Yes / To develop an efficient nanotechnology fluorescence-based method to track cell proliferation to avoid the limitations of current cell-labeling dyes. Material & methods: Synthesis, PEGylation, bifunctionalization and labeling with a fluorophore (Cy5) of 200 nm polystyrene nanoparticles (NPs) were performed. These NPs were characterized and assessed for in vitro long-term monitoring of cell proliferation. Results: The optimization and validation of this method to track long-term cell proliferation assays have been achieved with high reproducibility, without cell cycle disruption. This method has been successfully applied in several adherent and suspension cells including hard-to-transfect cells and isolated human primary lymphocytes. Conclusion: A novel approach to track efficiently cellular proliferation by flow cytometry using fluorescence labeled NPs has been successfully developed.

Chemokines are a family of chemotactic cytokines that play a multifaceted role in human biology. Chemokine receptor CCR7, activated by its ligands CCL19 & CCL21, is known to play a significant role in cancer metastasis. In view of the wider roles that chemokines play in the biology of the cell, we hypothesised that CCR7 could influence cancer progression through other mechanisms in particular increased proliferation and/or increased chemo- and radioresistance, and whether these effects may have a physiological/clinical relevance. Interestingly, CXCR4 involvement in cancer recurrence, metastasis and proliferation is already well established. Thus, it was used as a point of reference to compare whether CCR7 axis effect on cancer proliferation and chemoresistance is similar to that observed in CXCR4 axis. It is proven that hypoxia is a driving factor in increased CCR7 expression. This study shows that CCR7 expression is upregulated as a response to a number of other stress factors, in particular that caused by different chemotherapeutic treatments. In addition, we showed that CCL21, one of the two endogenous ligands for CCR7 is similarly produced under these conditions We used several techniques to establish the expression and functionality of CCR7 and CXCR4 in different cancer cell lines. We then showed that activation of the CCR7 axis by physiologically relevant concentrations of CCL21 induces cancer cell proliferation and chemo- and radio-resistance. Furthermore, these effects are abrogated by small molecule antagonists (ICT13069), neutralizing monoclonal antibody, or CCR7 knockdown. Our findings support the hypothesis that antagonising CCR7 receptor will not only inhibit cancer metastasis, as it is well-illustrated in the literature, but it would also lead to alternative therapeutic approaches as well as potential clinical endpoints. / University of Tobruk, and the Ministry of Higher Education of Libya

肺癌是一個世界性的健康難題。大量研究證據顯示，煙草及其致癌物NNK對環氧酶（COX）-2及其下游產物具有促進效應。血栓素（TxA）2是COX-2的關鍵性下游產物之一，該論文闡述了TxA2在NNK導致的肺癌增長中的可能作用。 / 我們發現相对于非吸烟者，吸煙者肺癌組織表达更高水平的TxA2合酶（TxAS）。NNK可以刺激培養的肺癌細胞TxA2合成。用TxAS抑制劑和TxA2受體（TP）拮抗劑分別阻抑TxA2的合成與功能可以引起細胞凋亡，從而有效抑制NNK導致的細胞增殖效應。在TxA2合成受抑制的情況下，TP激動劑U46619幾乎可以重建NNK效應，說明TP在NNK效應中的重要作用。研究還顯示，激活的TP可以通過PI3K/Akt和ERK通路進一步激活CREB，從而參與NNK對肺癌細胞的促生長效應。 / 緊接著，我們的研究顯示TP 可以調節NNK對COX-2 和TxA2的誘導，而且發現NNK刺激的TxA2合成主要依賴於COX-2活性。COX-2和TxA2功能抑製劑對NNK的促細胞生長作用具有相似的抑制效用。考慮到TP是TxA2的功能受體，該資料說明TP在NNK處理的肺癌細胞中傳遞了上游因子COX-2的促腫瘤作用。在使用COX-2小干擾RNA（siRNA）抑制NNK作用的情況下，TP激動劑U46619幾乎可以恢復NNK的效應證實了TP的傳遞者角色。研究還發現 TPα而不是TPβ在培養的肺癌細胞系中廣泛表達，並且過表達TPα具有促進腫瘤生長的作用。在用NNK處理細胞的條件下，TPα還具有促COX-2表達和TxA2生成的作用。 / 我們的研究進一步發現，在吸煙者肺癌組織中TPα表達增高，這與TxAS的表達相似。与此结果相一致，在經NNK處理的A/J小鼠肺癌組織中，TxAS和TP表達水準也是明顯上升的。在細胞培養實驗中，NNK能夠提高TxAS蛋白和信使RNA（mRNA）的表達水準。但是，在TP的兩個亞型TPα和TPβ中， NNK僅能促進TPα的蛋白表達，對它們的mRNA均無影響。NNK對TxAS的促表達作用是核轉錄因數(NF)-κB依賴性的。其他的幾個關鍵轉錄因數，諸如特異性蛋白(SP)-1，CREB和活化受體 （PPAR）γ均未參與NNK對TxAS和TPα的表達促進作用。進一步的，轉錄後機理被證實參與了NNK對TPα的作用。TPα而不是TPβ經鑒別在NNK的促NF-κB 激活 和 促TxAS 表達效應中起正向調節作用。 / 總之， 我們的研究說明TxA2相關通路在NNK的促肺癌細胞生長效應中起正向調節作用。我們的研究揭示了TPα的自我激活環路。通過該環路，TxA2，或者說TxAS和TPα參與了NNK的肺癌促生長效應。因此，我們的研究為肺癌的防治了提供了一個新的方向，即靶向TxAS和TPα是一種可能有效的策略。 / Lung cancer concerns a world-wide health problem. There is considerable evidence of that tobacco smoke and its carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) have the potential effects on the production of cyclooxygenase (COX)-2 and its downstream products in tumor cells. This thesis is constructed to describe the study focused on the role of thromboxane A2 (TxA2), one of the key downstream products of COX-2, in NNK-induced lung tumor growth. / We found that as compared to non-smokers, lung cancer tissues obtained from smokers tended to express more TxA2 synthase (TxAS). Moreover, NNK could stimulate TxA2 synthesis in lung cancer cells. Blockade of TxA2 synthesis and action by TxAS inhibitor and TxA2 receptor (TP) antagonist completely blocked NNK-promoted cell proliferation via inducing apoptosis. Moreover, TP agonist U46619 reconstituted a near full proliferative response to NNK when TxAS was inhibited, affirming the role of TP in NNK-induced cell growth. Furthermore, we revealed that the activated TP may then activate CREB through PI3K/Akt and ERK pathways, thereby contributing to the NNK-induced lung cancer cell growth. / We subsequently showed that TP could modulate the induction of COX-2 and TxA2 by NNK. The synthesis of TxA2 stimulated by NNK was found to be mainly dependent on COX-2 activity. Intriguingly, there are similar inhibitory effects on NNK-induced cell growth between pharmacological inhibition of COX-2 and the blockade of TxA2 synthesis and action. Because TP is the natural receptor of TxA2, these results suggest that TP may function as a mediator for the tumor-promoting effects of COX-2 upon NNK treatment, which was confirmed by the data showing that U46619 almost restored NNK effects in the presence of COX-2-siRNA. Importantly, TPα, but not TPβ was found to be widely expressed in lung cancer cells and be able to promote tumor growth, COX-2 expression and TxA2 synthesis upon NNK treatment. / We further demonstrated that in lung tumor tissues obtained from smoker, TPα protein was increased, which was similar to the change in TxAS protein. The increased levels of TxAS and TP proteins were also found in lung cancer tissues of A/J mice treated with NNK. In cell culture experiments, NNK could increase TxAS at both protein and mRNA levels. However, TPα rather than TPβ was increased by NNK at protein but not mRNA level. NNK-stimulated TxAS expression was dependent on nuclear factor (NF)-κB signaling. Other key transcriptional factors, such as specificity protein(SP)-1, CREB and peroxisome proliferator-activated receptor-gamma (PPARγ), were not involved in NNK-induced TxAS and TPα expression. Further experiments revealed that post-transcriptional mechanisms were responsible for NNK-induced TPα expression. TPα rather than TPβ was finally identified to have a positive role in NNK-induced NF-κB activation and TxAS expression. / Taken together, our study suggests that TxA2 pathway has a positive role in NNK-induced lung cancer cell growth. An auto-positive feedback loop of TPα activation to facilitate lung tumor growth in the presence of NNK is delineated by these results. Therefore, targeting TxAS or/and TPα may represent a promising strategy for prevention and treatment of lung cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Runyue. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 / Publications / Acknowledgement / Abbreviations / Table of contents / Chapter Chapter 1 --- General introduction--Tobacco smoking, COX-2 pathway and cancer / Chapter 1.1 --- Abstract --- p.1 / Chapter 1.2 --- Introduction --- p.2 / Chapter 1.3 --- Cyclooxygenase and prostanoids --- p.5 / Chapter 1.4 --- The effects of tobacco smoking on COX-2 pathway, and the related pathologies --- p.8 / Chapter 1.4.1 --- Smoking, PGE2, inflammation and immunosupression --- p.8 / Chapter 1.4.2 --- Smoking, TxA2, platelet activation, cell contraction and angiogenesis --- p.11 / Chapter 1.4.3 --- Smoking and PGI2 --- p.16 / Chapter 1.5 --- The role of cyclooxygenase-2 pathway in the progression of tobacco smoke-related cancers --- p.19 / Chapter 1.5.1 --- Lung cancer --- p.19 / Chapter 1.5.2 --- Gastrointestinal cancer --- p.23 / Chapter 1.5.3 --- Bladder cancer --- p.24 / Chapter 1.5.4 --- Head and neck squamous cell carcinoma --- p.25 / Chapter 1.5.5 --- The signaling mechanisms underlying the induction of COX-2 by smoking in tumors --- p.26 / Chapter 1.6 --- Summary, future directions and key questions --- p.28 / Chapter Chapter 2 --- NNK induces lung cancer cell growth by stimulating TxA2 and its receptor / Chapter 2.1 --- Abstract --- p.32 / Chapter 2.2 --- Introduction --- p.33 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.3.1 --- Cell lines and cell culture --- p.35 / Chapter 2.3.2 --- Chemicals and drug treatment --- p.35 / Chapter 2.3.3 --- Thromboxane B2 EIA assay --- p.36 / Chapter 2.3.4 --- MTT assay --- p.36 / Chapter 2.3.5 --- BrdU cell proliferation assay --- p.37 / Chapter 2.3.6 --- Flow cytometry for analysis of apoptosis --- p.37 / Chapter 2.3.7 --- Transfection of cells with CREB siRNA --- p.38 / Chapter 2.3.8 --- Western blot analysis and antibodies --- p.38 / Chapter 2.3.9 --- Statistical analysis --- p.39 / Chapter 2.4 --- Results --- p.41 / Chapter 2.4.1 --- High expression of TxAS in lung cancer tissues of smoker --- p.41 / Chapter 2.4.2 --- NNK stimulated TxA2 synthesis in lung cancer cells --- p.43 / Chapter 2.4.3 --- Blockade of TxA2 synthesis and action prevented NNK-induced cell growth --- p.44 / Chapter 2.4.4 --- TxA2 mimetic U46619 reconstituted NNK-enhanced cell proliferation under TxA2-inhibited condition --- p.47 / Chapter 2.4.5 --- Blockade of TxA2 synthesis or action induced the apoptosis of the NNK-exposed cells --- p.47 / Chapter 2.4.6 --- CREB is accountable for the key role of TxA2 in NNK-enhanced cell proliferation --- p.49 / Chapter 2.4.7 --- PI3K/Akt and ERK rather than JNK and p38 pathways were mediated by TxA2 in the NNK-exposed cells --- p.52 / Chapter 2.4.8 --- CREB is located downstream of the PI3K/Akt and ERK pathways in NNK-treated cells --- p.53 / Chapter 2.5 --- Discussion --- p.55 / Chapter Chapter 3 --- The positive role of TPα in the induction of COX-2, TxA2 and cell growth by NNK in human lung cancer cells / Chapter 3.1 --- Abstract --- p.62 / Chapter 3.2 --- Introduction --- p.63 / Chapter 3.3 --- Materials and methods --- p.65 / Chapter 3.3.1 --- Cell culture and chemicals --- p.65 / Chapter 3.3.2 --- Transient transfections --- p.66 / Chapter 3.3.3 --- TxB2 measurement --- p.66 / Chapter 3.3.4 --- Cell growth detection --- p.67 / Chapter 3.3.5 --- Analysis of apoptosis --- p.67 / Chapter 3.3.6 --- Western blot analysis and antibodies --- p.67 / Chapter 3.3.7 --- Statistical analysis --- p.68 / Chapter 3.4 --- Results --- p.70 / Chapter 3.4.1 --- Examination of TP as the modulator for induction of COX-2 and TxA2 by NNK --- p.70 / Chapter 3.4.2 --- The TxA2 generated in cells treated with NNK is mainly dependent on COX-2 activity --- p.72 / Chapter 3.4.3 --- Examination of TP as the key mediator for the tumor-promoting effect of COX-2 --- p.72 / Chapter 3.4.4 --- The expression and action of α and β isoforms of TP in human lung cancer cells --- p.77 / Chapter 3.4.5 --- the identification of positive role of TPα in NNK-induced COX-2, TxA2 and cell growth in lung cancer cells --- p.79 / Chapter 3.5 --- Discussion --- p.81 / Chapter Chapter 4 --- TP-α facilitates lung tumor growth through an autoregulatory feedback mechanism / Chapter 4.1 --- Abstract --- p.88 / Chapter 4.2 --- Introduction --- p.89 / Chapter 4.3 --- Materials and methods --- p.91 / Chapter 4.3.1 --- Human lung tissue and immunohistochemical analysis --- p.91 / Chapter 4.3.2 --- Animal treatment --- p.91 / Chapter 4.3.3 --- Cell culture and chemicals --- p.92 / Chapter 4.3.4 --- Transient transfection --- p.93 / Chapter 4.3.5 --- Real-time PCR --- p.93 / Chapter 4.3.6 --- Western blot analysis and antibodies --- p.94 / Chapter 4.3.7 --- Statistical analysis --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- The effects of smoking on the expression of TP in human lung cancer tissue --- p.96 / Chapter 4.4.2 --- The effects of NNK on the expression of TxAS and TP in lung tissues of A/J mice --- p.98 / Chapter 4.4.3 --- The effects of NNK on the expression of TxAS and TPα in lung cancer cells --- p.99 / Chapter 4.4.4 --- Identification of the roles of NF-κB, CREB and SP1 in NNK-induced TxAS and TPα expression --- p.101 / Chapter 4.4.5 --- The negative role of PPARγ in NNK-induced TxAS and TPα expression --- p.104 / Chapter 4.4.6 --- NNK-induced TPα expression via post-transcriptional mechanism --- p.105 / Chapter 4.4.7 --- Examination of TPα auto-activation mechanism in lung cancer cells stimulated with NNK --- p.106 / Chapter 4.5 --- Discussion --- p.109 / Chapter Chapter 5 --- Conclusion and future works / Chapter 5.1 --- Conclusion --- p.114 / Chapter 5.2 --- Future works --- p.115 / Chapter 5.2.1 --- The possible role of miR-34c in the auto-regulatory loop of TxAS expression or TPα activation --- p.116 / Chapter 5.2.2 --- The possible role of FOXO3a in the auto-regulatory loop of TxAS expression or TPα activation --- p.116 / References --- p.119

Cancer has been a disease, which has generated intense research interest for many years. Misexpression of two oncoproteins, TAL 1 and LMO 1, has been found to help induce a particular type of leukemia, called T-cell acute lymphoblastic leukemia (T-ALL). Presently, it is not completely understood how these proteins induce leukemogenesis or what other cellular proteins they interact with to drive this progression. In this study, a series of experiments were conducted to identify downstream targets of TALI and LMO1. Using retroviral gene transfer, both genes were introduced, either singly or in combination, into a murine T-cell line called AKR-DP-603. Empty vectors were introduced as controls. In order to assay the effects of TALI and LMO I expression on expression of other proteins, a series of Western blots were completed on all populations of engineered cells. It was determined that there were differences in expression of Bcl-2 and p16 as indicated by differences in band intensities on the blots. This is important because it implies an effect on protein levels by TAL 1 and LMO 1. However, there were no differences in protein expression levels for Bax or cyclin D1. This suggests that TAL1 and LMOI do not have any regulatory effects on these proteins. In addition, apoptotic assays were completed on all populations of cells. The results of both a TUNEL assay and ethidium bromide/acridine orange staining protocol showed TAL1- and LMO1expressing cells to have an increase in cell survival under starvation conditions and a lower frequency of apoptosis. Statistical analysis verified significant difference in the apoptosis assays. The data suggests an up-regulation of anti-apoptotic proteins. The finding of this research allow a clearer understanding of the process of leukemogenesis and may lead to a development of better cancer treatments. / Department of Biology

This study is designed to assess the effectiveness of the Nuclear Non-Proliferation Regime and analyze theories for effectively analyzing countries that are a risk for proliferating nuclear armaments. The initial phase of my research is designed to assess the existing literature and primary theoretical approaches for analyzing nuclear non-proliferation initiatives and potential nuclear proliferators. My main means of analysis will be to examine the national and international actors involved in each case. With this method, I plan to analyze a government at the level of each of its ruling institutions. Each of these institutions will be analyzed in the context of Joseph Cirincione's five drivers and barriers: security, prestige, domestic politics, technology, and economics. This study will then review multiple historical cases of countries and treaties related to the nuclear non-proliferation issue in the context of my method of analysis. In particular, each historical study will discuss major actors and institutions with respect to the five major proliferation concepts, as a means of demonstrating the validity of my method. The primary section of my thesis will be the application of my method to one of the preeminent nuclear proliferation threats today: Iran. After a discussion of the physical status of Iran's nuclear program, I will begin my analysis in terms of my concepts, and will examine the principal actors involved in the Iranian nuclear dispute. These will be the Iran's moderate and conservative factions, as well as the U.S., Israel, the EU-3, and IAEA, and they will be examined in the context of the five drivers and barriers. The final section will be my overall risk analysis for Iran. My preliminary analysis is that regime survival is the most critical issue when it comes to the principal motivations of a state to develop nuclear arms. If this is correct, policy options designed to take advantage of the actors' positions in Iran can be formulated based on the specific conditions that prevail in Iran.

No description available.

Der pRB/E2F-Signalweg ist ein wichtiger Schlüsselpunkt für die Wachstumskontrolle in Säugerzellen und in vielen Tumoren sind Komponenten dieses Signalweges dereguliert. Durch die Nullmutation von E2F3 in Mausembryonalen Fibroblasten (MEFs) und Mäusen konnte gezeigt werden, dass E2F3 essentiell für das zelluläre Wachstum ist und in der Maus organspezifisch sowohl als Tumorsuppressor als auch Onkogen agieren kann. Jedoch sind dafür die zugrunde liegenden Mechanismen noch nicht genau geklärt. Möglicherweise tragen verschiedene Signalwege, die durch den Verlust von E2F3 dereguliert werden, zu den Defekten bei. In dieser Arbeit wurde TGFbeta1, ein wichtiger Wachstumsregulator, in den E2f3-/- MEFs untersucht und es konnte zum ersten Mal eine direkte Verbindung zwischen der E2F3-Expression und der TGFbeta1-Signalwirkung gezeigt werden. Durch den Verlust von E2F3 werden Tgfb1 und die TGFbeta1-regulierten Gene PAI-1, p21, Vimentin und Fibronectin in MEFs dereprimiert. Darüber hinaus werden MEFs und humane Lungenkarzinomzellen durch den Verlust von E2F3 gegenüber TGFbeta1 sensibilisiert und reagieren verstärkt auf TGFbeta1-induzierte Genexpression und Prozesse wie Wachstumsarrest und EMT. Somit wird E2F3 nicht nur durch TGFbeta1 reguliert, sondern kann auch auf TGFbeta1 und die TGFbeta1-Signalwirkung Einfluss nehmen, was für die Tumorprogression weit reichende Auswirkung haben kann. Um die tumorsuppressiven Eigenschaften von E2F3 besser zu verstehen, wurden im zweiten Teil dieser Arbeit murine medulläre Schilddrüsentumore mit unterschiedlichem metastatischen Potential miteinander verglichen und es konnten neue E2F-Zielgene identifiziert werden. Die Untersuchung von humanen Struma nodosa-Biopsien und metastatischen medullären Schilddrüsentumoren ergab, dass die in den Mäusen gefundenen Gene künftig auch als humane Metastasemarker Verwendung finden können. / The pRB/E2F-pathway plays a key role in growth control and it is deregulated in many tumors. Previously, by analysing E2f3 deficient mouse embryonic fibroblasts (MEFs) and mice it has been shown that E2F3, a key downstream target of pRB, is essential for cellular proliferation and can act either as an oncogene or tumorsuppressor in mice depending on the organ. However, the underlying mechanism is still unclear. We suggest that specific pathways which are deregulated due to the deletion of E2F3 contribute to these defects. TGFbeta1, which is one of the most potent growth regulators for mammalian cells was analysed in E2f3-/- MEFs. In this study, we could establish a direct link between E2F3 expression and TGFbeta1 signalling. Loss of E2F3 in MEFs leads to de-repression of Tgfb1 and TGFbeta1-regulated genes like PAI-1, p21, vimentin and fibronectin. Moreover, loss of E2F3 in MEFs or in human lung carcinoma cells results in an increased sensitivity to TGFbeta1-induced gene expression and processes like growth arrest and epithelial mesenchymal transition. These data suggest that not only TGFbeta1 can act on E2F3 but also E2F3 can affect TGFbeta1 and the outcome of TGFbeta1-induced signalling. In order to understand the tumor suppressive properties of E2F3, we compared gene expression profiles of murine medullary thyroid carcinomas (MTCs) of different metastatic potential and could identify novel E2F-target genes. Analysis of human struma nodosa biopsies and human metastatic medullary thyroid tumors showed that the genes identified in the mouse model can also be used as metastasis markers in human tumors.

Zusammenfassung in PDF Zielstellung: Der biologische Botenstoff N ist von signifikanter Bedeutung für die Funktionsfähigkeit des Herz-Kreislauf-Systems. Pathologische Bedingungen sind oft auf eine veränderte Verfügbarkeit von N zurückzuführen. Das Verständnis der Regulierung der N-Bildung durch Endothelzellen (EC) und die nachfolgende N-Chemie im gesunden und kranken Organismus ist deshalb essentiell, jedoch längst nicht vollständig. Das Ziel dieser Dissertation war aus diesem Grund Untersuchungen zum Schutz von N vor der Wechselwirkung mit S (durch SOD-mimetische Nitroxide), sowie zum Effekt des Membranpotentials (Em) und des Zellwachstums (Proliferation) auf die Aktivität der endothelialen N Synthase (eNOS) an Kulturen von Atrium-Endothelzellen des Rindes (BAtEC). Methoden: Zum empfindlichen und spezifischen Nachweis von N wurde die ozon-vermittelte N-Chemolumineszenz (N-CL) evaluiert, modifiziert und an verschiedene in vitro Systeme angepaßt. Die N-CL wurde auf Grundlage des durch SOD hemmbaren Abbaus von N durch S als ein neuartiger Ansatz zur Bestimmung physiologisch niedriger Konzentrationen an S vorgeschlagen. Ergebnisse: Die Nitroxide -hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 3-carboxy-proxyl und 3-ethoxycar-bonyl-proxyl erhöhten die N-Konzentration durch ihre SOD-mimetische Wirkung in Modellsystemen von 3-morpholinosydnonimine (SIN-1) und Kulturen von BAtEC. Mittels ESR-Spin-Trap Untersuchungen an Lösungen von SIN-1 wurde die Bildung von H-Addukten durch Dismutierung von S bestätigt, Peroxynitrit wurde als Quelle ausgeschlossen. Modulierung des Em (durch Veränderung der extrazellulären K + -Konzentration) beeinflußte die endotheliale N-Freisetzung; Hyperpolarisierung erhöhte, Depolarisierung verminderte die N Produktion. Inhibierung der elektrogenen Na + -K + ATPase induzierte synchrone Oszillationen der endothelialen N-Freisetzung. Die systematische Untersuchung zu EC-Wachstum/Proliferation zeigte keine Änderung der N-Produktion (in Gegenwart von SOD). Jedoch war die Menge von verfügbarem N (in der Abwesenheit von SOD) niedrig in präkonfluenten, stark proliferierenden BAtEC. Die Expression der eNOS erhöhte sich mit der Kultivierungsdauer und erreichte ein Maximum bei Konfluenz. Die relative eNOS-Aktivität (N, 2-, und L-citrulline-Produktion pro eNOS-Protein) war am größten in präkonfluenten, stark proliferierenden BAtEC. Schlußfolgerungen: Die Wechselwirkung mit S ist kritisch für die Halbwertszeit von N. Aus der durch diese Studie gezeigten Erhöhung von N in biologischen und chemischen Modellsystemen kann geschlußfolgert werden, daß Nitroxide unter Bedingungen einer S-bedingten Verminderung von verfügbarem N pharmakologische Wirksamkeit besitzen könn-ten. Untersuchungen ergaben den Nachweis zur Em-abhängigen Modulierung der endothelialen N-Freisetzung. Dies liefert eine Erklärung für die Em-bedingten Veränderungen des Gefäßto-nus, nachgewiesen während physiologischer Zellstimulierung, aber auch unter pathologischen Bedingungen. Untersuchungen zu EC-Wachstum/Proliferation lieferten den Beweis für die Ver-minderung von verfügbarem N in präkonfluenten, stark proliferierenden BAtEC durch endogen gebildetes S. Die Expremierung von eNOS war proliferationsabhängig. Die spezifische eNOS-Aktivität war in proliferierenden BAtEC erhöht (wahrscheinlich durch post-translationale Verän-derungen) und in ruhenden BAtEC erniedrigt (wahrscheinlich durch Substrat- oder Kofaktormangel). / abstract in pdf Objective: The biological messenger n is of significant importance for the functionality of the cardiovascular system. Pathological conditions are often attributed to an altered availability of n. The understanding of the regulation of n formation by endothelial cells (EC) and the concomitant chemistry of n in health and disease is therefore essential but far from being complete. The objective of this dissertation was therefore to study in culture systems of bovine atrial endothelial cells (BAtEC) the protection of n from the interaction with S by SOD-mimetic nitroxides, and the effect of the membrane potential (Em) and cell growth/proliferation on the activity of the endothelial nitric oxide synthase (eNOS). Methods: For the sensitive and specific detection of n, the ozone-mediated n chemiluminescence (n-CL) was evaluated, modified and adapted to various in vitro systems. Due to the SOD-inhibitable degradation of n by S , the n-CL was proposed as a novel approach to quantify the physiological low levels of S . Results: The nitroxides 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl increased the n concentration by their SOD-mimetic action in a model system of 3-morpholinosydnonimine (SIN-1) and cultures of BAtEC. EPR spin trapping in SIN-1 solution revealed the formation of H adducts due to dismutation of S and not via decomposition of peroxynitrite. Changes in Em (by alteration of the extracellular K + concentration) affected the endothelial n release; membrane hyperpolarization increased, membrane depolarization decreased the n production. Inhibition of the electrogenic Na + -K + ATPase induced synchronous oscillations in endothelial n liberation. The systematic investigations on EC growth/proliferation showed no change in total n production (in the presence of SOD). However, the bioavailable n (in the absence of SOD) was low in preconfluent, highly proliferating BAtEC. Expression of eNOS increased with culture duration with a maximum at confluence. Relative eNOS activity (n, 2, and L-citrulline production per eNOS protein) was highest in preconfluent, highly proliferating BAtEC. Conclusions: The interaction of S is critical for the half-life of n. With the increase of n in biological and chemical model systems shown by this study, nitroxides might exert pharmacological potential under conditions of S -mediated diminution of bioavailable n. Experimental evidence was shown for the Em-dependent modulation of endothelial n formation. It supports an explanation for the Em-mediated changes of vascular tone, as demonstrated for physiological cell stimulation but also under pathological conditions. Investigations on the EC growth/proliferation provided evidence that BAtEC-derived S decreases bioavailable n in preconfluent, highly proliferating BAtEC, whereas expression of eNOS is proliferation-dependent. The specific eNOS activity is upregulated in proliferating BAtEC (probably via post-translational modifications) and down-regulated in quiescent BAtEC (probably via substrate or cofactor limitations).

Temporal control, inherent in all biological processes, relies on intrinsic systems to govern periodical behaviors and physiological responses. The circadian clock, a vital timekeeper, enables organisms to anticipate and adjust to daily environmental changes. In mammals, the circadian clock is organized hierarchically, with a central master clock in the hypothalamic suprachiasmatic nucleus regulating peripheral clocks distributed across the body. To maintain coherent circadian rhythms at the tissue level, peripheral oscillators exchange intercellular coupling factors by paracrine signaling pathways to synchronize. At the individual cell scale, the circadian clock interacts with another periodical biological process: the cell cycle. However, the mechanisms governing this interplay remain poorly elucidated. Here, we explore the influence of extracellular circadian synchronization on the intracellular coordination between the circadian clock and the cell cycle. To do so, we combined a mathematical model and long-term live-imaging recordings at the single-cell and population level of a human cell line. We show that the global circadian and cell cycle coordination within individual cells is disrupted when the extracellular circadian synchronization is lost, obstructing collective tissue growth. Populations with coherent circadian rhythms display rhythmic growth oscillations, uncovering a novel global regulator of tissue dynamics. Knocking down core circadian elements abolished these effects, revealing the fundamental role of circadian clock control as a timing mechanism. These findings advance our understanding of how biological systems maintain equilibrium and regulate proliferation in normal and pathological conditions. The circadian clock plays a crucial role in orchestrating cell proliferation, impacting tumor initiation, growth, and treatment responses. Recent research has reported significant changes in drug response for different administration hours throughout the day, highlighting the benefits of aligning treatment strategies to the inherent circadian rhythm. However, chronotherapy is still omitted in clinical practice, primarily due to a lack of understanding of the underlying mechanisms driving time-dependent drug responses. Currrently, no standardized protocols exist for identifying these temporal factors. Therefore, we developed a combined mathematical and experimental approach to identify the factors influencing time-dependent drug sensitivity in human cells. Our results show how circadian and drug properties independently shape time-of-day drug responses, offering novel insights into the time-dependent treatment outcome. This framework holds potential for developing personalized treatment schedules aligned with the internal circadian clock, optimizing cancer therapeutical strategies. On the other hand, tissue growth masks heterogeneous proliferation patterns at the single-cell level, potentially jeopardizing the treatment outcome, which cannot be exclusively attributed to circadian clock regulation. Clustering the cells upon their overall number of divisions, the proliferative patterns remain strikingly constant across different tissues, a phenomenon reported by several recent studies. This consistency implies the existence of a common underlying mechanism that is currently unknown. Proliferation control relies on a set of checkpoint mechanisms that accurately and quickly detect DNA damage. The onset of cellular stress triggers the activation of the p53 protein, orchestrating the expression of hundreds of genes responsible for cell cycle regulation or apoptosis, among other functions. Here, we present evidence that changes in cellular stress levels contribute to the gradual proliferation variability. Specifically, different DNA damage levels are encoded quantitively into signal parameters of p53 and p21 proteins in a gradual manner, shaping proliferation activity proportionally. These results propose a novel function of the p53-p21 signaling network in deciphering and decoding the magnitude of DNA damage to adjust and control proliferation. / This study examines how extracellular circadian synchronization affects the coordination between the circadian clock and the cell cycle. By combining mathematical modeling and long-term live imaging of human cells, we show that loss of synchronization disrupts global circadian and cell cycle coordination in individual cells, hindering tissue growth. When circadian rhythms are coherent, rhythmic growth oscillations occur, indicating a global tissue dynamics regulator. Knocking down key circadian elements abolished these effects, emphasizing the circadian clock's timing role. These findings enhance our understanding of biological balance and proliferation regulation in both normal and pathological states. The circadian clock is also vital in cell proliferation, influencing tumor growth and treatment responses. Drug responses vary depending on the time of day, highlighting the importance of aligning treatments with circadian rhythms. However, chronotherapy is not widely used in clinical practice due to insufficient understanding of the underlying mechanisms. Our approach identifies factors affecting time-dependent drug sensitivity, offering insights into personalized treatment schedules. Tissue growth masks single-cell proliferation patterns, essential for effective treatment. By clustering cells based on division numbers, we found consistent proliferation patterns across tissues, suggesting an unknown underlying mechanism. The p53-p21 signaling network regulates proliferation by quantifying DNA damage and adjusting cell cycle responses. This study reveals how p53-p21 signaling decodes DNA damage levels to control proliferation. / Diese Studie erforscht, wie extrazelluläre zirkadiane Synchronisation die Abstimmung zwischen der zirkadianen Uhr und dem Zellzyklus beeinflusst. Durch mathematische Modellierung und langfristige Live-Bildgebung an menschlichen Zellen zeigt sie, dass der Verlust der Synchronisation die zirkadiane und Zellzykluskoordination stört und Gewebewachstum hemmt. Bei kohärenten zirkadianen Rhythmen entstehen rhythmische Wachstumsoszillationen, die eine globale Steuerung der Gewebedynamik andeuten. Das Ausschalten zirkadianer Elemente beseitigt diese Effekte und verdeutlicht die zeitliche Rolle der zirkadianen Uhr. Diese Ergebnisse liefern Einblicke in die biologische Balance und Regulierung der Proliferation in normalen und pathologischen Zuständen. Die zirkadiane Uhr beeinflusst die Zellproliferation, das Tumorwachstum und die Wirkung von Behandlungen. Arzneimittelreaktionen variieren tageszeitabhängig, was die Relevanz der Chronotherapie unterstreicht – der Anpassung von Therapien an zirkadiane Rhythmen. Allerdings wird Chronotherapie selten klinisch genutzt, da die zugrundeliegenden Mechanismen nicht ausreichend verstanden sind. Diese Studie identifiziert Faktoren, die die zeitabhängige Arzneimittelempfindlichkeit beeinflussen, und bietet Perspektiven für personalisierte Therapien. Gewebewachstum verdeckt Proliferationsmuster einzelner Zellen, die für Behandlungen entscheidend sind. Durch Clusteranalysen der Zellteilungen zeigten sich konsistente Muster in Geweben, was auf unbekannte Mechanismen hindeutet. Das p53-p21-Signalnetzwerk reguliert die Proliferation, indem es DNA-Schäden bewertet und Zellzyklusreaktionen anpasst. Die Studie zeigt, wie dieses Netzwerk DNA-Schäden interpretiert, um Zellwachstum zu steuern.

Connaître en temps réel la réponse et le comportement des cellules et organismes modèles suite à des changements de leur environnement, ou à des modulations de leurs fonctions biologiques est devenu essentiel dans les sciences du vivant. Ces réponses nous permettent ensuite de comprendre les mécanismes qui régissent le fonctionnement des cellules vivantes, avec des implications en recherche fondamentale, appliquée et biomédicale. Un des plus gros défis technologiques reste le contrôle des paramètres environnementaux en microscopie haute résolution. De nos jours, aucun système ne permet de réguler un ensemble complexe de paramètres de manière précise, dynamique et simultanée tout en observant les cellules dans leur environnement. L’objectif de ma thèse est de mettre au point un tel dispositif permettant a minima une régulation fine de la température, de la composition du milieu, et notamment de la concentration de divers drogues. Ce système doit être compatible avec les applications les plus poussées en microscopie photonique. Mon approche au cours de ma thèse pour élaborer un tel système est l’utilisation de la microfluidique. En effet, c’est la seule technologie qui puisse de réaliser un tel multiplexage. Elle permet de manipuler des petites quantités de fluide à travers un système contenant des canaux de dimensions allant du micromètre au centimètre. Cet ordre de grandeur des canaux constitue un atout majeur (réduction de la consommation des réactifs, réduction des couts, cinétiques des réactions chimiques et biologiques élevées, temps de diffusion court, etc.) et permet d’allier les expériences biologiques à la microscopie. Mon objectif est de concevoir une puce microfluidique qui représentera un pas technologique majeur et ouvrira de nouvelles possibilités de recherche. / Monitoring in real-time the response of cells and model organisms to the changes in their environment or to modulations of their biological functions has become essential in life sciences. One of the main technical challenges for biologists is the precise and dynamic control of various environmental parameters while doing high-resolution microscopy. My thesis consists of building a robust and versatile system, dedicated to live-cell imaging that will be compatible with adherent and non adherent models, that could provide a precise and simultaneous control of 1) the temperature, 2) the media exchanges and 3) the drug concentration while doing photonic microscopy. My approach is to use microfluidics, which is the best candidate in order to achieve this system and provides all the necessary controls of micro-scaled volumes for culturing, maintaining or analyzing cells. It produces miniaturized systems used as tools for biological experiments, in which channels of a micro-scaled dimension are used for the fluid circulation. The laminar flow in these chips allows fast molecule diffusion as well as fast temperature diffusion. Because of the high surface to volume ratio, the consumption of reagents is reduced, and media switches can be fast. This system will represent a major technical and beneficial step and will open new possibilities of research in biology.