The mechanism of ILEI cleavage and its physiological relevances

Metastasierung ist ein sehr häufiger Prozess bei Krebserkrankungen und das entscheidende Kriterium für durch Krebs verursachte Todesfälle. Metastasierende Tumorzellen wandern aktiv im Gewebe, gehen ins Blut/Lymphe und werden invasiv, d.h. wandern in entfernte Organe aus, wo sie Tochtergeschwülste (Metastasen) bilden. Dies wird durch genomische Veränderungen, die Tumormikroumgebung mit erhöhter Protease-Aktivität und modifiziertem ECM und Stroma unterstützt. Infolgedessen brechen die Zellen durch Gewebebarrieren und Basalmembranen und es werden sekundäre Tumore in entfernten Organen gebildet. Bei den ersten Schritten der frühen Metastasierung, bevor sich die Zellen aus dem primären Tumor lösen und sich verbreiten, tritt ein Prozess namens Epitheliale mesenchymale Transition (EMT) auf, bei dem die Zellen ihre epitheliale Polarität und Fähigkeit zur Zellkontaktbildung verlieren. Sie weisen dann den Phänotyp von Bindegewebszellen (Fibroblasten) auf. EMT erfordert eine kooperative Wirkung von aktiviertem Ras und TGFß Signalling. Eine Studie über Expressionsanalyse, um EMT spezifische , durch TGFß induzierte Targetgene in einem epithelialen Maus- Brustkrebs- Zellmodell zu finden , hat ein neues, Zytokin ähnliches Protein namens ILEI (Interleukin like EMT-Inducer) identfiziert. TGFß reguliert ILEI auf der translationalen Ebene hoch. Dauerhafte Überexpression von ILEI in einigen Zelllinien verursacht EMT und verstärkt Tumorwachstum und Metastasierung. ILEI ist ein fakultativ sezerniertes Protein und seine sekretierte Form wird durch Serinproteasen wie Plasmin spezifisch an einer R / S Site am N-Terminus gespalten. Die physiologische Rolle der ILEI Spaltung in der Tumorgenese wurde bei tumorigenischen aber nicht metastatischen EpC40 Zellen, die verschiedene mutierte Formen des Proteins überexprimieren, analysiert. Das Protein wurde mutiert; entweder durch Einführung von Punktmutationen auf der Spaltstelle (cleavage mutant), welche das proteolytische Spalten durch Proteasen inhibieren, oder das Propeptid an dem N-Terminus wurde deletiert (Δ-pro peptid mutant), was zu einem konstitutiv gespaltenen, reifen ILEI Protein führt. (bei dem Protease-Spaltung nicht mehr benötigt wird.) Das Fehlen des Propeptides am N-Terminus gewährleistet.eine hohe Sekretion des Proteins. In vivo Tumorigenese Experimente haben demonstriert, dass die cleavage Mutante überexprimierte EpC40 Zelle kleine Tumoren und sehr wenige Metastasen im Vergleich zu den wt-ILEI überexprimierten Zellen gebildet haben. Daneben beschleunigten die Δ-pro Peptid ILEI überexprimierten Zellen die Tumorentwicklung sogar stärker als die wt-ILEI überexprimierten Zellen und wiesen erhöhte Metastasierung auf. Wir untersuchten die Notwendigkeit der ILEI Spaltung für ein verstärktes Tumorwachstum im Tumorigenese-Testen bei denen ein nicht spezifischer Proteaseninhibitor, Aprotinin verwendet wurde. 10 Wir erhielten eine signifikant verminderte Tumorwachstumsrate und Tumorgröße nur bei wt-ILEI überexprimierten Zellen im Vergleich zu den unbehandelten Tiere. Wie erwartet, hatte Aprotinin bei durch die Δ-pro peptid Mutante erzeugten, schnellwachsenden Tumoren keine Wirkung, da dieses Proteiun ja bereits gepalten ist. In vitro Experimente wießen darauf dass, die ILEI Sekretion durch exogenes Plasmin stimuliert werden kann. In weiteren Analysen zur Erklärung der Ursache dieses Phänomenes, wurde getestet, ob die ILEI-Sekretion durch die Aktivierung von Plasmin – uPAR Signalling reguliert werden könnte und nicht durch einen „autocrine Feed-back“ Mechanismus von ILEI, bei dem die gespaltene reife ILEI-Form seine eigene Sekretion ausgelöst haben könnte. Im zweiten Teil dieser Diplomarbeit wurde ein doxycyclin induzierbarer (TET-ON) ILEI transgenischer Mausstamm in vitro und in vivo auf die Induzierbarkeit und die Expressions-Stärke des durch das ILEI Transgen kodierte Protein bestimmt. Zwei Transgene, TetrTA und tetO-ILEI, wurden durch homologe Rekombination in spezifische Loci (ROSA26 and Col1A1) des Mausgenoms eingeführt, um Nebenwirkungen von zufälliger Integrationen, wie Zerstörung von endogenen Genen, zu vermeiden. Doxycyclin induzierte ILEI Expression wurde in allen untersuchten Geweben außer in der Lunge detektiert. Die höchsten Expression Levels wurden im Dünndarm und Dickdarm gefunden. Dieses induzierbare ILEI transgenische System ermöglicht das Studieren der physiologischen Funktion von ILEI und seiner Rolle in der Krebsentwicklung.Metastasis is the main reason for cancer related deaths. Epithelial tumor cells become invasive and motile, aided by genomic alterations and the tumor microenvironment with increased protease activity and modified ECM and stroma. As a result, the cells break down tissue barriers and establish secondary tumors at distant organs. During the early steps of metastasis, when tumor cells start to disseminate from the primary cancer, a process called epithelial to mesenchymal transition (EMT) occurs, in which cells loosen cell-cell contacts and deregulate their epithelial polarity. EMT requires a cooperative action of activated Ras and TGFb signaling. Expression profiling studies to identify TGFb-induced, EMT specific target genes in a murine mammary epithelial cell model has revealed a novel cytokine like protein, named ILEI (interleukin like EMT inducer). TGFb upregulates ILEI at the translational level. Stable overexpression of ILEI in several cell lines caused EMT, elevated tumor growth and aspects of metastasis. ILEI is a secreted protein and its secreted form is cleaved by serine-type proteases, like plasmin, specifically at an R/S site at its N terminus. The physiological role of ILEI processing in tumorigenesis was analyzed using tumorigenic but non-metastatic EpC40 cells overexpressing (OE) different mutant forms of the protein. The protein was mutated either by introducing point mutations at the cleavage site, which inhibited proteolytic cleavage by proteases, or the pro-peptide at the N-terminus was deleted, creating an artificially matured protein not requiring proteolytic cleavage. This lack of the pro-peptide at the N-terminus caused constitutive, abnormally high secretion of the protein. In vivo tumorigenesis experiments demonstrated that cleavage mutant overexpressing EpC40 cells formed small tumors and very few metastases compared to the wt-ILEI OE cells. In addition, Δ-pro peptide ILEI OE cells accelerated tumor development even more than the wt-ILEI OE cells and exhibited enhanced metastasis. We examined the necessity of ILEI cleavage for generating elevated tumor growth in tumorigenesis assay using a non-specific serine protease inhibitor, aprotinin. Mice treated with this inhibitor showed significantly decreased tumor growth rates and tumor sizes induced by wt-ILEI OE cells, as compared to animals not treated with inhibitor. In vitro assays indicated that ILEI secretion can be stimulated by exogenous plasmin. Further analyses in attempts to explain the cause of this phenomenon suggested that it might occur via the activation of plasmin - uPAR signaling, and not through an autocrine feedback mechanism of ILEI, where the processed form would trigger its own secretion. In the second part of this diploma thesis, a doxycyclin inducible (TET-ON) ILEI transgenic mouse strain was characterized in vitro and in vivo for the inducibility and expression levels of the ILEI transgene. The two transgenes, TetrTA and tetO-ILEI were introduced into specific loci (ROSA26 and Col1A1) by homologous recombination, eliminating the side effects of random integration such as disruption of endogenous genes. Doxycyclin responsive ILEI expression was detected in all examined tissues except lung. Highest expression levels were found in small and large intestines. This inducible ILEI transgenic system allows studying the physiological role of ILEI and its role in cancer development

Port of Famagusta: Development of a Mediterranean port from 13th to 20th Century

Kıbrıs’ın doğu kıyılarında konumlanmış olan Gazimağusa Limanı, doğal korunma niteliklerinden dolayı tarih boyunca adadaki en iyi demirleme imkânlarını sağlamıştır. Liman, bir iç liman ve bir dış limandan oluşmaktadır. Dış Liman 1,5 km uzunluğundadır ve doğal resiflerle çevrilmiştir. İç Liman ise, deniz surları boyunca, İç Kale ve Arsenal Kulesi arasında uzanan, karşısında bulunan üç ada dolayısıyla da çok güvenli ve korumalıdır. 13. yüzyılda, ‘Lüzinyan Krallığının’ adadaki yönetiminin başlamasının ardından, Gazimağusa ana limana dönüşmüştür. Gazimağusa’nın tercih edilme sebepleri, Kutsal Topraklardaki limanlara ve Küçük Ermenistan’a yakınlığı ve coğrafi üstünlükleridir. Limanın savunması için 1232 yılında kullanılmış olan bir kuleden söz eden F. Amadi, kentteki ve limandaki bir yapıyla ilgili en erken tarihli bilgileri vermiştir. Gazimağusa Limanı’nın ihtiyaçları olan tersane ve bir kale 1300 yılından önce buradaki yerini almıştır. 1308 yılında, İç Kale ve ‘Torion del Arsenale’ arasındaki deniz surları ve Deniz Kapısı’nın inşaatının devam ettiği bilinmektedir. Kısa bir süre sonra kale karşıdaki doğal resiflerle bir zincirle birleştirilmiş, daha sonraları resiflerin üzerine bir kule inşa edilmiştir. 14. yüzyıl sonuna kadar limanın çoğunlukla savunmaya yönelik olan fiziksel öğelerinin büyük bir kısmı tamamlanmıştır. Savunma ve limanın işletilmesi için gerekli fiziksel öğelerden sonra, kara tarafındaki kentsel mekânlar ve yapılar yerlerini almışlardır. Bu çalışmanın amacı, Gazimağusa Limanı’nın, tersane, tersane kapısı, Deniz kapısı/kapıları, kale/iç kale, kuleler/gözetleme kulelerinden oluşan fiziksel öğelerinin, 1250 yılından 1950 yılına kadar süren 700 yıl boyunca sürekliliğini ve değişimini ortaya koymayı hedeflemektedir.  Anahtar Kelimeler: Akdeniz kenti, Gazimağusa, liman, kentsel dönüşüm.Cyprus, in the middle of Eastern Mediterranean, is an island whose economy laid on sea trade since Prehistoric times. Its ports, with changing significances through history were the main scenes of its past. Among its port cities, Famagusta is a more recent one comparing with the others. Its name began to emerge only after 10th century, parallel to decline of Salamis/Constantia, facing with many problems like earthquakes, Arab invasions and its silted up harbour. Port of Famagusta, which is located at the back of Famagusta Bay on the east shores of Cyprus, had supplied the best anchorage possibilities in the island throughout its history, because of its natural protection. The port consists of an outer and an inner port. The outer port is 1.5 km long and surrounded by natural reefs. The inner port, lying along the sea walls, between Citadel and Arsenal Tower, is very safe and protected due to three small islands on the sea side. In 13th century, after the reign of a noble crusader family "Lusignan", Famagusta became the main port. Famagusta was favourable because of its closeness to the ports of Holy Land and Ayas in Lesser Armenia and its geographical advantages. F. Amadi, mentioning a tower used for the defence of the port in his "Chronicle", gave the earliest remarks on a building on the port and in the town in 1232. The essentials of the port of Famagusta, an arsenal and a castle were settled soon before 1300. In 1308, its sea walls and sea gate were under construction between the castle and "Torion del Arsenale". After a short while, the castle had been connected with the opposing end of the natural reefs with a chain, and afterwards a tower had been built on the reefs. So, the port was divided into two: outer harbour and inner harbour. The development of the port of Famagusta had been extensively completed until the end of the 14th century. The physical elements of the port mostly had defence functions. Presenting the importance of the port as a defence line, these elements preserved their significance until military technology changed in the 19th century. The chain was still there at the beginning of 20th century. The urban spaces and buildings along the land side of the port have been located, after the development of physical features needed for the defence and operation of the port. After Lamberto di Sambuceto?s notes, many important public buildings were constructed between the Citadel and Arsenal Tower, in the early 14th century. The buildings known to be located along the port, from the Citadel to the Arsenal Tower are the Genoese Loggia, the Venetian Loggia, the Customs Buildings, St. Anthony Church and Hospital, and the Fish Market. On the south-west of the Citadel, one of the oldest buildings of the city, St. George of the Latins Church is located. These buildings have generated a dense axis along the port in this period. So, the administrative, religious and commercial elements had been settled after the military ones, along the port. After 1373 Genoese period started and lasted for 90 years in the city. During this period the activities of the port of Famagusta had declined. It is known that the Genoese put their efforts more on defense improvement works. After a short period of Lusignans after Genoese in the city, Venetians managed the sovereignty of the island. During Venetians port development gained importance. Arsenal, sea-gate, citadel on sea-side, sea walls and towers were all improved/ rebuilt together with other parts of fortifications. The improvements to the port were the last for the pre-modern times. There had been done very little for the port during Ottoman Period after 1571, as the east port of island Larnaca became the main port after the Ottoman conquest. British rule, taking into account the capacity of port of Famagusta, made projections for Famagusta port. Famagusta harbour development projects were realised in three phases in 1905, 1933, and 1965. This study aims to search the continuity and change of physical elements of the port during a 700 years period, from 1250 to 1950 comprising arsenal, arsenal gate, sea-gate/gates, castle/citadel, towers/watch-towers, chain, custom-houses, reefs, and its borders. The physical changes of the port in centuries are represented on maps. Keywords: Mediterranean city, Famagusta, port, urban transformation

A newborn with diabetic ketoacidosis and thalassemia major: A rare case

Diabetic ketoacidosis is a systemic situation caused byabsolute insulin deficiency and characterized by hyperglycemia,ketonemia, acidemia, glycosuria and ketonuria.Thalassemia Major is a very serious hereditary blooddisorder due to low levels or absence of “beta globulin”chain, characterized by requiring a blood transfusion from3-4. month of life due to the relatively short life of red cells.We, herein presented a rare case of 20 day-old newbornwith anemia, hyperglycemia, vomiting, acidosis being diagnosedas thalassemia major that required blood transfusionin the early period of life and diabetic ketoacidosiswithout ketonuria who born from 24 year old father carrierof thalassemia and 23-year-old mother with carrier of thalassemiaand gestational diabetes.The case was presented in order to emphasize that diabeticketoacidosis can occur in newborns without ketonuriaand thalassemia major may cause anemia in the earlyperiod of life due to hyperglycemia and acidosis

Structural insights from lipid-bilayer nanodiscs link α-Synuclein membrane-binding modes to amyloid fibril formation

Peer reviewe

Development of a New largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions