Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Robert Gilbert, Dept. of Mathematical Sciences. Includes bibliographical references.

The compact structure of the eukaryotic genome dictates the accessibility to genes, and therefore adds an additional layer of regulation for gene expression. A specialized class of proteins called "chromatin remodelers" facilitates this process in the cell. The imitation switch (ISWI) subfamily of chromatin remodelers is a well studied class of proteins affecting gene expression. Its member ISW2 was recently shown to behave differently from other chromatin remodeling proteins. For instance, the ISW2 complex has been shown to be stimulated by ~5-6 fold in its ATPase activity when bound to a nucleosome rather than to a DNA molecule. Nucleosome remodeling by ISW2 has even been shown to depend on the N-terminal tail of histone H4 and therefore, the octamer of a nucleosome might be playing a significant role in nucleosome remodeling by the ISW2 complex. The aim in this investigation was to delineate the protein-protein interactions that the ISW2 complex establishes with the octamer upon binding to a nucleosome. Several histones with unique cysteines engineered at specific positions were refolded with other wild type histones to produce histone octamers with a single cysteine in one of the four histones. Based on previous reports from site-specific DNA photoaffinity cross-linking and hydroxyl-radical footprinting experiments, it was inferred that the SHL2, entry-exit position and the extranucleosomal linker DNA were contacted by the ISW2 complex on a nucleosome1. Considering these critical regions and taking into account the accessibility of residues in close proximity to these regions, five discrete positions were selected on the octamer surface for scanning the face of the nucleosome. The five sites were residues 19, 89 and 113 of histone H2A (H2A-19, H2A-89, H2A-113), residue 109 of histone H2B (H2B-109), and residue 80 of histone H3 (H3-80). Initially, octamers with cysteine at one position in one of the four histone proteins were reconstituted on a 0N70 DNA (where `N' represents the 147bp 601 DNA sequence, and the lengths of the linker DNA is represented by the numbers 0 and 70). Nucleosomes were modified with the protein-protein cross-linker- MAB (methanethiosulfonate-tetrafluorophenylazide-biotin) reagent. This reagent makes a disulfide bond with the cysteines in the octamer of a nucleosome. The MAB reagent had a distance of ~11.1Aº between its photoreactive tetrafluorophenylazide group and the disulfide forming methanethiosulfonate group. The ISW2 complex was bound to the modified mononucleosomes and cross-linked by irradiating with UV-light. Under reducing conditions the biotin moiety was transferred from the nucleosome to the ISW2 complex. The subunit of the remodeler that was photocross-linked at these positions on the nucleosome was blotted onto nitrocellulose and detected with streptavidin conjugated to horseradish peroxidase (HRP). The catalytic subunit-Isw2 of the ISW2 complex was cross-linked at all five positions but with the following order of intensity from most to least- H2A-89, H3-80, H2B-109, H2A-19, and H2A113. Mass spectrometry was used to decipher these residues, motifs or domains of the catalytic subunit- Isw2 that interacted with the octamer at each position. The ISW2 complex was digested with trypsin, and the biotinylated peptides were enriched using monomeric avidin affinity chromatography. The largest subunit of the ISW2 complex, Itc1, did not get cross-linked at any of the positions.

Malgré une prévalence importante, les varices de la veine saphène sont peu étudiées. Le but de notre étude a été de comprendre le rôle de la prostaglandine (PG) E₂ et du sulfure d’hydrogène (H₂S) dans cette pathologie. La PGE₂ est un lipide bioactif qui peut contrôler le tonus vasculaire et induire l’activation des métalloprotéinases matricielles (MMP). Des études récentes ont montré que la production de cette PG peut être régulée par l’H₂S. Ce gaz endogène serait fortement impliqué dans le système cardiovasculaire. Nos études ont montré que la PGE₂ est largement produite par les parois des veines saphènes saines (SV) et par leurs graisses périvasculaires ; de plus, elle induit la vasorelaxation de ces veines en activant le récepteur EP4. Dans les varices, nous avons mesuré l’absence ou des concentrations réduites de nombreux marqueurs de l’inflammation en comparaison des SV. Ainsi, les contenus en PGE₂ et du récepteur EP4 sont diminués dans les varices. Cette dérégulation entraîne un déficit de l’activation des MMP et induit l’expression de leurs inhibiteurs endogènes (TIMP). Dans la paroi des varices, la diminution du ratio MMP/TIMP calculé à partir de nos résultats peut expliquer l’accumulation des collagènes. Finalement, nos expériences ont montré que le taux d’H₂S endogène est deux fois plus élevé dans les varices par rapport aux SV et que ce gaz inhibe la synthèse de PGE₂. En conclusion, l’H₂S serait responsable de l’épaississement de la paroi des veines saphènes variqueuses. Il pourrait jouer un rôle protecteur en renforçant la paroi vasculaire pour résister à la stase veineuse. Ce remodelage permettrait d’éviter l’ectasie veineuse et une rupture de la paroi. / Despite its high prevalence, the varicose vein pathology is poorly studied. The aim of our study was to understand the role of prostaglandin (PG) E₂ and hydrogen sulfide (H₂S) in this pathology. PGE₂ is involved in the control of the vascular tone and this bioactive lipid participates to the vascular wall remodeling by activating metalloproteinases (MMP). Recent studies have shown that hydrogen sulfide (H₂S), an endogenous gas, could be involved in the cardiovascular system and can regulate PGE₂ production. Our experiments have shown that PGE₂ is significantly produced by vascular wall or by the perivascular adipose tissue of human healthy saphenous veins (SV). Furthermore, PGE₂induces a potent vasorelaxation after the activation of its EP4 receptor. In varicose veins, we have measured the absence or a weak concentration of several inflammatory markers in comparison to SV. PGE₂ and EP4 contents were decreased in varicose veins. This regulation involves a lack of MMP activation and induces MMP inhibitor expression (TIMP). Our results indicate that the MMP/TIMP ratio is decreased involving an extracellular matrix accumulation and in particular of collagen in the vascular wall of varicosity. Furthermore, our experiments show that in the pathological veins H₂ S content is up-regulated (2 fold) and this gas inhibits the PGE₂ production. In conclusion, H₂ S could be responsible for the vascular wall thickening in human varicose veins. This mechanism could be protective, H₂ S effect could strengthen the vascular wall in order to resist at the venous stasis. This vascular wall remodeling could avoid ectasic segment formation and a venous wall rupture.

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La galectine-3 (Gal-3) est biologiquement liée au processus d'inflammation et de fibrose dans l’insuffisance cardiaque (IC). Le système rénine-angiotensine-aldostérone (SRAA) a été largement démontré comme exerçant des effets profibrotiques, prohypertrophiques et pro-inflammatoires dans la pathophysiologie de l’IC. En outre, il existe des preuves expérimentales que Gal-3 et SRAA peuvent interagir dans le développement des dommages cardiovasculaires dans des modèles d’hypertension. Cependant, une telle interaction n'a pas été étudiée dans un contexte de dysfonction ventriculair gauche (VG). Nous avons donc cherché à tester les hypothèses suivantes: 1. La Gal-3 participe aux mécanismes de la fibrose cardiaque et du remodelage tissulaire médiés par l'aldostérone dans un modèle murin de dysfonction du VG; 2. Le niveau de Gal-3 est associé au développement du remodelage du VG et de la fibrose et peut prédire les effets des l'antagonistes neurohormonaux chez les patients atteints d'IC chronique due à une cardiomyopathie dilatée non ischémique (CMDNI). Méthode 1. Des souris mâles adultes atteintes d'hyperaldostéronisme cardiaque spécifique ont été soumises à des injections sous-cutanées d'isoprotérénol (200 mg/kg x 2/jour pendant deux jours), puis randomisées pour recevoir un placebo, un inhibiteur de Gal-3 (modified citrus pectin, MCP), un antagoniste de récepteurs aux minéralocortïcoides (canrenoate de potassium) ou l’association de deux (MCP+canrenoate) pendant 14 jours. 2. Nous avons recruté 150 patients avec un diagnostic de CMDNI. Tous les patients ont bénéficié d'une évaluation clinique et biohumorale, incluant le dosage de Gal-3, l'échocardiographie, et une imagerie par résonance magnétique cardiaque (IRM) contrastée pour l'évaluation des volumes et de la fibrose par le late gadolinium enhancement (LGE). Une plus petite cohorte de 70 patients a également reçu un dosage de soluble suppression of tumorigenicity protein 2 (sST2) à l’inclusion, et une IRM de suivi à 24 mois pour l'évaluation du remodelage inverse du VG, définie comme une augmentation de >10% de la fraction d’ejection du VG ou une diminution de 10% du volume diastolique du VG indexé. Résultats L’isoprotérénol a induit une diminution rapide et persistante de la fonction systolique du VG chez la souris, qui a été nettement améliorée par le traitement au MCP ou canrenoate. Le MCP et le canrenoate ont également prévenue l'hypertrophie et la fibrose cardiaques ainsi que l’augmentation de l'expression de gènes impliqués dans la fibrogénèse (Coll-1 et Coll-3) et l'infiltration de macrophages (CD-68 et MCP-1). L'utilisation combinée du MCP et canrenoate a entraîné des effets additifs sur l'hypertrophie cardiaque, l'inflammation et la fibrose, comparativement au MCP ou au canrenoate seul. Dans notre cohorte de patients avec CMDNI, la valeur médiane de Gal-3 était de 14,4 ng/mL; le LGE a été observée chez 106 patients. Les patients ayant un LGE positif avaient un taux de Gal-3 plus élevée que ceux qui n'en avaient pas (p=0,006). Après analyse multivariée suel la Gal-3, le sexe, l’ancienneté del la cardiopathie et la fraction d’éjection du ventricule droit sont des facteurs prédicteurs indépendants de la présence de LGE. L’IRM de suivi a montrée que 35 patients présentaient un remodelage inverse. L’analyse multivariée met en evidence Gal-3 comme un prédicteur indépendant du remodelage inverse contrairement au sST2. Conclusion La Gal-3 participe à des mécanismes de lésions myocardiques médiées par l'aldostérone dans un modèle murin de IC. De même, dans un cadre clinique de CMDNI, Gal-3 semble être associée à la fibrose du VG et à l'évolution du remodelage cardiaque, en identifiant éventuellement le sous-ensemble de patients avec une réponse plus prononcée à l'antagonisme neuro-hormonal pharmacologique. / BackgroundGalectin-3 (Gal-3) is biologically linked to the process of inflammation and fibrosis in heart failure (HF). The renin-angiotensin-aldosterone system (RAAS) has been largely demonstrated to exert profibrotic, prohypertrophic and proinflammatory effects in the pathophysiology of HF. Further, there is initial experimental evidence that Gal-3 and RAAS may interplay in the development of cardiovascular damage in hypertensive models. However, such interaction has not been investigated in the setting of left ventricular (LV) dysfunction and HF. We aimed therefore to test the following hypotheses:1. Gal-3 participates in the mechanisms of aldosterone mediated cardiac fibrosis and tissue remodeling in a murine model of LV dysfunction;2. Gal-3 level is associated with the development of LV remodeling and fibrosis and can predict the effects of neurohormonal antagonism in patients with chronic HF due to non-ischemic dilated cardiomyopathy (NIDCM).Methods1. Adult male mice with cardiac-specific hyperaldosteronism (AS) underwent isoproterenol subcutaneous injections (200 mg/kg x 2/day over two days), to be then randomized to receive placebo, a Gal-3 inhibitor (modified citrus pectin, MCP), an aldosterone antagonist (potassium canrenoate), or MCP+canrenoate for 14 days.2. We enrolled 150 patients with a diagnosis of NIDCM. All patients underwent a comprehensive clinical and biohumoral evaluation, including Gal-3 assay, 2-D echocardiography, and a contrast-enhanced cardiac magnetic resonance (CMR) for the assessment of LV volumes and fibrosis by the late gadolinium enhancement (LGE) technique. A smaller cohort of 70 patients also received soluble suppression of tumorigenicity protein 2 (sST2) assay at baseline as well as a follow-up CMR after 24 months for the evaluation of LV reverse remodeling, defined as a >10 percentage units increase in LV ejection fraction or a >10% decrease in LV end-diastolic volume indexed.ResultsIsoproterenol induced a rapid and persistent decrease in LV systolic function which was markedly improved by treatment with either MCP or canrenoate. MCP and canrenoate also reduced cardiac hypertrophy and fibrosis and the expression of genes involved in fibrogenesis (Coll-1 and Coll-3) and macrophage infiltration (CD-68 and MCP-1). The combined use of antagonists of Gal-3 and aldosterone resulted in enhanced effects on cardiac hypertrophy, inflammation, and fibrosis, when compared to MCP or canrenoate alone. In our cohort of NIDCM patients, median Gal-3 value was 14.4 ng/mL; LGE was detected in 106. Patients with LGE had higher Gal-3 than those without (p=0.006). Among univariate predictors of LGE, Gal-3 maintained its predictive value at multivariate analysis, together with sex, hypertension, disease duration and right ventricular ejection fraction. At follow-up CMR, 35 patients showed reverse remodeling. Gal-3, but not sST2 resulted as an independent predictor of LV reverse remodeling at multivariate analysis.ConclusionGal-3 participates in mechanisms of aldosterone-mediated myocardial damage in murine model of HF. Likewise, in the clinical setting of NIDCM, Gal-3 seems to be associated with LV fibrosis and to the evolution of cardiac remodeling, possibly identifying the subset of patients with a more pronounced response to pharmacological neurohormonal antagonism.

Nach einem Myokardinfarkt werden ventrikuläres Remodeling und myokardiale Funktion unter anderem durch die ablaufenden Reaktionen des angeborenen Immunsystems beeinflusst. Von zentraler Bedeutung für die Regulation dieser Immunreaktion ist der Transkriptionsfaktor Nuclear Factor kappa B. Tiere, bei denen NF-κB durch das Fehlen seiner Untereinheit p50 global inaktiv ist, wei- sen einen Schutz vor linksventrikulärem Remodeling auf. Bisher ist jedoch un- klar, welche Zellen für diesen protektiven Effekt verantwortlich sind. Vorange- gangene Studien konnten zeigen, dass die Protektion nicht auf die fehlende NF- κB Aktivierung in Kardiomyozyten zurückzuführen ist. Aus Ischämie- Reperfusions-Experimenten an NF-κB-defizienten Tieren ergaben sich Hinwei- se, dass v.a. die Hemmung von NF-κB in Entzündungszellen die protektiven Effekte vermittelt. Durch Kreuzung von LysMCre- mit lox-IKKβ-Tieren erzeugten wir Tiere, denen makrophagenspezifisch IκB-Kinase β (IKKβ) fehlt. IKK deaktiviert den Inhibitor von NF-κB und ist somit essentiell für eine NF-κB-Aktivierung. Als Modell der Herzinsuffizienz diente der chronische Myokardinfarkt. Die Nachbeobachtung erfolgte über 56 Tage. Die Knockout-Tiere (KO) hatten im Vergleich zu den Wildtyp-Tiere (WT) eine signifikant bessere Überlebensrate (KO vs. WT, 100% vs. 49%, p < 0,01). Präoperativ sowie postoperativ an den Tagen 1, 21 und 56 wurden transthora- kale Echokardiographien durchgeführt. Bei gleicher Infarktgröße zeigten die KO-Tiere eine deutlich geringere linksventrikuläre Dilatation. Es konnte moleku- larbiologisch keine Reduktion der humoralen Entzündungsreaktion nachgewie- sen werden, ebenso blieb das Entzündungszellinfiltrat immunhistochemisch unverändert. Auch bezüglich Apoptoserate und Neovaskularisation zeigte sich kein Unterschied zwischen den Gruppen. Allerdings zeigten die LysM-IKKβ-KO-Tiere 56 Tage nach Myokardinfarkt einen deutlich erhöhten septalen Kollagen- gehalt als Hinweis auf ein verändertes extrazelluläres Remodeling. Aus diesen Ergebnissen kann geschlossen werden, dass die protektiven Effek- te der globalen NF-κB-Hemmung durch die fehlende NF-κB-Aktivierung in Ma- krophagen und Granulozyten, nicht aber in Kardiomyozyten vermittelt wurden. Die durch die makrophagenspezifische NF-κB-Hemmung vermittelten Verände- rungen im Remodeling der extrazellulären Matrix führen zu einer Verbesserung der Überlebensrate, besseren funktionellen Ergebnissen und einem insgesamt verminderten linksventrikulären Remodeling nach Myokardinfarkt. / Improvement of left ventricular remodeling by inhibition of Nuclear Factor kappa B in macrophages and granulocytes

Blood-borne biochemical markers were used to track comparative rates of bone turnover in horses fed differing amounts of Ca, P and Mg. Bone turnover was tracked by serum osteocalcin, bone resorption by the carboxyterminal telopeptide of type I collagen (ICTP), and bone formation by the carboxyterminal propeptide of type I procollagen (PICP). Twenty-four long-yearling Quarter Horses were blocked by gender and age, randomly assigned to one of four diets and subjected to 128 d of race training. Horses entered the study at approximately 20 months of age. The study was conducted in 32-d periods, each consisting of 28 d of race training followed by a 4-d fecal and urine collection, or a 4-d rest period. Blood samples were taken weekly during the training period. Serum and plasma samples were analyzed for concentrations of osteocalcin, the carboxyterminal telopeptide of type I collagen (ICTP) and the carboxyterminal propeptide of type I procollagen (PICP). Urine was collected for analysis of deoxypyridinoline (DPD) and creatinine. Onset of training resulted in elevated concentrations of ICTP, PICP and osteocalcin. Horses consuming the highest levels of Ca, P and Mg exhibited higher concentrations of PICP and lower concentrations of ICTP indicating greater bone formation coupled with lesser amounts of bone resorption. Further, ICTP, PICP and osteocalcin concentrations decreased dramatically following 4-d of confinement and relative inactivity. Therefore it appears that feeding minerals at levels greater than current NRC recommendations provided a protective effect on the developing skeleton of the young racehorse. Additionally, the biochemical markers used in this study were sensitive enough to track daily changes in bone activity resulting from daily changes in stress to the skeleton.

Shopping centres play an important role of Hong Kong people’s lifestyle, as they are not just places for shopping but also for community and social activities. They can provide products and services including entertainment, supermarket, dining, daily products and also other special services. However, the overall standard for shopping centre declines over time. To deal with aged properties, shopping renovation project is a possible solution. In this paper, the needs and importance of the shopping centre renovations are assessed. From the source of existing literature, the needs of shopping centre renovations come from the failure to meet the customer satisfactions and the lack of competitiveness against other shopping centres. Renovation projects can provide improvement to the existing facilities and enhance the overall value of the shopping centres. Renovation works do not only extend the building life and make the building more desirable and economically valuable, but they can be treated as a tool to fulfill some special objectives decided by the developers. For the next objective, upon the completed renovation projects by the Link REIT, their outcomes are assessed. To achieve this objective, seven criteria leading to shopping centre success are introduced. They are accessibility, visibility, anchor tenants, design and layout, size, tenant mix, trade mix and environment. The overall performance for pre renovation and pro renovation are measured based on these criteria and their substantial factors. For quantitative measurement, questionnaires are used for the measurement and there is an overall improvement recorded as shown by increment of scores in the seven criteria. For qualitative measurement, in-depth interviews are applied and detail ways for improvement are collected. Finally, this research investigates the area of improvement and further suggestions will be advised in future projects. They include regular communication with target groups (tenants and shoppers), continuous minor improvements applied to the completed projects to slow down performance backslide and also the enhancement of social responsibility towards different stakeholders. / published_or_final_version / Housing Management / Master / Master of Housing Management

Improving the energy performance of existing building refurbishment has been identified as one of the key measures to reduce greenhouse gases (GHGs) emissions and combat climate change. According to Environmental Protection Department, buildings in Hong Kong take up almost 90% of urban electricity consumption. Sustainable building refurbishment not only can help decrease energy consumption but may also improve building’s overall condition, and thus prolong its life, uplift the living conditions, ensure better health and safety as well as minimize the negative effects to environment. To respond to the energy emission reduction, many researchers focus on technical improvements through various refurbishment methods. However, there is a research gap in determining the appropriate refurbishment solutions for high-rise residential buildings in developed cities like Hong Kong. The challenge is aggravated as there are a number of owners and occupants in multi-storey residential buildings and their behaviour can be very different. Albeit more and more attentions have been attributed to human behaviours and occupant satisfaction, little has been done to examine their effects to the choices and success of sustainable refurbishment solutions. This study aims to systematically analyse the effect of human behaviour to the satisfaction of sustainable refurbishment by setting up a unified model so as to maximize the opportunity for emission reduction without sacrificing the satisfaction of owners and occupants. Literature review was first conducted to attain the knowledge of sustainable refurbishment and human behaviour. Then, a list of potential sustainable building refurbishment method items was compiled under five criteria through literature review. In order to further examine the suitability of sustainable building refurbishment methods in Hong Kong scenario from the perspective of owners and occupants, a questionnaire survey was administered. Following that, literature review and interviews were carried out to identify a set of critical success factors (CSFs) of electing sustainable refurbishment strategies as well as key performance indicators (KPIs) of a sustainable building refurbishment scheme. Based on that, another questionnaire survey was conducted to examine the occupants’ perception to the relative importance of the identified CSFs and KPIs. Finally, a structural equation model was set up to unveil the relationships between occupants’ satisfaction and project success, and the findings were validated through expert interviews. The results shows that disruption is the most important factors affecting occupants’ decisions, followed by Management and Organization; Comfort; Cost; and Health and Safety. The technological and environmental accomplishments are proven to be the most important KPIs to the success of a sustainable building refurbishment project. The model developed can help decision-makers select on suitable sustainable building refurbishment methods to meet the social expectations of occupants while achieving the carbon emission target. / published_or_final_version / Civil Engineering / Master / Master of Philosophy

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