Zellkultursysteme mit Krebszelllinien werden seit Langem zur Untersuchung der Interakti-on zwischen Pathogenen und ihren Wirtszellen eingesetzt. Diese Systeme eignen sich aufgrund der reduzierten Komplexität für die Analyse einzelner Faktoren, spiegeln jedoch nicht den Zustand primärer Zellen oder die komplexe Gewebestruktur wieder. Um die Beschränkungen zu umgehen, wurden in dieser Arbeit neue Modelle etabliert auf der Grundlage von reversibel immortalisierten humanen Primärzellen und ex vivo Kultur von intaktem humanem Eileitergewebe. Infektionen mit dem humanpathogenen Bakterium Chlamydia trachomatis, welches chronische Schmerzen oder Unfruchtbarkeit auslösen kann, wurden in diesen Modellen untersucht. Reversible Immortalisierung wurde mit pri-mären human Eileiterzellen (FT Zellen) und humanen Nabelschnurzellen (HUVEC) durchgeführt. Das System basiert auf lentiviralem Gentransfer und dem Cre-lox-System. HUVEC Zellen wurden mit Kombinationen der Onkoproteine hTERT, SV40T und Bmi1 immortalisiert. Immortalisierung von FT Zellen wurde mit SV40T und Bmi1 erreicht. Eine Analyse der FT Zelllinien zeigte Veränderungen des Karyotyps durch die Immortalisie-rung. Bemerkenswerterweise konnten die Stammzellmarker CD44 und Oct4 in FT Zellen nachgewiesen werden. Ex vivo Gewebekultur humaner Eileiter wurde als stabiles Infekti-onsmodel für Chlamydia trachomatis etabliert. Mittels hochauflösender Konfokal-mikroskopie wurde gezeigt, dass die Infektion mit C. trachomatis tiefgreifende Verände-rungen im Epithel der Mukosa auslöst und zum Verlust der Zelladhäsion und Zellpolarität führt. Ein erhöhter Anteil apoptotischer Zellen wurde nach Infektion mit Serovar D beo-bachtet, einem klinischen Isolat des Genitaltraktes. Dieses Ergebnis steht im Gegensatz zu Infektionen mit dem Laborstamm Serovar L2. Phänotypische Veränderungen in nicht infizierten Zellen weisen auf die Existenz parakriner Signalwege während der akuten In-fektion und Veränderung der epithelialen Homeostase hin. / Cell culture systems with cancer-derived cell lines have long been used to study the interaction between pathogens and their host cells. Due to reduced complexity these systems are convenient for the analysis of single factors; however, they do not represent the condition of primary cells or the complex tissue structure. To circumvent these limitations new models were established in this study on the basis of reversibly immortalized human primary cells and ex vivo culture of intact human fallopian tube tissue. Infections with the human pathogenic bacterium Chlamydia trachomatis, which can lead to chronic pain or infertility, were analyzed in these models. Reversible immortalization was applied to primary human fallopian tube (FT) cells and human umbilical vein cells (HUVEC). This system is based on lentiviral gene transfer and the Cre-lox-system. HUVEC cells were immortalized with a combination of two of the oncoproteins hTERT, SV40T and Bmi1. Immortalization of FT cells was achieved with SV40T and Bmi1. Analysis of FT cell lines revealed changes of the karyotype induced by immortalization. Remarkably, the stem cell markers CD44 and Oct4 were detected in FT cells. Ex vivo tissue culture of human fallopian tubes was established as stable and reliable infection model for Chlamydia trachomatis. Via high resolution confocal analysis the infection with C. trachomatis was discovered to trigger profound changes in the epithelial mucosa, causing loss of cell adhesion and polarity. Interestingly, an increase in the rate of apoptotic cells was observed after infection with serovar D, a clinical genital tract isolate. This finding is in contrast to infections with serovar L2, a laboratory strain. Phenotypic changes in non-infected cells suggest the existence of paracrine signalling during acute infection and change in epithelial homeostasis.

Introdução: Chlamydia trachomatis (CT) é causa da infecção sexualmente transmitida de origem bacteriana mais comum. Na mulher, a infecção genital pela CT pode causar cervicite, uretrite, endometrite, salpingite. Infecções persistentes ou recorrentes provavelmente representam um importante fator de risco para o desenvolvimento de sequelas associadas, como dor pélvica crônica, gravidez ectópica e infertilidade por fator tubário. A lectina ligadora de manose (MBL), componente doo sistema imune inato, tem importante papel na defesa antimicrobiana, reconhecendo vírus, fungos e patógenos bacterianos. O gene que codifica para a MBL é polimórfico, e a substituição de um único nucleotídeo resulta na produção de uma proteína instável, que é rapidamente degradada. Objetivo: Avaliar se mulheres brasileiras portadoras de um polimorfismo do gene da MBL apresentam diferentes susceptibilidades para a ocorrência de obstrução tubária, na presença ou ausência de uma infecção prévia por Chlamydia trachomatis. Métodos: Em estudo caso-controle, foram avaliadas 75 pacientes com obstrução tubária e 75 pacientes com tubas pérvias, atendidas na Divisão de Ginecologia do Hospital das Clínicas da Universidade de São Paulo (HCFMUSP). Anticorpos IgG anti-CT foram mensurados através de um ensaio de imunoabsorção enzimática para investigar uma infecção prévia pela CT. Para o polimorfismo, realizou-se coleta de células bucais e o DNA extraído foi analisado através de reação em cadeia da polimerase (PCR), digestão de endonuclease e gel de eletroforese, utilizando pares de primers específicos para a região polimórfica. Todo material foi mantido a -80°C e enviado em gelo seco para a Division of Immunology and Infectious Diseases da Weill Cornell Medicine em Nova York. Associações entre genótipos de MBL ou alelos e permeabilidade tubária foram analisadas pelo teste de qui-quadrado de Pearson com ou sem correção de Yates. Resultados: Não houve diferença na detecção de anticorpos da CT entre os grupos. Mulheres com tubas obstruídas tiveram uma prevalência maior do genótipo AB (36%) versus (16%), resultado estatisticamente significativo (p < 0,01). De maneira semelhante, a distribuição do alelo A e do alelo variante B também apresentaram diferenças significantes entre os grupos (p < 0,01). Conclusão: Os achados sugerem, que embora a exposição à Chlamydia trachomatis tenha sido semelhante em ambos os grupos, a presença do alelo variante B do gene que codifica para a MBL aumenta o risco de desenvolvimento da obstrução tubária, subsequente à infecção pela CT ou outros agentes infecciosos. Nas mulheres brasileiras avaliadas a presença de tal polimorfismo genético aumentou a probabilidade de obstrução tubária em consequência de uma infecção do trato genital / Introduction: Chlamydia trachomatis (CT) is the cause of the most common bacterial sexually transmitted infection. In women, genital CT infection may cause cervicitis, urethritis, endometritis, salpingitis. Persistent or recurrent infections probably represent an important risk factor for the development of associated sequelae, such as chronic pelvic pain, ectopic pregnancy and tubal factor infertility. Mannose-binding lectin (MBL), a component of the innate immune system, has an important role in antimicrobial defense, recognizing viral, bacterial and fungal pathogens. The gene coding for MBL is polymorphic and a single nucleotide substitution results in production of an unstable protein, that is rapidly degraded. Objective: To evaluate whether Brazilian women with a polymorphism in the MBL gene present different susceptibilities to the occurrence of fallopian tube damage, in the presence or absence of a previous infection by CT. Method: In a case-control study, 75 patients with tubal obstruction and 75 patients with patent tubes were studied, all seen at the Gynecology Division of the Hospital das Clínicas of the University of São Paulo (HCFMUSP). IgG anti-CT antibodies were measured by enzyme-linked immunoassay to investigate a previous CT infection. For the polymorphism analysis, buccal cells were collected and the extracted DNA was analyzed by polymerase chain reaction (PCR), endonuclease digestion and gel electrophoresis using primer pairs specific for the polymorphic region. All material was maintained at -80 ° C and sent on dry ice to the Division of Immunology and Infectious Diseases at Weill Cornell Medicine in New York. Associations between MBL genotypes or alleles and tubal permeability were analyzed by the Pearson chi-square test with or without Yates correction. Results: There was no difference in CT antibody detection between the two groups. Women with obstructed tubes had a higher prevalence of being positive for the heterogenous genotype AB (36%) versus (16%) (p < 0.01). Similarly, the distribution of the normal A allele and variant B allele were also significant different between the two groups (p < 0,01). Conclusion: The findings suggest that while exposure to CT was similar in both groups of women the presence of the variant MBL B allele increases the risk for development of tubal obstruction, subsequent to a CT or other infection. In the Brazilian women evaluated possession of this genetic polymorphism increased the likelihood that blocked fallopian tubes will be a consequence of a genital tract infection

Introdução: Chlamydia trachomatis (CT) é causa da infecção sexualmente transmitida de origem bacteriana mais comum. Na mulher, a infecção genital pela CT pode causar cervicite, uretrite, endometrite, salpingite. Infecções persistentes ou recorrentes provavelmente representam um importante fator de risco para o desenvolvimento de sequelas associadas, como dor pélvica crônica, gravidez ectópica e infertilidade por fator tubário. A lectina ligadora de manose (MBL), componente doo sistema imune inato, tem importante papel na defesa antimicrobiana, reconhecendo vírus, fungos e patógenos bacterianos. O gene que codifica para a MBL é polimórfico, e a substituição de um único nucleotídeo resulta na produção de uma proteína instável, que é rapidamente degradada. Objetivo: Avaliar se mulheres brasileiras portadoras de um polimorfismo do gene da MBL apresentam diferentes susceptibilidades para a ocorrência de obstrução tubária, na presença ou ausência de uma infecção prévia por Chlamydia trachomatis. Métodos: Em estudo caso-controle, foram avaliadas 75 pacientes com obstrução tubária e 75 pacientes com tubas pérvias, atendidas na Divisão de Ginecologia do Hospital das Clínicas da Universidade de São Paulo (HCFMUSP). Anticorpos IgG anti-CT foram mensurados através de um ensaio de imunoabsorção enzimática para investigar uma infecção prévia pela CT. Para o polimorfismo, realizou-se coleta de células bucais e o DNA extraído foi analisado através de reação em cadeia da polimerase (PCR), digestão de endonuclease e gel de eletroforese, utilizando pares de primers específicos para a região polimórfica. Todo material foi mantido a -80°C e enviado em gelo seco para a Division of Immunology and Infectious Diseases da Weill Cornell Medicine em Nova York. Associações entre genótipos de MBL ou alelos e permeabilidade tubária foram analisadas pelo teste de qui-quadrado de Pearson com ou sem correção de Yates. Resultados: Não houve diferença na detecção de anticorpos da CT entre os grupos. Mulheres com tubas obstruídas tiveram uma prevalência maior do genótipo AB (36%) versus (16%), resultado estatisticamente significativo (p < 0,01). De maneira semelhante, a distribuição do alelo A e do alelo variante B também apresentaram diferenças significantes entre os grupos (p < 0,01). Conclusão: Os achados sugerem, que embora a exposição à Chlamydia trachomatis tenha sido semelhante em ambos os grupos, a presença do alelo variante B do gene que codifica para a MBL aumenta o risco de desenvolvimento da obstrução tubária, subsequente à infecção pela CT ou outros agentes infecciosos. Nas mulheres brasileiras avaliadas a presença de tal polimorfismo genético aumentou a probabilidade de obstrução tubária em consequência de uma infecção do trato genital / Introduction: Chlamydia trachomatis (CT) is the cause of the most common bacterial sexually transmitted infection. In women, genital CT infection may cause cervicitis, urethritis, endometritis, salpingitis. Persistent or recurrent infections probably represent an important risk factor for the development of associated sequelae, such as chronic pelvic pain, ectopic pregnancy and tubal factor infertility. Mannose-binding lectin (MBL), a component of the innate immune system, has an important role in antimicrobial defense, recognizing viral, bacterial and fungal pathogens. The gene coding for MBL is polymorphic and a single nucleotide substitution results in production of an unstable protein, that is rapidly degraded. Objective: To evaluate whether Brazilian women with a polymorphism in the MBL gene present different susceptibilities to the occurrence of fallopian tube damage, in the presence or absence of a previous infection by CT. Method: In a case-control study, 75 patients with tubal obstruction and 75 patients with patent tubes were studied, all seen at the Gynecology Division of the Hospital das Clínicas of the University of São Paulo (HCFMUSP). IgG anti-CT antibodies were measured by enzyme-linked immunoassay to investigate a previous CT infection. For the polymorphism analysis, buccal cells were collected and the extracted DNA was analyzed by polymerase chain reaction (PCR), endonuclease digestion and gel electrophoresis using primer pairs specific for the polymorphic region. All material was maintained at -80 ° C and sent on dry ice to the Division of Immunology and Infectious Diseases at Weill Cornell Medicine in New York. Associations between MBL genotypes or alleles and tubal permeability were analyzed by the Pearson chi-square test with or without Yates correction. Results: There was no difference in CT antibody detection between the two groups. Women with obstructed tubes had a higher prevalence of being positive for the heterogenous genotype AB (36%) versus (16%) (p < 0.01). Similarly, the distribution of the normal A allele and variant B allele were also significant different between the two groups (p < 0,01). Conclusion: The findings suggest that while exposure to CT was similar in both groups of women the presence of the variant MBL B allele increases the risk for development of tubal obstruction, subsequent to a CT or other infection. In the Brazilian women evaluated possession of this genetic polymorphism increased the likelihood that blocked fallopian tubes will be a consequence of a genital tract infection

Bislang wurden Bullen in der Milchrinderzucht anhand des Töchterleistungsvergleiches bewertet, was zur Folge hatte, dass die Zuchtwertschätzung erst nach fünf Jahren vorlag (REENTS UND REINHARDT, 2007). Durch die 2010 eingeführte genomische Zuchtwertschätzung werden Bullenkälber bereits in den ersten Lebensmonaten geprüft und die erfolgreichen Bullen ab einem Alter von 12 Monaten zur Spermaproduktion von den Besamungsstationen eingesetzt. Zu bedenken ist dabei, dass Jungbullen unter 2 Jahren lediglich ein durchschnittliches Ejakulatvolumen von 2-4 ml aufweisen und die Spermienkonzentration deutlich unter der von Altbullen liegt. Es müssen daher entweder mehr Jungbullen zum Einsatz kommen oder die Besamungsportionen müssen in ihrer Spermienkon-zentration reduziert werden. Eine am Institut für Nutztiergenetik Mariensee ent-wickelte Methode, der Sperm-Intra-Fallopian-Transfer (SIFT®), ermöglicht es, minimal dosierte Spermaportionen unchirurgisch in den Eileiter zu übertragen (GROSSFELD ET AL., 2011B). Ein wesentlicher Vorteil dieser Methode ist die Re-duzierung des Besamungsvolumens unter Beibehaltung des Volu-men/Mengenverhältnisses. Zielsetzung dieser Arbeit war es, eine in Volumen und Spermienzahl reduzierte Spermaportion neu zu konfektionieren und ein angepasstes Kühl- sowie Gefrierprotokoll zu entwickeln. Hierbei sollten vergleichbare Auftauqualitäten wie in einer üblichen Besamungsportion erreicht werden. Die Qualität des Spermas wurde dabei sowohl unter Laborbedingungen als auch in Testbesamungen an zufällig ausgewählten Färsen und Kühen evaluiert. In ersten Screeningversuchen wurde zunächst ein Tiefgefrierverfahren für gering dosierte Verpackungssysteme (Nanostraw) mit einem Volumen bis 50 µl entwickelt. Dabei sollten vergleichbare Auftauqualitäten üblicher Besamungs-portionen erreicht werden. Es wurden sechs verschiedene Kühlkurven (A-D, D+ und E) entwickelt. Dabei zeigte sich, dass aufgrund der Stickstoff-volumeneinströme ein Haltepunkt bei -8°C in keiner der Einfrierkurven für kleine Volumina zu etablieren war. Dies ist u.a. auf die bauartbedingten Vorgaben des verwendeten Einfrierautomaten zurückzuführen. Ebenso wurde der Zusatz von 7,5% Glyzerin im Verdünner bei dem Gefrierprotokoll D+ aufgrund der Datenlage des Vorversuchs für den Hauptversuch verworfen. Die Proben wurden bullenindividuell erhoben, zur besseren statistischen Absicherung jedoch für die Auswertung zusammengefasst. Aus den Screeningversuchen erschien das Kryokonservierungsprotokoll D+ für den Nanostraw unter Laborbedingungen am geeignetsten und wurde im Thermoresistenztest, der den Bedingungen im Hinblick auf die Temperatur im Uterus bzw. Eileiter ähnlich ist, getestet. Weiterhin fand ein Vergleich der spermatologischen Qualitätsparameter nach dem Auftauen zwischen der Kontroll- und der Nanostrawgruppe mit der Kühlkurve D+ im Thermoresistenztest statt. Im praktischen Teil der Arbeit wurde die Methode SIFT® zunächst anhand eines Versuchs mit geschlechtsdifferenziertem Frischsamen praktiziert. Dabei wurden 20 Färsen mittelseiner Prid®α 1, 55g Progesteron-Spirale und zehn Kühe mit einem Ovsynch Programm synchronisiert. In einem weiteren Besamungsversuch konnte das Sperma aus den Nanostraws verwendet werden, das vorher kryokonserviert worden ist. Es wurden nur spontan brünstige Färsen und Kühe in den Eileiter besamt. Die Untersuchungen führten zu folgenden Ergebnissen: 1) In den Screeningversuchen zeigte sich, dass der Anteil motiler Spermien signifikant (p≤0,05) abhängig von der Gefriergeschwindigkeit der Kühlkurve war. Der höchste Anteil motiler Spermien aus dem Nanostraw war in Kühlkurve D+ mit einem Anteil von 52,9% zu verzeichnen. Eine signifikante Zunahme des Anteils membranintakter Spermien von Kurve A zu B und D zu D+ war zu beobachten. Die Hälfte der Nanostrawspermien zeigten in der Kühlkurve D+ eine intakte Membranintegrität. Die Kontrollspermien lagen bei 56,9%. In Kurve D+ zeigten 66,6% der Spermien aus dem Nanostraw keine morphologischen Veränderungen, in der Kontrollgruppe waren dies 75,4%. Die Änderung der Glyzerinkonzentration im Verdünnungsmedium zeigte keine signifikante Verbesserung der Spermaqualitätsparameter nach dem Auftauen. 2) Im Thermoresistenztest erreichten die Spermien aus dem Nanostraw in allen gemessenen Parametern die Mindestanforderungen an die Spermaqualität nach Rath et al. (2009). So belief sich der Anteil membranintakter Spermien aus dem Nanostraw nach dreistündiger Inkubation bei 37°C auf 56,4%, die Kontrollstrawspermien lagen bei einem Anteil von 67,2%. Dabei zeigte der Faktor Bulle einen signifikanten Effekt (p≤0,01) auf den Anteil motiler und progressiver Spermien sowie den Anteil PI-negativer Spermien. Mögliche Effekte des Bullen auf die Qualität und Befruchtungsfähigkeit der Spermien nach dem Auftauen zeigten andere Versuchsanstellungen und Forschungshypothesen (JANUSKAUSKAS ET AL., 1996, KATHIRAVAN ET AL., 2011). 3) In dem ersten Besamungsversuch wurden 20 Färsen und zehn Kühen geschlechtsdifferenziertes Sperma mit der Methode SIFT® in den Eileiter über-tragen. 37,5% der Färsen und 33% der Kühe wurden tragend. Dabei konnte ein bullenindividueller dosisabhängiger Effekt festgestellt werden. 50% der Kühe, die mit 500.000 Samenzellen inseminiert wurden und 16% der Kühe, die mit 250.000 Samenzellen besamt wurden, wurden tragend. 4) Im Besamungsversuch mit dem kryokonservierten Sperma aus dem Na-nostraw wurden sechs spontan brünstige Rinder mittels SIFT®-Spermien in den Eileiter übertragen. Alle Tiere wurden als Kälber für die Follikelpunktion in einem anderen Versuch verwendet. Fünf Tiere wurden mit dem Bullen A besamt. 40% der Tiere wurden tragend. Lediglich ein Tier wurde mit dem Bullen B besamt. Das Trächtigkeitsergebnis war negativ. Die Kühe wurden mit dem Bullen A besamt, dieser erzielte eine Trächtigkeitsrate von 33%.

This thesis focuses on gynecopathology. It consists of a collection of seven papers published in pathology journals with impact factor. Introduction section contains selection of examples showing scientific application of molecular genetic methods. Further on the aims of individual research projects are described. The first project comprises histomophologic study of skin endometriosis addressing "mullerian" differentiation. A case report of a rare tumor namely borderline papillary serous tumor of the fimbriated end of the fallopian tube follows with molecular genetic analysis of KRAS, BRAF and p53 gene mutation status. Prospective longitudinal study on high grade squamous dysplasia (HSIL) of the cervix in HPV vaccinated women, so called DAV (dysplasia after vaccination), aims to elucidate pathogenesis of this phenomenon. Two other studies focus on incidence of fumarate hydratase deficient leiomyomas of the uterus and hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC). The aim of those studies is to improve our diagnostic capability and increase detection rate of the patients with HLRCC syndrome. Finally a new subtype of HSIL namely bizarre cell dysplasia is described in two separate studies. Conclusion remarks contemplate the role of molecular genetics in surgical pathology.

This thesis focuses on gynecopathology. It consists of a collection of seven papers published in pathology journals with impact factor. Introduction section contains selection of examples showing scientific application of molecular genetic methods. Further on the aims of individual research projects are described. The first project comprises histomophologic study of skin endometriosis addressing "mullerian" differentiation. A case report of a rare tumor namely borderline papillary serous tumor of the fimbriated end of the fallopian tube follows with molecular genetic analysis of KRAS, BRAF and p53 gene mutation status. Prospective longitudinal study on high grade squamous dysplasia (HSIL) of the cervix in HPV vaccinated women, so called DAV (dysplasia after vaccination), aims to elucidate pathogenesis of this phenomenon. Two other studies focus on incidence of fumarate hydratase deficient leiomyomas of the uterus and hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC). The aim of those studies is to improve our diagnostic capability and increase detection rate of the patients with HLRCC syndrome. Finally a new subtype of HSIL namely bizarre cell dysplasia is described in two separate studies. Conclusion remarks contemplate the role of molecular genetics in surgical pathology.

Motile cilia play critical roles in the human body, including expelling mucus from the lungs and facilitating the transport of oocytes and sperm through the fallopian tubes. Understanding the complex structure and motility of cilia, as well as the diseases associated with them, is of big importance. This study investigates the proteins expressed in ciliated cells from both respiratory and reproductive tissues using multiplex immunofluorescence. We determined the subcellular localization of 134 proteins in the fallopian tube, endometrium, cervix, nasopharynx, and bronchus, focusing on five subcellular regions: the cilia tip, transition zone, basal body, cytoplasm, and nucleus. This analysis was conducted using an automated image analysis method developed specifically for this project. Our findings revealed a high correlation in protein expression across all tissues, although several proteins exhibited distinct expression patterns between different tissues. Notably, the fallopian tube showed a higher correlation with the nasopharynx and bronchus than with the endometrium and cervix. Within these proteins, six gene clusters were identified, with the two largest clusters being strongly associated with ciliary structure. This study enhances our understanding of motile ciliary structures and ciliated cells, identifying key proteins for further research into cilia motion, function, and related diseases.