The overall aim of my PhD research work is to design, develop, and evaluate a new practical environment to generate separated representations of arterial and venous trees in non-contrast pulmonary CT imaging of human subjects and to extract quantitative measures at different tree-levels. Artery/vein (A/V) separation is of substantial importance contributing to our understanding of pulmonary structure and function, and immediate clinical applications exist, e.g., for assessment of pulmonary emboli. Separated A/V trees may also significantly boost performance of airway segmentation methods for higher tree generations. Although, non-contrast pulmonary CT imaging successfully captures higher tree generations of vasculature, A/V are indistinguishable by their intensity values, and often, there is no trace of intensity variation at locations of fused arteries and veins. Patient-specific structural abnormalities of vascular trees further complicate the task. We developed a novel multi-scale topo-morphologic opening algorithm to separate A/V trees in non-contrast CT images. The algorithm combines fuzzy distance transform, a morphologic feature, with a topologic connectivity and a new morphological reconstruction step to iteratively open multi-scale fusions starting at large scales and progressing towards smaller scales. The algorithm has been successfully applied on fuzzy vessel segmentation results using interactive seed selection via an efficient graphical user interface developed as a part of my PhD project. Accuracy, reproducibility and efficiency of the system are quantitatively evaluated using computer-generated and physical phantoms along with in vivo animal and human data sets and the experimental results formed are quite encouraging. Also, we developed an arc-skeleton based volumetric tree generation algorithm to generate multi-level volumetric tree representation of isolated arterial/venous tree and to extract vascular measurements at different tree levels. The method has been applied on several computer generated phantoms and CT images of pulmonary vessel cast and in vivo pulmonary CT images of a pig at different airway pressure. Experimental results have shown that the method is quite accurate and reproducible. Finally, we developed a new pulmonary vessel segmentation algorithm, i.e., a new anisotropic constrained region growing method that encourages axial region growing while arresting cross-structure leaking. The region growing is locally controlled by tensor scale and structure scale and anisotropy. The method has been successfully applied on several non-contrast pulmonary CT images of human subjects. The accuracy of the new method has been evaluated using manually selection of vascular and non-vascular voxels and the results found are very promising.

Retinal imaging enables the visualization of a portion of the human microvasculature in-vivo and non-invasively. The scanning laser ophthalmoscope (SLO), provides images characterized by an ultra-wide field of view (UWFoV) covering approximately 180-200º in a single scan, minimizing the discomfort for the subject. The microvasculature visible in retinal images and its changes have been vastly investigated as candidate biomarkers for different types of systemic conditions like cardiovascular disease (CVD), which currently remains the main cause of death in Europe. For the CARMEN study, UWFoV SLO images were acquired from more than 1,000 people who were recruited from two studies, TASCFORCE and SCOT-HEART, focused on CVD. This thesis presents an automated system for SLO image processing and computation of candidate biomarkers to be associated with cardiovascular risk and MRI imaging data. A vessel segmentation technique was developed by making use of a bank of multi-scale matched filters and a neural network classifier. The technique was devised to minimize errors in vessel width estimation, in order to ensure the reliability of width measures obtained from the vessel maps. After a step of refinement of the centrelines, a multi-level classification technique was deployed to label all vessel segments as arterioles or venules. The method exploited a set of pixel-level features for local classification and a novel formulation for a graph cut approach to partition consistently the retinal vasculature that was modelled as an undirected graph. Once all the vessels were labelled, a tree representation was adopted for each vessel and its branches to fully automate the process of biomarker extraction. Finally, a set of 75 retinal parameters, including information provided by the periphery of the retina, was created for each image and used for the biomarker investigation.

Various diseases, including cancer, stroke and heart attack, are associated with disruption of the vascular system. However, lack of a profound understanding of the transcription regulation during vascular development hinders the formation of effective molecular intervention strategies targeting angiogenesis. Here we describe an enhancer of Neuropilin1 (Nrp1) from the second intron of the gene that directs arterial and coronary endothelial cell-specific expression. Mice transgenic for either human or mouse sequences of the Nrp1in2 enhancers drove expression of the LacZ reporter gene specifically in the endothelial cells within the arterial compartment from early in development, while no expression was detected in veins. In addition, the hNrp1in2 enhancer directed expression to the endothelial cells in the developing coronary vasculature, with the initial expansion from around the sinus venosus at E11.5, and eventually contributed to the capillary, venous and arterial compartments of the coronary vessels but not the endocardium. This expression pattern is consistent with that reported in the Apelin-nlacZ line (Red-Horse et al., 2010), making the Nrp1 enhancer the first identified mammalian regulating enhancer of the coronary endothelial cell. Phylogenetic footprinting, and a tissue culture reporter assay suggested that this enhancer contains a 184bp minimal core region hNrp1in2peakA2 that recapitulates the expression profile of the full length enhancer. hNrp1in2peakA2 has conserved and in vitro validated recognition sites for Gata, Ets, and Fox. The validated Fox and Ets sites form a functional FOX:ETS motif, and the FOX:ETS motif is responsible for synergistic activation ofthe enhancer by FoxC2 and Etv2 in reporter assays. Mutation introduction to the functional Ets sites or compound ablation of the Gata and Fox site in hNrp1in2peakA2 result in total loss of vascular expression, in terms of both arterial and coronary expression. The Fox, Ets and Gata recognition sites may be sufficient to achieve arterial- and coronary- specific expression of the hNrp1in2peakA2.

Arterien und Venen sind aufgrund ihrer Funktion im Körper morphologisch, funktionell und genetisch unterschiedlich. Schon die großen Blutgefässe auskleidende Endothelzellen zeigen eine arteriovenöse Determinierung. Ausgehend von einer Mikroarray-Analyse der mRNA-Expression arterieller und venöser Endothelzellen der Nabelschnur wurde im Rahmen dieser Arbeit auf Moleküle des Notch-Signalwegs, Dll-4, Notch-4, Hey-1 und Hey-2 fokussiert, die präferenziell bis exklusiv arteriell exprimiert werden. Weitere Gene mit einem arteriellen Expressionsmuster, die im Rahmen der vorliegenden Arbeit analysiert wurden, sind Angiopoietin-2 und CD44s. Trotz der genetisch definierten Unterschiede ließ der Vergleich physiologisch relevanter Funktionen, wie Proliferation oder die Interaktion mit monozytären Zellen keinen vom endothelialen Zelltyp abhängigen Unterschied erkennen. Die im Matrigel ausgebildeten kapillar-ähnlichen Strukturen sind durch homogene, eng beieinander liegende Netzwerke charakterisiert. Studien über den Einfluss von Wachstumsfaktoren und Schubspannung auf die arteriovenöse Expression von Angiopoietin-2 deuten auf eine schubspannungsvermittelte Regulation der Gefäßstabilität und Differenzierung hin. Die in der Zellkultur durchgeführten Manipulationen, wie der Einfluss verschiedener Wachstumsfaktoren oder die Applikation unterschiedlicher Schubspannungen, erreichten in Bezug auf die Expression der untersuchten Markergene keine tiefer greifende Redifferenzierung des jeweiligen endothelialen Phänotyps. Der evolutionär hoch konservierte Notch-Signalweg ist präferenziell arteriell exprimiert, wobei Hey-2 ausschließlich arteriell exprimiert wird. In adulten Endothelzellen erfolgt die Hey-2-Regulation jedoch Notch-unabhängig. Die arteriovenöse Genexpression von Molekülen des Notch-Signalwegs ist unabhängig von der Schubspannung. Unter hypoxischen Bedingungen verringerte sich die Expression von Dll-4, Hey-1 / 2 dramatisch, ohne das jedoch physiologische Beeinträchtigungen zu beobachten waren. Die Expression des venösen COUP-TF II wird in arteriellen Endothelzellen nicht durch den Notch-Signalweg reguliert. Die Auswertung der Daten in der vorgelegten Arbeit lässt vermuten, dass die einmal festgelegte genetische Determinierung adulter Endothelzellen fixiert und äußeren Einflüssen gegenüber stabil und unumkehrbar ist. Dennoch ist eine gewisse Anpassungsfähigkeit der Endothelzellen an bestimmte Situationen möglich, die zwar die Ausprägung von Merkmalen des jeweilig anderen Phänotyps beinhaltet, jedoch nicht eine vollständige Redifferenzierung.

Η μικροχειρουργική τεχνική και η μεταφορά ελεύθερων αγγειούμενων κρημνών για την αποκατάσταση εκτεταμένων και σύνθετων ελλειμμάτων ογκολογικής ή τραυματικής αιτιολογίας της κεφαλής και του τραχήλου αποτελεί μέθοδο επιλογής. Με την τεχνική αυτή επιτυγχάνεται στον ίδιο χρόνο με την εκτομή, η λειτουργική και αισθητική αποκατάσταση με μειωμένο ποσοστό επιπλοκών και επίπτωση από την δότρια περιοχή. Στην παρούσα μελέτη καταγράφουμε και αναλύουμε την εμπειρία μας σε ασθενείς που αντιμετωπίστηκαν με μικροχειρουργική τεχνική και ελεύθερους αγγειούμενους κρημνούς. Την περίοδο 2003 έως 2010, σε 48 ασθενείς πραγματοποιήθηκαν 56 ελεύθεροι αγγειούμενοι κρημνοί. Οι 34 ασθενείς από τους 48 υπεβλήθηκαν συγχρόνως σε ογκολογική εκτομή λόγω Ca και σε αποκατάσταση, ενώ σε 14 από τους 48 ασθενείς αφορούσε έλλειμμα τραυματικής αιτιολογίας. Η τοπογραφία του ελλείμματος αφορούσε το τριχωτό της κεφαλής και του μετώπου σε 12 ασθενείς (25%), το μέσο τριτημόριο του προσώπου και τα παραρίνια σε 9 ασθενείς (18.7%), το κάτω τριτημόριο του προσώπου και τραχήλου σε 27 ασθενείς (56.25%). Σε 7 ασθενείς χρησιμοποιήθηκαν διπλοί ελεύθεροι αγγειούμενοι κρημνοί και σε 41 ασθενείς μονήρεις κρημνοί. Χρησιμοποιήθηκαν συνολικά 56 ελεύθεροι αγγειούμενοι κρημνοί. Σε 16 ασθενείς χρησιμοποιήθηκε φλεβικό μόσχευμα (33.3%). Οι κρημνοί επιλογής ήταν ο κερκιδικός κρημνός 28.5%, ο κρημνός της περόνης 17.8%, ο προσθιοπλάγιος κρημνός του μηρού 14.2%, ο μυοδερματικός κρημνός του ορθού κοιλιακού(VRAM) 12.5% και εγκάρσιος(ΤΡΑΜ) 5.3%, ο κρημνός του πλατέως ραχιαίου 8.9%, ο κρημνός του ισχνού προσαγωγού 7.1% και ο πρόσθιος οδοντωτός 1.7%. Οι μικροαγγειακές αναστομώσεις έγιναν στα αγγεία του τραχήλου σε ποσοστό 96.2%. Η επιβίωση των κρημνών ήταν σε ποσοστό 92.8% (52/56) και 4 κρημνοί απορρίφθηκαν (7.1%) λόγω θρόμβωσης. Ένας ασθενής απεβίωσε στην μετεγχειρητική περίοδο λόγω σοβαρών συστηματικών επιπλοκών. Ο σωστός σχεδιασμός, η επιλογή του κατάλληλου κρημνού, η εμπειρία στην μικροχειρουργική και η συνεργασία των εμπλεκόμενων ιατρικών ομάδων είναι αυτά που διασφαλίζουν το υψηλό ποσοστό επιτυχίας στην επανορθωτική μικροχειρουργική με πολύ καλό λειτουργικό και αισθητικό αποτέλεσμα. / Microsurgical free tissue transfer considered as the best choice for the reconstruction of head and neck extended and complex tissue defects due to tumor resection or trauma. A total of 48 patients underwent free tissue transfer between 2003-2010. There were 34 patients underwent one stage tumor resection and microsurgical free flap reconstruction and 16 patients for free flap reconstruction due to head and neck trauma. The defect in 12 patients 25% was on the scalp and forehead, the middle third in 9 patients 18.7% lower third in neck in 27 patients 56.25%. We used a combination of double free flaps for reconstruction in 7 cases and in 41 patients a single free flap. Vane grafts were used in 16 cases (33.3%). We used in total 56 free flaps with success rate 92.8% (52/56). Four flaps were lost due to anastomotic thromboses. Work horse flaps in our series include the radial forearm 28.5%, fibula 17.8%, ALT 14.2%, VRAM 12.5%, TRAM 5.3%, latissimus dorse 8.9%, gracilis 7.1% and serratus anterior 1.7%. The neck recipient vessels were used in 96.2%. One patient died in post surgical period after systemic complications. Preoperative surgical and reconstruction plan, flaps selection, high microsurgical experience and team collaboration are essential for the good functional and aesthetic results in microsurgery reconstruction of head and neck tissue defects.