Universiẗat, Diss., 2008--Kassel.

Die vorliegende Arbeit beschäftigt sich mit der Identifikation neuer immunologischer Indikatoren (Biomarker) für den allgemeinen Gesundheitszustand von Hochleistungsmilchkühen. Diese Biomarker sollen möglichst einfach und schnell mittels eines Stalltests nachweisbar sein, weshalb die gelösten Proteine in der Milch im Fokus standen. Die neuen Biomarker sollten nicht nur Mastitis, sondern vor allem auch Entzündungen außerhalb des Euters anzeigen können. Zu Beginn sollte das Gesamtspektrum an Immunkomponenten erfasst werden, weshalb zunächst auf Proteinexpressionsebene angesetzt wurde. Das schloss die Analyse von vorhandenen Immunzellpopulationen in Blut- und Milchproben ein, um einen Überblick über potentielle Produzenten der immunologischen Indikatoren zu erhalten. Es konnte erstmals Cluster of Differentiation (CD) 25 (alpha-Kette des Interleukin-2-Rezeptors, IL2R) auf bovinen polymorphnukleären, neutrophilen Granulozyten (PMN) aus peripherem Blut nachgewiesen werden. Die Expression (mittlere Fluoreszenzintensität, MFI) von CD25 stieg dabei mit dem Schweregrad der entzündlichen Erkrankung an. Die Ergebnisse konnten auf Transkript- wie auch auf Proteinexpressionsebene bestätigt werden. Gleiche Tendenzen waren auch für Milchzellen erkennbar. In der statistischen Analyse zeigte CD25 auf PMN im peripheren Blut ein hohes Abgrenzungsvermögen für erkrankte Kühe. Die Messung von CD25 auf PMN könnte somit zur Bestimmung des allgemeinen Gesundheitszustandes von Hochleistungsmilchkühen genutzt werden.

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Abnormal epigenetic control is a common early event in tumour progression, and aberrant acetylation in particular has been implicated in tumourigenesis. Histone deacetylases (HDACs) are enzymes that regulate acetylation of chromatin and a variety of other non-histone substrates. Significantly, HDAC inhibitors are potent anti-proliferative agents and exhibit clinical activity in lymphoproliferative and haematological maligancy. However, the mechanistic details by which HDAC inhibitors affect proliferation remain to be elucidated. I have explored the cellular processes affected by HDAC inhibitors, and begun to illuminate a new pathway, regulated by HDAC, which impinges on the cellular effect of HDAC inhibitors. My results suggest that the proteins HR23B and Myd88 are important sensitivity determinants for HDAC inhibitor treatment, and that their interplay with HDAC6 dictates cell fate choice between survival by autophagy or apoptosis.

EVALUATION OF REDOX POTENTIAL AS A NOVEL BIOMARKER OF OXIDATIVE STRESS, INFLAMMATORY RESPONSE, AND SHOCK USING NANOPOROUS GOLD ELECTRODES Background: Redox potential is a chemical species’ affinity for electrons. Increased oxidant concentration is associated with disease1,2, yet there is not a way to measure systemic redox status.3 Redox potentiometry uses metal electrodes that do not work in blood because protein molecules adhere on the metal surface, blocking electron exchange. Methods: Nanoporous gold electrodes have large surface areas that allowed electron exchange to continue in blood.4 Redox potential was measured in blood with ascorbic acid, in cardiac bypass patients and pigs undergoing hemorrhagic shock and resuscitation. Results: Blood redox decreased with ascorbic acid addition, both in vitro and in vivo. It was more positive in patients undergoing cardiac surgery compared to healthy volunteers. Conclusions: Preliminary studies were limited, but appear to show correlation to disease processes and medical therapies. More work needs to be done to further examine the relation of redox to disease and treatment.

The Defender against apoptotic cell death (DAD1) is a negative regulator of programmed cell death that was initially identified in the temperature sensitive tsBN7 cell line. It has been shown to be an essential subunit of oligosaccharyltransferase and is localized to the Endoplasmic reticulum (ER) in a normal physiological state. However, our data suggests that DAD1 localizes outside of the cell and alters the apoptotic signaling via FAS ligand to give cancer cells a survival advantage. Although the mechanism is poorly understood, increased expression of DAD1 has been associated with increasing Gleason score in prostate cancer (PCa) patient tumors. Based on the aforementioned evidence, our study attempts to unravel cellular localization and the underlying mechanisms by which DAD1 mediates prostate cancer cell survival, and explore its potential as a biomarker in prostate cancer. As evidenced by qRTPCR, immunocytochemistry, immunohistochemistry, Co-ip, ELISA, and immuno-blot analysis, cancer cells down regulate the expression of the binding partner of DAD1 responsible for retention of DAD1 in the ER, which allows DAD1 to exit the ER and be exocytosed. The exocytosed DAD1 binds to FAS L and prevents apoptotic signaling. Treatment with DAD1 antibody induces significantly higher cell death in prostate cancer cells compared to the non-tumorigenic cells. Combination of DAD1 antibody with currently used chemotherapeutic agents like Docetaxel and Doxorubicin can be used to achieve higher cell death at a lower dose of these drugs to minimize side effects. Further, our immunohistochemistry data in tumor microarray suggests that DAD1 could serve as a potential biomarker marker in PCa. In addition to the tissue, we also examined the expression of DAD1 in prostate cancer patient serum samples using sandwich ELISA; our results indicate DAD1 is a more sensitive and specific prognostic marker for prostate cancer compared to PSA. Our data suggests that localization of DAD1 outside of the cells is crucial to the survival of PCa cells and this phenomenon can be exploited to specifically target prostate cancer cells in therapy and serve as a biomarker in prostate cancer. / 1 / Nobel Bhasin

LASP1 spielt eine Schlüsselrolle in verschiedenen physiologischen und pathologischen Prozessen, wie etwa in der Entwicklung, Zellstruktur, Zellkommunikation, Tumorgenese und Metastasierung. Die Vielseitigkeit von LASP1 ist hauptsächlich durch seine besondere Proteinstruktur bedingt, die eine Interaktion mit vielen verschiedenen Bindepartnern ermöglicht. Effekte von LASP1 werden aber wahrscheinlich nicht nur durch cytosolische Interaktion mit Bindepartnern vermittelt, sondern auch, in Folge einer Translokation in den Zellkern, durch nukleäre Interaktion, evtl. als transkriptioneller Co-Faktor. Besonders die Rolle von LASP1 in diversen Krebserkrankungen stand in den letzten Jahren im Fokus der Forschung. Sowohl in Karzinomen, als auch in Medulloblastom und Leukämien wächst die Evidenz für eine LASP1-Überexpression, die vor allem durch fehlende microRNA Regulation und Mutationen im p53 Tumorsuppressor bedingt scheint. Die hohe LASP1-Expression konnte in vielen in vitro und in vivo Studien mit vermehrter Proliferation, Migration und/ oder Invasion von Krebszelllinien in direkten Zusammenhang gebracht werden. Dieser Effekt von LASP1 auf Tumoraggressivität ist eine mögliche Erklärung für die mit hoher LASP1-Expression korrelierte schlechtere Prognose in verschiedenen Krebserkrankungen. Das Transitionalzellkarzinom ist die fünfhäufigste Krebserkrankung des Menschen und weist eine hohe Rezidivrate auf. Daher sind regelmäßige Nachsorgeuntersuchungen notwendig. Angesichts bisher fehlender verlässlicher Biomarker für das Transitionalzellkarzinom ist die Zystoskopie weiterhin der Goldstandard in der Nachsorge. Diese wird aber von Patienten als unangenehm empfunden, ist mit einem Infektionsrisiko verbunden, von der Erfahrung des Untersuchers abhängig und kostenintensiv. Tatsächlich ist das Transitionalzellkarzinom eine der teuersten Krebserkrankungen in der Nachsorge, weshalb die Entwicklung alternativer Diagnostikverfahren auch gesundheitsökonomische Relevanz hat. LASP1 wurde als ein vielversprechender Biomarker des Transitionalzellkarzinom-Rezidivs identifiziert, der durch einfache Proteinmengenbestimmung mittels Western Blot im Urinpellet evaluiert werden kann. Zum damaligen Zeitpunkt gab es außerdem bereits erste Hinweise auf eine funktionelle Relevanz von LASP1 im Blasenkarzinom in vitro. Angesichts dieser Erkenntnisse wurden als Ziele dieser Arbeit formuliert, 1) die Generierung von stabil transfizierten, induzierbar LASP1 spezifische shRNA exprimierenden Transitionalzellkarzinomzelllinien, 2) die funktionelle Charakterisierung eines LASP1-Knockdowns in selbigen in vitro, und 3) der Vergleich von Eigenschaften von LASP1 im Transitionalzellkarzinom mit denen in anderen Karzinomen. Für die zwei Transitionalzellkarzinomzelllinien T24 und RT4 konnte eine 4-5-Fache LASP1-Überexpression, verglichen mit normalem Urothel, gezeigt werden. Beide Zelllinien wurden erfolgreich mit einem induzierbar shRNA gegen LASP1 exprimierenden Konstrukt transduziert, sodass ein 50 % LASP1-Knockdown durch Doxycyclin induziert werden kann. Bei der Evaluierung des Effektes des LASP1-Knockdowns auf die Adhäsion, Proliferation und Migration dieser Zelllinien in vitro konnte eine signifikante Reduktion der Migration in beiden Zelllinien nachgewiesen werden. Passend dazu ergab eine GSEA von TCGA Daten zum Blasenkarzinom eine Korrelation von LASP1-Expression mit diversen Gen-Sets, die mit dem Phänotyp Metastasierung annotiert sind. Des Weiteren konnte für T24 und RT4 eine nukleäre LASP1-Lokalisation nachgewiesen werden, die abhängig von der Serin-146 Phosphorylierung war. Bioinformatische Analysen ergaben eine hochsignifikante, negative Korrelation von LASP1-Expression und miR-203 im Blasenkarzinom. Eine Korrelation von LASP1-Expression mit Prognose konnte mittels TCGA Daten für das Blasenkarzinom nicht festgestellt werden. Jedoch lagen lediglich Expressionsdaten auf mRNA Level vor, die meisten LASP1 mit Prognose assoziierenden Studien basieren hingegen auf Immunhistochemie, also der Expression auf Proteinlevel, welche in Blasenkrebszelllinien von der Expression auf mRNA Level abweichen kann. Die generierten Zelllinien wiesen nach lentiviraler Transduktion, Selektion und Sorten im Vergleich zum Wildtyp teilweise veränderte Zelleigenschaften auf, und ein Verlust des Fluoreszenzsignals des der shRNA vorangestellten tRFP wurde beobachtet. Daher müssen die Zellen bei weiterer Verwendung regelmäßig mit Puromycin nachselektioniert werden und die Validität dieser Zellen als Modell für das Transitionalzellkarzinom, besonders im Xenograft Mausmodell, ist kritisch zu hinterfragen. Entsprechend sind die Ergebnisse dieser Arbeit im Einklang mit bisherigen Studien zu LASP1. Damit unterstreicht diese Arbeit einmal mehr die Relevanz von LASP1 in diversen Krebserkrankungen. Weitere Studien zum Wert von LASP1 als prognostischer oder gar diagnostischer Marker erscheinen daher vielversprechend. / The LIM and SH3 protein 1 (LASP1) plays key roles as a nucleo-cytoplasmic shuttling protein and a putative transcriptional co-factor in various physiological and pathological processes including development, cell structure, cell signaling, tumorigenesis, and metastasis. Moreover, LASP1 is overexpressed in a broad range of cancers. In several studies, the overexpression of LASP1 has been linked to increased tumor aggressiveness in vitro and in vivo. Furthermore, in various tumor entities, like breast carcinoma and colorectal carcinoma LASP1 expression correlates with worse prognosis. Recently, LASP1 has been evaluated as a marker for the transitional cell carcinoma (TCC), especially for TCC recurrence due to limitations in case of hematuria. TCC is the fourth most common cancer in men, with a high recurrence rate. To date, there is no established bona fide marker for predicting recurrence. Hence, expensive cystoscopy is still the gold standard in followup examinations. Earlier work, however, demonstrated that the LASP1 protein concentration in urinary cell pellets has good predictive values for TCC recurrence. Here, the functional role of LASP1 in TCC was investigated. Thus, the invasive TCC cell line T24 and the non-invasive TCC cell line RT4, which both presented a fourfold higher LASP1 protein expression than normal urothelium, were lentiviral transduced with an inducible shRNA expression system directed against LASP1. With those cell lines the impact of LASP1 knockdown on proliferation, migration, and adhesion was assessed. At a knock down efficiency of around 50%, adhesion and proliferation remained unchanged, but migration was significantly reduced. Moreover, cytoplasmic and nuclear localization of LASP1 was observed in protein extracts from the TCC cell lines. Interestingly, serine 146 phosphorylated LASP1 was primarily located in the nucleus. Furthermore, analysis of microRNA expression data from The Cancer Genome Atlas project indicated a potential regulation of LASP1 expression by mir-203 in TCC. Hence, those findings are consistent with previous reports on LASP1 in other cancer entities and confirm again the important role of LASP1 in cancers. In conclusion, LASP1 is highly overexpressed in TCC and has impact on migration, possibly due to its nuclear localization and intranuclear interactions. Future studies have to identify the nuclear interaction partners and to validate the value of LASP1 as a diagnostic and prognostic biomarker in the TCC.

Chk2 is a cell cycle checkpoint kinase that is essential for initiating the DNA damage response in the presence of genetic damage. Its role is highly conserved from budding yeast (where it is named Rad53) to humans. Very few cell cycle checkpoint proteins have ever been studied in fish and the role of Chk2 has never been characterized. Oncorhynchus mykiss (Rainbow trout) was chosen for this project due to its importance in the commercial aquaculture industry and the availability of rainbow trout cell cultures at the University of Waterloo. This study was the first to clone the CHK2 gene in a teleost species, verified through both genomic and cDNA cloning. A section of the CHK2 gene, specifically the forkhead associated domain (FHA), was used to express recombinant Chk2 protein and generate polyclonal anti-Chk2 antibodies. A southern blot was performed and CHK2 was found to exist as a single copy number in the rainbow trout genome. The tissue specificity of Chk2 was also examined both at the mRNA transcript and protein level. Interesting tissue specific differences were discovered with transcript levels moderately low in gill and higher in brain, while protein levels were extremely high in gill and lower in brain tissues. Protein levels were verified in both whole fish tissue samples and in cell culture suggesting that cell cultures accurately reflect the state of checkpoint proteins in vivo. These tissue specific differences suggest that in gill, Chk2 is maintained at a high protein level to combat any toxins in the water attempting to transverse this barrier tissue and gain access to the fish’s circulatory system. Meanwhile, the blood brain barrier offers protection to the highly sensitive brain tissue, suggesting that high levels of Chk2 protein are not constitutively required, but instead remain in a transcript reservoir able to be quickly translated in the event of DNA damage. To determine whether Chk2’s checkpoint role is conserved in O. mykiss, both gill and brain cell cultures were treated with low and high doses of bleocin (a commercially available form of bleomycin) known to cause high levels of double-strand breaks, the most deleterious type of DNA damage and a specific activator of the Chk2 DNA damage response (DSB). Results showed that bleocin had no effect on levels of Chk2 in gill cells, confirming that the protein is constitutively active in this tissue always on alert against potential genetic insult. In contrast, brain cells were able to upregulate Chk2 in a dose-dependent manner to bleocin induced DNA damage demonstrating that Chk2 can act as a biomarker for genetic damage in brain cells. In conclusion, the tissue specific expression of Chk2 and its ability to respond to DNA damage suggests that checkpoint proteins may serve as suitable biomarkers for DNA damage in O. mykiss and other fish species.

Chk2 is a cell cycle checkpoint kinase that is essential for initiating the DNA damage response in the presence of genetic damage. Its role is highly conserved from budding yeast (where it is named Rad53) to humans. Very few cell cycle checkpoint proteins have ever been studied in fish and the role of Chk2 has never been characterized. Oncorhynchus mykiss (Rainbow trout) was chosen for this project due to its importance in the commercial aquaculture industry and the availability of rainbow trout cell cultures at the University of Waterloo. This study was the first to clone the CHK2 gene in a teleost species, verified through both genomic and cDNA cloning. A section of the CHK2 gene, specifically the forkhead associated domain (FHA), was used to express recombinant Chk2 protein and generate polyclonal anti-Chk2 antibodies. A southern blot was performed and CHK2 was found to exist as a single copy number in the rainbow trout genome. The tissue specificity of Chk2 was also examined both at the mRNA transcript and protein level. Interesting tissue specific differences were discovered with transcript levels moderately low in gill and higher in brain, while protein levels were extremely high in gill and lower in brain tissues. Protein levels were verified in both whole fish tissue samples and in cell culture suggesting that cell cultures accurately reflect the state of checkpoint proteins in vivo. These tissue specific differences suggest that in gill, Chk2 is maintained at a high protein level to combat any toxins in the water attempting to transverse this barrier tissue and gain access to the fish’s circulatory system. Meanwhile, the blood brain barrier offers protection to the highly sensitive brain tissue, suggesting that high levels of Chk2 protein are not constitutively required, but instead remain in a transcript reservoir able to be quickly translated in the event of DNA damage. To determine whether Chk2’s checkpoint role is conserved in O. mykiss, both gill and brain cell cultures were treated with low and high doses of bleocin (a commercially available form of bleomycin) known to cause high levels of double-strand breaks, the most deleterious type of DNA damage and a specific activator of the Chk2 DNA damage response (DSB). Results showed that bleocin had no effect on levels of Chk2 in gill cells, confirming that the protein is constitutively active in this tissue always on alert against potential genetic insult. In contrast, brain cells were able to upregulate Chk2 in a dose-dependent manner to bleocin induced DNA damage demonstrating that Chk2 can act as a biomarker for genetic damage in brain cells. In conclusion, the tissue specific expression of Chk2 and its ability to respond to DNA damage suggests that checkpoint proteins may serve as suitable biomarkers for DNA damage in O. mykiss and other fish species.

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