The pulmonary vein (PV) constricts to hypoxia however little is known about the underlying mechanisms. Hypoxic PV constriction is proposed to recruit upstream capillary beds and optimise gas exchange in healthy humans and may play a role in high altitude pulmonary oedema. The PV is also intrinsic to disease states including pulmonary hypertension and pulmonary veno-occlusive disease. Blood vessel culture can be a powerful tool to enable assessment of the impact of environmental factors on vessel function and as a disease model. However culture conditions alone affect vessel contractility; the effect of culture conditions on PV function remained to be established. The aim of this project was to investigate hypoxic responses of porcine PVs including the impact of maintenance in culture. Maintenance of PVs in culture conditions for 24 hours increased contraction to hypoxia and inhibited hypoxic relaxation post-contraction. These changes to PV hypoxic responses were thought to result from endothelial dysfunction. However, the endothelial nitric oxide synthase inhibitor L-NAME inhibited PV hypoxic contraction and enhanced relaxation. The impact of K+ channel inhibitors on hypoxic contraction was also investigated. Penitrem A, 4AP, DPO-1, ZnCl2 and glyburide had no significant effect however TEA and BDM inhibited the hypoxic contraction. This suggested that TASK, KV1.5, BKCa and KATP do not play a role in the mechanism of hypoxic pulmonary venoconstriction however KV channels containing KV2.1 α subunits may modulate the response. Results with L-NAME suggested endothelial dysfunction may not fully account for the change in PV function after exposure to culture. Therefore the impact of PV maintenance in culture was further explored using an isolated PV smooth muscle cell (PVSMC) model. Maintenance of PVs in culture conditions had minimal impact on morphology and electrical properties of PVSMCs. Notably, resting membrane potential and hypoxia-induced depolarisation were not significantly different. Based on the findings of this study, the endothelium in PVs appears to a) play a major role in modulation of the hypoxic response b) be sensitive to short-term exposure to culture conditions. K+ channels appear to play a minor role in PV hypoxic contraction and SMCs isolated from PVs maintained in culture conditions have similar morphological and electrophysiological characteristics to freshly isolated PVSMCs. Taking all this into account, endothelial regulation of contractility should be a key focus for future PV research.

Foi realizado o estudo morfométrico e o estudo hemodinâmico da veia porta em vinte cães normais, de idade igual e inferior a 120 dias, e em cinco cães portadores de desvio portossistêmico, de idades entre 90 e 360 dias. Dois animais do grupo de cães portadores de desvio portossistêmico foram submetidos ao tratamento cirúrgico (colocação de anel ameróide) e avaliações subseqüentes ao procedimento cirúrgico foram realizados. Nos cães do grupo normal, as margens hepáticas apresentaram-se entre 1,50cm e 3,00cm depois da margem costal. As médias dos diâmetros médios da veia porta (VP), veia cava caudal (VCC) e aorta abdominal (AO) obtidas foram respectivamente, 0,38cm, 0,37cm e 0,41cm. As proporções entre os diâmetros médios VP/VCC e VP/AO apresentaram médias de 1,10 e 0,94, respectivamente. As médias das áreas de VP, VCC e AO mediram respectivamente, 0,12cm2, 0,11cm2 e 0,14cm2. No estudo hemodinâmico de VP destes animais, utilizando-se o ultra-som Doppler, a velocidade média de fluxo sangüíneo portal (VMFSP) mediu 17,77cm/s. A média de fluxo sangüíneo portal (FSP) mediu 83,11ml/min/kg. O índice de congestão (IC) apresentou média de 0,009. Para o grupo de cães portadores de desvio portossistêmico, o fígado apresentou redução de seu volume, sendo visibilizado entre 1,00cm e 2,00cm antes da margem costal. No estudo morfométrico, as médias dos diâmetros médios obtidos de VP, VCC e AO mensuraram respectivamente, 0,52cm, 0,79cm e 0,58cm. As proporções entre os diâmetros médios VP/VCC e VP/AO mediram respectivamente, 0,62 e 0,84. As médias das áreas de VP, VCC e AO mediram respectivamente, 0,22cm2, 0,56cm2 e 0,27cm2. Ao ultra-som Doppler a VMFSP mediu 26,10cm/s e a média do IC obtido foi de 0,009. Nos animais do grupo de cães portadores de desvio portossistêmico submetidos ao procedimento cirúrgico, foi observado aumento de volume hepático na semana seguinte à colocação do anel ameróide e a VMFSP manteve-se inferior a 19,50cm/s em todos exames subseqüentes à cirurgia no cão 1. / The morphometry and haemodynamic aspects of portal vein were studied in 20 normal dogs with less than 120 days of age and in 5 dogs presenting portosystemic shunting with ages between 90 and 360 days. 2 dogs of the group of animals with portosystemic shunting were submitted to surgical treatment, using a specialized device (ameroid constrictor). Subsequent evaluations were made after the surgical procedure. In the normal group the hepatic margins were seen 1.50cm to 3.00cm below de costal margin. Collected data indicated that the mean diameter of portal vein (VP), caudal vena cava (VCC) and abdominal aorta (AO) measured respectively, 0.38cm, 0.37cm and 0.41cm. The VP/VCC and VP/AO mean ratios were respectively, 1.10 and 0.94. The average of VP, VCC and AO areas were respectively, 0.12cm2, 0.11cm2 and 0.14cm2. The haemodynamic of portal vein was studied by ultrasound Doppler and the mean velocity of portal blood flow (VMFSP) measured was 17.77cm/s. It was verified that portal blood flow (FSP) average was 83.11ml/min/kg and the congestion index (IC) average was 0.009. In the group of animals presenting portosystemic shunting, the hepatic margins were seen 1.00cm to 2.00cm above the costal margin. The morphometry of VP, VCC and AO presented a mean diameter of 0.52cm, 0.79cm and 0.59cm, respectively. The VP/VCC and VP/AO mean ratios were respectively, 0.62 and 0.84. The average of VP, VCC and AO areas were respectively, 0.22cm2, 0.56cm2 and 0.27cm2. The haemodynamic study demonstrated that the VMFSP measured was 26.10cm/s and de IC average was 0.009. In the group of animals with portosystemic shunting which were submitted to surgical treatment, an increase of the liver size was seen from the first ultrasonographic evaluation. The measurements of VMFSP collected in the post surgical period were <= 19.50cm/s in dog 1.

5‐羟色胺（5-HT）是一种强有力的血管活性神经递质，被广泛的应用在调节血管张力。当5‐HT 被释放后，会被单胺氧化酶（MAOs）催化的酶促反应代谢，从而产生不同的代谢产物，比如5‐HIAA，5‐HTOL 和过氧化氢（H₂O₂）。然而，5‐HT对于内皮细胞活性氧物种（ROS）的产生作用以及5‐HT 转运体，5‐HT 受体，MAOs和ROS 的产生伴随着细胞内钙变化是否参与了其中的信号传导尚未被阐明。所以，这个研究最初的目的是考查外源性加入的5‐HT 对于脐静脉内皮细胞中ROS产生的影响以及其潜在的生物机理。 / 数据清楚的显示在没有L‐NAME（一种抑制一氧化氮（NO）产生的抑制剂）预处理的情况下，5‐HT 并不能在脐静脉内皮细胞内产生显著性的ROS。然而，在L‐NAME 预处理的情况下，NO 的产生被完全抑制，我们观察到明显的显著性的线粒体内的ROS 产生。5‐HT 产生的线粒体ROS 可以被clorgyline（一种MAO‐A 抑制剂），indatraline（一种5‐HT 转运体阻断剂），LY272015（一种5‐HT‐2B 受体拮抗剂），ketanserin（一种5‐HT2A 受体拮抗剂），XeC（一种IP3 受体拮抗剂），Gd³⁺（一种非选择性TRP 通道阻断剂），BAPTA（一种强效钙离子螯合剂），PEG‐Catalase，U73122（一种选择性PLC 抑制剂）以及没有钙离子的培养基所阻止。同时，5‐HT介导的胞内钙离子变化被XeC, Gd³⁺, BAPTA, U73122, ketanserin, LY272015 以及没有钙离子的培养基所阻止。另外，MAO‐A 基因敲除抑制了5‐HT 导致的线粒体ROS的产生却对5‐HT 介导的胞内钙离子变化没有影响。基于以上所述的结果，我们可以得出结论，通过5‐HT 转运体，5‐HT 被摄取入细胞内，然后通过MAO‐A 介导的酶促代谢反应，产生钙离子依赖性的线粒体内ROS 的产生，这一结论对于解释血小板聚集而引起的内皮细胞功能性障碍起到非常重要的作用。 / 根据前人所述，内皮细胞内产生的ROS 对于内皮细胞通透性变化有着重要的作用，但是5‐HT 诱导的脐静脉内皮细胞ROS 的增加是否会对内皮通透性有所影响并没有被说明。在这项研究中，我们设计了实验旨在测试平面细胞表面积（ PCSA ）， 跨内皮电阻（ TER ）， 细胞高度， 肌球蛋白轻链磷酸化（MLCphosphorylation）和肌动蛋白细胞骨架（F‐actin cytoskeleton）水平的变化。此外，b‐catenin 在ROS 引起的F‐actin cytoskeleton 重组中的作用也在我们的讨论范围之内。 / 数据表明，在L‐NAME 预处理的情况下，5-HT 降低了脐静脉内皮的PCSA，TER 以及细胞高度，却增加了MLCP 和与b‐catenin 表达负相关的F‐actincytoskeleton 的水平。这些作用明显被PEG‐Catalase 预处理和MAO‐A 基因敲除减弱，证明了5‐HT 通过MAO‐A 介导产生的H₂O₂ 可以增加内皮细胞的通透性。 / 据文献报道，不论内源性还是外源性的低浓度的H₂O₂ 都可以激活导致血管生成的信号通路。文献进一步表明5‐HT 可以通过特定的5‐HT 受体亚型促进各种类型的内皮细胞的血管生成。然而，5‐HT 诱导的H₂O₂ 对于脐静脉内皮的血管生成作用并没有被报道。我们通过最初的实验先验证5‐HT 对于内皮细胞增殖和迁移的影响，然后我们才去验证H₂O₂ 在其中的作用及其潜在的机理。 / 实验结果表明，在L‐NAME 预处理的情况下，不论是急性（30 分钟）还是慢性（24 小时）的5‐HT 的处理都可以导致脐静脉内皮细胞的迁移，而这个作用会被5‐HT‐2 受体拮抗剂ketanserin，LY272015，ROS 清除剂PEG‐Catalase 以及PI3K的抑制剂wortmannin 所抑制。同时，在L‐NAME 预处理下，5‐HT 增加了cortactin,p‐Akt 和 p‐eNOS 的蛋白表达量而并没有影响Akt, eNOS 和p‐cortactin 的蛋白表达量。而5‐HT 增加的p‐Akt 和p‐eNOS 的蛋白表达被wortmannin 和PEG‐Catalase所抑制。不论是在Cyuant 细胞增殖检测还是在BrdU 细胞增殖检测中，5‐HT 诱导了一种非显著性的DNA 合成的增加，并且再BrdU 细胞增殖检测中，增加了的DNA 合成被PEG‐Catalase 显著性降低。总结以上实验结果，我们可以得出结论，通过一种5‐HT‐2 受体介导的PI3K 依赖性通路，而不是cortactin 磷酸化依赖性的信号通，路5‐HT 可以引导内皮细胞迁移。 / 除此之外，ROS 也被印证可以加剧内皮细胞的炎症反应和加速内皮细胞的老化。因此，我们也观察了5‐HT 对于粘附蛋白比如ICAM‐1 和VCAM‐1 以及抗老化因子SIRT‐1 的表达是否有影响。数据表明，在L‐NAME 预处理的情况下，30 分钟的5‐HT 处理显著的增加了ICAM‐1，SIRT‐1 而不是VCAM‐1 的表达。同时，这些作用均可以被PEG‐Catalase 所抑制表明了5‐HT 通过诱导H₂O₂ 的产生来形式其促进炎症反应和抗衰老的作用。 / 最后，总结以上，通过抑制NO 的产生，5‐HT 可以通过MAO‐A 介导的酶促代谢反应在人体脐静脉内皮细胞线粒体诱导ROS 的产生。同时，5‐HT 诱导的H₂O₂参与了改变内皮细胞通透性，促进血管生成（内皮迁移）及炎症反应的过程。 / 5-Hydroxytryptamine (5-HT), a potent vasoactive neurotransmitter, is involved in the regulation of vascular tone. After its release, 5-HT is terminated at the nerve terminals via enzymatic metabolism catalyzed by monoamine oxidases (MAOs), resulting in the generation of different metabolites (e.g. 5-HIAA, 5-HTOL and H₂O₂). Our lab demonstrates for the first time that 5-HT-induced ROS production indeed occurs and therefore, the aim of this study is to investigate exogenously added 5-HT on ROS generation in human umbilical vein endothelial cells (HUVECs), in order to understand the mechanisms involved in 5-HT-induced ROS production. / Our results clearly demonstrated that in the absence of L-NAME(a NO production inhibitor), there wasno apparent ROS production induced by 5-HT. However, after the inhibition of NO synthesis by L-NAME, 5-HT caused a significant increase in mitochondrial H₂O₂ production. The 5-HT-induced mitochondrial H₂O₂ generation was sensitive to clorgyline (a MAO-A inhibitor), indatraline (a 5-HT transporter blocker), LY272015 (a 5-HT2B antagonist) and ketanserin (a 5-HT2A antagonist), Xextospongin C（XeC，a IP3 receptor antagonist), Gd³⁺ (a non-selective TRP channel blocker), BAPTA (a potent Ca²⁺ ions chelator), PEG-Catalase, U73122 (a selective PLC inhibitor), and in [Ca²⁺]o-free medium. Concurrently, 5-HT-mediated [Ca²⁺]i changes were sensitive to XeC, Gd³⁺, BAPTA, U73122, ketanserin, LY272015, and in [Ca²⁺]o-free conditions. In addition, gene knockdown of MAO-A suppressed 5-HT-elicited H₂O₂ production with no effects on [Ca²⁺]i changes. Based on all the results above, we can conclude that 5-HT caused a Ca²⁺-dependent mitochondrial H₂O₂ generation via MAO-A-mediated metabolism with the pre-requisite uptake of 5-HT into HUVECs through 5-HT transporter. / ROS derived from endothelial cells have been implicated in changes in endothelial permeability, but whether 5-HT-induced H₂O₂ generation could alter endothelial cells permeability has as yet not been demonstrated. Here, we measured the planar cell surface area (PCSA), transendothelial electrical resistance (TER), cell height, myosin light chain phosphorylation and F-actin cytoskeleton level in response to 5-HT challenge to investigate the change of endothelial permeability. Moreover, the participation of β-catenin in regulation of F-actin cytoskeleton remodeling in ROS-modulated alteration in endothelial permeability was also investigated. Results indicated that in the presence of L-NAME, 5-HT reduced the PCSA, TER and cell height in HUVECs. In contrast, 5-HT (with L-NAME) increased myosin light chain phosphorylation (MLCP) expression and F-actin cytoskeleton level, which are negatively associated with β-catenin expression. All of these effects were ameliorated by pre-treatment of PEG-Catalase or gene knockdown of MAO-A, implying 5-HT can consistently elicit the increase in endothelial permeability via MAO-A mediated H₂O₂ generation. / Low dose of ROS from exogenous or endogenous source can activate signaling pathway that lead to angiogenesis.5-HT can promote endothelial angiogenesis through specific 5-HT receptor subtype in various endothelial cell types, but the concomitant ROS generation had not previously been indicated to play a role in the process. In this study, we seek to test out the effects of 5-HT on endothelial cells migration, and should there be a functional role for ROS in the process. / Our results revealed that in the presence of L-NAME, both acute (30 min) and chronic (24 hr) treatment of 5-HT caused HUVECs migration, the effects of which were reversed by pre-incubation of 5-HT-2 receptor antagonists, ketanserin, LY272015, ROS scavenger PEG-Catalase or selective PI3K inhibitor wortmannin. With L-NAME, 5-HT consistently increased cortactin, p-Akt and p-eNOS expression without affecting total Akt, eNOS and p-cortactin protein expression whereas the increased p-Akt and p-eNOS expression are suppressed by pre-treatment of wortmanin or PEG-Catalase. Both in Cyuant cell proliferation assay and BrdU assay, 5-HT caused a trend but non-significant increase in DNA synthesis whereas the pre-treatment of PEG-Catalase significantly suppressed cell proliferation in the BrdU assay. Based on these results, we can conclude that 5-HT elicits endothelial migration via 5-HT-2 receptor-mediated H₂O₂ generation in a PI3K-dependent pathway. Under this circumstance, cortactin phosphorylation-dependent pathway was excluded. / Besides, ROS is notorious for effects like aggravation of inflammation and acceleration aging processes. The investigation extends to looking at alterations ofexpression of adhesion protein including ICAM-1 and VCAM-1 in the inflammatory response pathway and also to looking at the major aging parameter SIRT-1 in the presence of 5-HT in endothelium. Our data showed that in the presence of L-NAME, 30 min treatment of 5-HT significantly increased ICAM-1 and SIRT-1 expression without altering VCAM-1 expression and the up-regulation of ICAM-1 and SIRT-1 expression was prevented by PEG-Catalase. / In conclusion, with the eradication of the influence of NO, 5-HT induced mitochondrial H₂O₂ production via MAO-A-mediated metabolism in HUVECs. At the same time, 5-HT-induced H₂O₂ generation was involved in increasing endothelial permeability, inflammation and angiogenesis (cell migration). / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Qian. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 326-450). / Abstracts also in Chinese. / Zhang, Qian.

Background: Current guidelines give a class I recommendation to use of embolic protection devices (EPD) for saphenous vein graft (SVG) intervention; however, studies have shown conflicting results. The objective of this meta-analysis is to compare all-cause mortality, major adverse cardiovascular events, myocardial infarction (MI), or target vessel revascularization in SVG intervention with and without EPD. Methods and Results: Literature was searched through October 2016. Eight studies (n=52 893) comparing SVG intervention performed with EPD (n=11 506) and without EPD (n=41 387) were included. There was no significant difference in all-cause mortality (odds ratio [OR], 0.79; confidence interval [CI], 0.55–1.12; P=0.19), major adverse cardiovascular events (OR, 0.73, CI, 0.51–1.05; P=0.09), target vessel revascularization (OR, 1.0; CI, 0.95–1.05; P=0.94), periprocedural MI (OR, 1.12; CI, 0.65–1.90, P=0.69), and late MI (OR, 0.80; CI, 0.52–1.23; P=0.30) between the 2 groups. Sensitivity analysis excluding CathPCI Registry study showed no difference in periprocedural MI, late MI, and target vessel revascularization; however, it favored EPD use in all-cause mortality and major adverse cardiovascular events. Further sensitivity analysis including only observational studies revealed no difference in all-cause mortality, major adverse cardiovascular events, target vessel revascularization, and late MI. Additional analysis after excluding CathPCI Registry study revealed no difference in outcomes. Conclusions: This study including 52 893 patients suggests no apparent benefit in routine use of EPD during SVG intervention in the contemporary real-world practice. Further randomized clinical trials are needed in current era to evaluate long-term outcomes in routine use of EPD, and meanwhile, current guideline recommendations on EPD use should be revisited.

Abnormalities in the vascular pattern of a retina, such as morphologic changes in vessel shape, branching pattern, width, tortuosity, or the appearance of retinal lesions, may be associated with the occurrence of retinopathies or cardiovascular diseases. Thus, an automated quantitative analysis of changes in vessel morphology may help indicating the clinical signs of aforementioned retinopathies, describing their early occurrence or severity. The responses obtained from different types of retinal vessels, i.e., arteries and veins, may be variable to retinopathies and their measurement may lead to a more precise diagnosis compared to that by the average response accounted for the entire vessel network. I propose a set of automated methods in order to analyze the retinal vessel network and to quantify its morphologic properties with respect to arteries and veins, in two-dimensional color fundus images. The analytical methods include; 1) Forma- tion of a well connected vessel network, 2) Structural mapping of a vessel network, 3) Artery-venous classification, and 4) Blood vessel hemorrhage detection. The quan- tification methods include vessel morphology analysis based on the measurement of tortuosity, width, branching angle, branching coefficient, and fractal dimension. The aforementioned morphologic parameters are measured with respect to arteries and veins separately in a vessel network. The methods are validated with the manually annotated retinal fundus images as a ground truth. The major contribution of this thesis includes the development of automated methods for; 1) Identification and separation of retinal vessel trees for individual vessel analysis, 2) Automated quantification of morphologic characteristics of retinal vessels for quick and precise measurement, 3) Automated quantification of vessel morphology with respect to arteries and veins, and 4) Analysis of two datasets, a) malarial retinopathy subject dataset, b) longitudinal study dataset. The ability of the automated methods to quantify the retinal vessel specific properties may enable the individual vessel analysis as an alternative to a time- consuming and subjective clinical evaluation, or to a quantitative morphology char- acterization averaged over the entire vessel network. The objective evaluation may indicate the progression of retinopathies precisely and may help characterizing nor- mal and abnormal vascular patterns with respect to arteries and veins. This may enable a quick diagnosis, treatment availability, prognosis, and facilitation of clinical health-care procedures in remote areas.

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.

<p>The primary aim of this thesis was to modify saphenous vein (SV) harvesting technique and evaluate its clinical importance. A new "no touch" (NT) technique of SV preparation was developed where the vein is harvested with a pedicle of surrounding tissue, which protects the vein from spasm therefore obviating the need for distension.</p><p>Firstly, a prospective randomised study in 156 patients who underwent coronary artery bypass grafting was done to compare this new "no touch" technique to two others, the conventional (C) and the intermediate (I) techniques. A morphological study of the endothelium showed an endothelial integrity of 97% in NT vessels while about half of endothelial surface of veins harvested by the other two techniques was devoid of endothelium. At angiographic follow up, the patency for NT was 95.4%, 88.9% for grafts in group C and 86.2% for grafts in group I. A statistically significant difference in patency rate was found between the NT group and group C (p=0.025) and the poorest result was observed in group I.</p><p>Secondly, the immunohistochemistry assessment using CD31-antibody confirmed a better-preserved endothelium for NT vessels. Putative NOS was identified by NADPH-diaphorase histochemistry and autoradiographic localization of [3H] L-nitroarginine (NOARG) binding. NADPH staining was almost continuous on the luminal aspect and was also present in the intact adventitia of NT vessels, which was markedly reduced in conventionally harvested veins. Autoradiographic analysis of specific NOARG binding showed greater binding in the no-touch vessels, confirming the histochemistry results. All three NOS isoforms were identified in the media of SV grafts. In NT, NOS I was abundant in adventitial nerves; NOS II was found in adventitial vasa vasorum and NOS III was associated with endothelial cells lining both the vessel lumen and microvessels within the adventitia.</p><p>In conclusion, this study demonstrated that the endothelial integrity and NOS activity are better maintained when using the no-touch technique for vein graft harvesting. The vasorelaxant and thromboresistent activities of NO may be responsible for the reduced venospasm and improved early patency rates observed. Furthermore, the mechanical properties provided by the cushion of surrounding tissue in grafts harvested by NT technique may contribute to the observed high patency rate.</p>

It has been proposed that the various 14-3-3 isotypes and isoforms present in all eukaryotes are largely functionally equivalent. However, this is not consistent with the conservation of multiple isoforms and isotypes, especially in vertebrates with seven 14-3-3 encoding genes and nine isotypes. The hypothesis tested in this thesis is that both isoform-specific and overlapping functions are likely mediated through tissue specific expression, colocalization and dimerization of 14-3-3 proteins occur in vivo. Drosophila melanogaster was selected because it offers a simple, but representative system to study these proteins functionally. This thesis focuses primarily on D14-3-3?, although the expression pattern and phenotypes associated with all three Drosophila 14-3-3s were determined. I first determined the expression pattern of the three different 14-3-3 isotypes (leoI, leoII and D14-3-3?) and described developmental phenotypes associated with mutations in 14-3-3 isotypes in Drosophila. I found that there is partial redundancy with respect to lethality. Both LEO and D14-3-3? appear required for normal germ-line and somatic gonadal development. However, they do not appear to be functionally equivalent with respect to this phenotype since LEO is unable to compensate for the loss of D14-3-3?. I also determined that D14-3-3? mutants have unique phenotypes including deficits in adult cross-vein formation and rapid habituation to olfactory and footshock stimuli. To further understand the unique role that D14-3-3? plays in the adult CNS, I mapped the areas in the brain involved in olfactory and footshock habituation. I found that although the mushroom bodies (MBs) are necessary to inhibit premature habituation such as that exhibited by D14-3-3? mutants, D14-3-3? expression specifically in the MBs is not sufficient to rescue premature habituation. Although the loss of either LEO or D14-3-3? appears to cause a deficit in olfactory associative learning, premature habituation is the cause of the deficit seen in D14-3-3? mutants. As leo mutants do not exhibit a premature habituation phenotype, it appears that within the MBs LEO and D14-3-3? are not functionally equivalent. Therefore, the data supports the hypothesis that 14-3-3s have functional specificity and redundancy likely to represent use of homo and heterodimers in different processes within the tissues of an organism.

The primary aim of this thesis was to modify saphenous vein (SV) harvesting technique and evaluate its clinical importance. A new "no touch" (NT) technique of SV preparation was developed where the vein is harvested with a pedicle of surrounding tissue, which protects the vein from spasm therefore obviating the need for distension. Firstly, a prospective randomised study in 156 patients who underwent coronary artery bypass grafting was done to compare this new "no touch" technique to two others, the conventional (C) and the intermediate (I) techniques. A morphological study of the endothelium showed an endothelial integrity of 97% in NT vessels while about half of endothelial surface of veins harvested by the other two techniques was devoid of endothelium. At angiographic follow up, the patency for NT was 95.4%, 88.9% for grafts in group C and 86.2% for grafts in group I. A statistically significant difference in patency rate was found between the NT group and group C (p=0.025) and the poorest result was observed in group I. Secondly, the immunohistochemistry assessment using CD31-antibody confirmed a better-preserved endothelium for NT vessels. Putative NOS was identified by NADPH-diaphorase histochemistry and autoradiographic localization of [3H] L-nitroarginine (NOARG) binding. NADPH staining was almost continuous on the luminal aspect and was also present in the intact adventitia of NT vessels, which was markedly reduced in conventionally harvested veins. Autoradiographic analysis of specific NOARG binding showed greater binding in the no-touch vessels, confirming the histochemistry results. All three NOS isoforms were identified in the media of SV grafts. In NT, NOS I was abundant in adventitial nerves; NOS II was found in adventitial vasa vasorum and NOS III was associated with endothelial cells lining both the vessel lumen and microvessels within the adventitia. In conclusion, this study demonstrated that the endothelial integrity and NOS activity are better maintained when using the no-touch technique for vein graft harvesting. The vasorelaxant and thromboresistent activities of NO may be responsible for the reduced venospasm and improved early patency rates observed. Furthermore, the mechanical properties provided by the cushion of surrounding tissue in grafts harvested by NT technique may contribute to the observed high patency rate.

I assessed the accuracy of two clinical prediction rules, the d-dimer blood test and point of care ultrasound for diagnosing lower limb deep vein thrombosis. Emergency physicians were trained in ultrasound and prospectively scanned emergency department patients with suspected deep vein thrombosis. Accuracy of the Wells and AMUSE rules and the ultrasound result was compared to radiology-performed ultrasound and a 90-day clinical outcome. Univariate and multivariate analyses were performed assessing which factors were associated with the outcome. The sensitivity and specificity of the Wells score for the clinical outcome was 85.7% and 68.5%; the AMUSE score 85.7% and 54.4%. Ultrasound had a sensitivity of 91.7% and specificity of 91.7% for radiology-diagnosed thrombus and 78.6% and 95.0% for clinical outcome. The odds ratio of a positive outcome with a positive ultrasound was 65.1. After receiving the ultrasound training program, emergency physicians were unable to demonstrate sufficient accuracy to replace current diagnostic strategies.