Inicialmente os resultados mostraram que o loop que corresponde à região entre os subdomínios D2 e D3 da cadeia α6 discrimina ligantes por interações eletrostáticas, e a partir disso, que a integrina α6β1 possui interação preferencialmente com peptídeos do tipo ECDF. Foi mostrado que o domínio LG1 de laminina 111 interage com a integrina α6β1 pelo contato da fita β H com o β-propeller de α6. Além disso, por seu caráter eletrostático e proximidade à fita β H , o resíduo Asp82 de LG1 se adere ao íon Mg+2 do MIDAS da integrina α6β1, e que esta interação é indispensável à ligação entre as duas proteínas.