Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes. / Master of Science

<p dir="ltr">Angiosarcoma (AS) is a rare, understudied cancer that arises from endothelial cells with an extremely poor prognosis. More research is necessary to understand AS pathogenesis, which will lead to the development of novel therapies to improve patient survival. Evidence from our lab highlights the importance of miRNAs in disease progression by demonstrating that endothelial cell-specific loss of mature miRNAs drives AS in mice. Furthermore, individual miRNAs have been characterized as tumor suppressors in AS. Taken together, this underscores the role of miRNAs in AS pathogenesis and suggests that they are an underexplored therapeutic strategy that could be efficacious in treating this cancer. Due to the evidence that miRNA loss is a driver of AS, we hypothesized that global miRNA enhancement would reduce cancer phenotypes. We interrogated this question through the use of a small molecule enhancer of RNAi, enoxacin. We found that enoxacin robustly reduced cancer phenotypes, particularly in AS models driven by miRNA loss, and that enoxacin increased the expression of mature tumor-suppressing miRNAs. We then thoroughly characterized miR-497 in angiosarcoma, demonstrating that miR-497 overexpression ablated tumor formation in mice through the regulation of a network of target genes. Furthermore, we identified <i>Vat1</i> as a novel target gene of miR-497, and found that genetic and pharmacologic inhibition of <i>Vat1</i> reduced cell migration in AS. Overall, this work further highlights the important roles miRNAs play in AS pathogenesis, and points toward miRNAs as an exciting therapy that should be explored further.</p>

La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D’autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l’inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre…), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les β-arrestines (β-arrs), des protéines d’échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l’interaction entre PTEN et les β-arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D’une part, les β-arrs augmentent l’activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d’aval ROCK activent PTEN, et ceci se fait par l’intermédiaire des β-arrs. La stimulation du récepteur à l’acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/β-arrs et permet le recrutement du complexe à la membrane. Par l’effet positif sur l’activité phosphatase lipidique de PTEN, les β-arrs participent à l’inhibition d’Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l’inverse dans les gliomes U373, les β-arrs lèvent l’inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l’activation de RhoA induite par blessure du tapis cellulaire, les β-arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les β-arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l’activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d’une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d’un biosenseur de PTEN basé sur le transfert d’énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l’activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l’augmentation de l’activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d’activité de PTEN dans les cellules vivantes. L’axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L’ensemble de nos travaux permet d’ajouter un degré de compréhension dans la régulation de PTEN, tant par les β-arrs que par l’interaction intramoléculaire et les changements conformationnels. / The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium…), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, β-arrestins (β-arrs), interact with PTEN.Our studies demonstrate that β-arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. β-arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires β-arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of β-arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, β-arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, βarrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, β-arrs show increased association with PTEN, and rescue glioma cell migration. β-arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity.

Further studies were done to investigate the mechanism responsible for 1-BI-induced apoptosis in NCI-H460. It was found that 1-BI stimulated the expression of pro-apoptotic p53, Bax and cytosolic NF-kB p65 subunit but decreased pERK in NCI-H460 cells. The active forms of caspase 3 and caspase 9 were detected by Western blot, accompanied by an increase in caspase 3 activity. Reactive oxygen species (ROS) was highly generated at 24 hours after the treatment and the mitochondrial membrane potential was significantly decreased at 48 and 72 hours. The application of either N-acetyl cysteine (NAC) or glutathione (GSH) attenuated the cell growth inhibition caused by 1-BI. NCI-H460 cells pretreated with NAC showed a decrease in ROS production and p65 protein but an increase in pERK. / Taken together, these findings suggest that the inhibition of THXS suppresses lung cancer cell growth by promoting either G1 cell cycle arrest or apoptosis. The status of p53 is critical for both cell cycle arrest and apoptosis in 1-BI-mediated growth inhibition, which is evident by enhanced apoptosis detected in p53-transfected NCI-H23 and DMS 114 cells and G1 cell cycle in lung cancer cells treated with PFT-alpha. The 1-BI-induced growth-inhibitory pathway is associated with the generation of ROS, alteration of mitochondrial membrane potential, down-regulation of pERK and p65. / The result showed that THXS expressed in all of the three lung cancer cell lines (NCI-H23, DMS 114 and NCI-H460). The activity of THXS was also reflected by the presence of THXS metabolite thromobxane B2 (TXB2) in the cells, which was detected by ELISA. 1-Benzylimidazole (1-BI), a specific THXS inhibitor, suppressed the lung cancer cell proliferation measured by MTT assay. 1-BI treatment caused G1 phase arrest and enhanced the level of cyclin dependent kinase inhibitor p27 in a time-dependent manner in NCI-H23 and DMS 114 cells. It markedly increased DNA fragmentation in NCI-H460 cells. The findings suggest that 1-BI inhibits cell growth by arresting cell cycle and inducing cell death. Annexin V/PI staining revealed that the cell death induced by I-BI was mainly in the format of apoptosis. Further experiments showed that the I-BI-induced apoptosis could be enhanced by the introduction of p53 into NCI-H23 and DMS 114 cells, and such enhancement was associated with a decrease in mitochondrial membrane potential. This result suggests that the p53 may play a positive role in apoptosis induced by 1-BI through changing of the mitochondrial membrane potential. The role of p53 in I-BI-mediated apoptosis was further confirmed by the experiment of the p53 inhibition. Pifithrin-alpha hydrobromide (PFT-alpha), a p53 specific inhibitor, suppressed the 1-BI-induced p53 protein expression and increased G1 cell cycle arrest. / Thromboxane A2 (TXA2) is a potent arachidonate metabolite in the cyclooxygenase-2 (COX-2) pathway, which is produced by a member of cytochrome P450 (CYP) superfamily called thromboxane synthase (THXS). Recent studies have showed that thromboxane and THXS are associated with cancer cell migration, angiogenesis, tumor metastasis and cancer proliferation but there is limited information on their role in lung cancer development. This thesis is to test the hypothesis that inhibition of THXS could alter lung cancer cell growth. / Leung, Kin Chung. / Adviser: George G. Chen. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3319. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 130-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 70-81.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Restricted until October 2005. Bibliography: leaves 104-113.

Les néoplasmes myéloprolifératifs classiques sont des maladies clonales dues à des mutations acquises de JAK2V617F ou de la Calréticuline (CALRm) au niveau des cellules souches hématopoïétiques (CSH) et conduisant à une surproduction de cellules myéloïdes. L’interféron alpha (IFNa) est le seul traitement curatif qui induit une réponse hématologique et moléculaire. Le but de notre projet est de comprendre son mécanisme d’action en utilisant une cohorte de 47 patients, des lignées cellulaires et des modèles de souris JAK2V617F. Ainsi, nous avons constaté que l’IFNa cible plus rapidement les CSH que les cellules matures chez les patients JAK2V617F, ce qui semble différent des patients CALRm. Grâce aux lignées cellulaires et aux souris, nous avons montré que JAK2V617F induit une pré-activation des voies de l’IFNa et une inhibition du cycle cellulaire des CSH. La découverte complète de ce mécanisme d’action conduira à l’amélioration du traitement. / Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only curative treatment that induces not only a hematological response of ET, PV and early MF but also a molecular response both on JAK2V617F or CALR mutated cells. In this study, we wanted to know how and with what kinetics IFN impacts the different mutated hematopoietic compartments. Thus, we have performed a prospective study with a cohort of 50 patients treated by IFNα for 3-5 years. The MPN diseases distribution was 44% ET, 45% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, we have also determined the clonal architecture by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors). After a median follow-up of 30 months, we observed that IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p<.05). Moreover, homozygous JAK2V617F clones responded more rapidly than heterozygous clones in all hematopoietic cell compartments showing that the intensity of JAK2V617F signaling is correlated with the efficacy of IFNα. These observations were slightly increased after a median follow-up of 51 months. In contrast, with a median follow-up of 30 months for CALR mutated patients, IFNα targeted similarly the HSPC and the mature cells. Moreover, IFNα induced a less rapid response to target CALR-mutated HSPC than the JAK2V617F HSPC (p<.05). The role of associated mutations at diagnosis was also investigated in the IFNα-mediated HSPC molecular responses using a NGS targeted myeloid panel. While in JAK2V617F-mutated patients, we found that the number of associated mutations did not impact the HSPC molecular response of the JAK2V617F clone, in CALR-mutated patients, even if the number of cases was low, the only molecular responders were those not associated with other mutations. Using Ba/F3-MPL cellular models and primary cells, we observed that JAK2V617F was more prone to sensitize to IFNα signaling (increased Phospho-STAT1 and IFN-stimulating genes (ISGs)) compared to controls or CALRdel52 mutated cells.Altogether, our results show that IFNα differentially targets the human JAK2V617F- and CALR-mutated HSPC and mature cells. Moreover, the molecular response was dependent not only on the JAK2V617F or CALR mutated status but also on the presence of other associated mutations.

Cardiomyocyte proliferation plays a crucial role in the developing mammal heart, as it is required for normal morphogenesis and in determining the appropriate heart size. Postnatally, the decrease in cell cycle activity is concomitant with the increase of cell cycle variants, such as endoreduplication and acytokinetic mitosis, which contribute to the hypertrophic growth of the heart. Although the adult mammal heart retains the ability to generate new cardiomyocytes, the extent of cardiomyocyte renewal is insufficient to compensate for the large-scale tissue loss associated with ischemic events. Indeed, ischemic events such as myocardial infarction, lead to a permanent loss of ventricular cardiomyocytes, formation of collagen-containing scar, and consequently cardiac remodeling. The development of therapies able to hamper cardiac remodeling by promoting cardiomyocyte turnover is one of the primary goals in the cardiovascular field. In the present study, we generated a human induced pluripotent stem cell (iPSC) line containing the fluorescence ubiquitination-based cell cycle indicator (FUCCI) under the Troponin T2 (TNNT2) promoter. To gain information about the cell cycle dynamics of human cardiomyocytes, we visualized cell cycle progression in TNNT2-FUCCI human iPSCs-derived cardiomyocytes. Notably, we revealed cardiomyocytes' cell cycle dynamics of cells undergoing proliferation, binucleation, and polyploidization and identified G2 cell cycle arrest in cardiomyocytes undergoing polyploidization. To demonstrate the versatility of the TNNT2-FUCCI human iPSCs line, we developed a live cell screening platform to identify pro-proliferative compounds within an autophagy compound library. We identified Clonidine, an alpha2-adrenergic receptor and imidazoline agonist, as an enhancer of cell cycle activity in TNNT2-FUCCI hiPSC-derived cardiomyocytes. Finally, we investigated the ability of Clonidine to promote cell cycle progression in hiPSC- derived cardiomyocytes and in in vivo and in vitro mouse neonatal cardiomyocytes. We showed that while Clonidine stimulated cardiomyocytes' polyploidization and multinucleation, respectively in in vitro in in vivo mouse cardiomyocytes, the treatment of hiPSC-derived cardiomyocytes with Clonidine enhanced their proliferative capability. In conclusion, we showed that the TNNT2-FUCCI system is a versatile tool for characterizing cardiomyocyte cell cycle dynamics and identifying pro-proliferative molecular candidates with regenerative potential in the mammalian heart.:1. Introduction 1.1 Heart function and composition 1.2 Human cardiac development 1.2.1 Cardiac organogenesis 1.2.2 Metabolic changes in the developing heart 1.3 Cardiomyocytes cell cycle activity 1.3.1 Cardiomyocytes cell cycle regulators and cell cycle arrest 1.3.2 CM multinucleation and polyploidization 1.4. The regenerative capabilities of the mammal heart 1.4.1 Model systems for heart regeneration 1.4.2 Stimulation of cardiomyocyte proliferation as a goal to preserve heart function 1.4.3 Assessment of cardiomyocytes proliferation 1.5 Human-induced pluripotent stem cells to model cardiac development and disease 2. Aim 3. Materials and methods 3.1 TNNT2-FUCCI hiPSC line generation 3.2 hiPSC culture and maintenance 3.3 hiPSC differentiation into CMs 3.4 Imagestream-X Analysis 3.5 TNNT2 expression assessment of hiPSC-derived CMs by immunohistochemistry 3.6 CDK1 immunohistochemistry expression assessment of hiPSC-derived CMs 3.7 Cell area and sarcomere spacing measurement of hiPSC-derived CMs 3.8 Live imaging and timelapse imaging analysis of TNNT2-FUCCI hiPSC 3.9 Murine neonatal CMs cell culture 3.10 Mouse nCM timelapse imaging and analysis 3.11 TNNT2-FUCCI hiPSC-derived CMs culturing and screen conditions 3.11.1 TNNT2-FUCCI screen image acquisition 3.11.2 TNNT2-FUCCI screen automated image analysis 3.11.3 TNNT2-FUCCI screen data analysis 3.12 Primary mouse nCM compound validation and immunohistochemistry 3.13 Mouse nCM immunohistochemistry for AurKB expression assessment 3.14 hiPSC-derived CMs immunohistochemistry for AurKB expression assessment 3.15 Analysis of CM ploidy and binucleation 3.16 in vivo Clonidine treatment of neonatal mice 3.17 Analysis of ploidy and binucleation in in vivo mouse nCMs 3.18 Cell cycle activity assessment in P7 neonatal mouse hearts after Clonidine treatment by immunohistochemistry 4. Results 4.1 TNNT2-FUCCI human iPSC line generation and validation 4.2 TNNT2-FUCCI marks proliferating and non-proliferating CMs 4.3 Live imaging identification of CM cell cycle activity 4.3.1 Cell cycle progression of TNNT2-FUCCI CMs 4.3.2 Cell cycle progression of mouse neonatal cardiomyocytes 4.4 TNNT2-FUCCI live-imaging identifies CM cell cycle activators 4.5 Compound validation in mouse nCMs 4.6 Clonidine elicits cycling activity via alpha1 adrenergic receptor and imidazoline receptor interaction 4.7 Clonidine stimulates hiPSC-derived CM proliferation 4.8 Clonidine stimulates CM polyploidization in mouse nCMs 4.9 Clonidine mediates in vivo CM cell cycle activity in the neonatal mouse 5. Discussion 5.1 TNNT2-FUCCI hiPSC: a new technology to identify cycling CMs 5.2 Study of CM cell cycle activity using TNNT2-FUCCI 5.3 TNNT2-FUCCI hiPSC in combination with a live screening platform revealed enhancer of CMs cell cycle activity. 5.4 Initial validation of pro-proliferative compounds in mouse nCMs reveals Clonidine and Dihydrocapsaicin as enhancers of cell cycle progression 5.5 Clonidine stimulates cell cycle activity in different model systems 5.5.1 Clonidine treatment stimulates proliferation in hiPSC-derived CMs 5.5.2 Clonidine treatment stimulates polyploidization in in vitro- and multinucleation in in vivo mouse nCMs 5.6 Conclusions and future outlooks 6. Appendix 7. Summary 8. Zusammenfassung Acknowledgements References Anlage 1 Anlage 2

Acute myeloid leukaemia (“AML”) is a clonal disease characterised by increased, uncontrolled abnormal white blood cells and the accumulation of leukaemia immature cells in the bone marrow and bloodstream. Chromosomal rearrangements have been detected in almost half of AML cases. It has been proven that the chromosomal rearrangements constitute a marker for the diagnosis and prognosis of AML and have therapeutic consequences. The discovery of these rearrangements has led to a new World Health Organization (“WHO”) classification system. However, small regions of cryptic chromosomal rearrangements have been identified among these cases. Such cryptic rearrangements can be explained by the identification of small regions which cannot be found by conventional chromosome banding techniques. Moreover, approximately 50% of AML cases have been found with normal karyotypes. The improvement of cytogenetic techniques, including fluorescence in situ hybridization (“FISH”) and single nucleotide polymorphism (“SNP”) platforms, have allowed the detection of small rearranged regions (such as copy number changes) both in normal and abnormal karyotype AML. This study identifies: (i) cryptic chromosomal translocations in leukaemia cells of AML patients; (ii) DNA copy number changes in patients with known chromosomal translocations; and (iii) the proliferating state of leukemic cells harbouring chromosomal abnormalities within a series of patients. In the initial study, the FISH technique was performed on 7 AML patient samples to validate a novel three colour probe for the detection of t(7; 12). The results demonstrated that the new three-colour FISH approach used in this study has enabled the detection of a cryptic t(7;12) translocation as part of a complex rearrangement in one patient previously been described as having t(7;16) and ETV6-HLXB9 fusion transcript at the molecular level. To date there are only two cases of a cryptic t(7; 12) translocation reported in the literature. Additionally, the new three-colour FISH approach also enabled identification of t(7; 12) in a new seven year-old AML patient (the first case of childhood leukaemia with an onset after infancy to be found positive for t(7; 12)). In the second study the FISH technique was used to validate three colour probe sets for the detection of 7(q22-q31) and 7(q22-q36.1) regions on several myeloid cell lines. The results indicate that the probes found chromosome 7 rearrangements in myeloid cell lines with complex rearrangements. The three colour probe sets enabled detection of a new rearrangement in the k562 cell line, described as a duplication of 7q36 region, followed by intrachromosomal insertion of long arm material into the short arm of chromosome 7. The intrachromosomal insertion identified in k562 cell line is an uncommon form of chromosomal rearrangement in myeloid leukaemia which has not been previously reported. In the third study, the Illumina BeadArray approach was used to assess copy number alterations (“CNAs”) and copy number loss of hertrozygosity (“CN-LOH”) regions in 22 AML patients samples with inv(16)(p13;q22) and t(8;21)(q22;q22) rearrangements. In order to distinguish between true CNAs and false-positive findings as well as to verify whether CNAs are present in the same clone harbouring inv (16), FISH was used on fixed chromosome and cell suspensions from the same patients. The results showed a low number of copy number losses and copy number gains in 17 (77.27%) out of 22 cases, with an average of 1.86 CNAs per case as well as copy neutral-LOH with an average of 6.7% per patient. Furthermore, interphase FISH was carried out on four cases showing a 7q36.1 deletion, 4q35.1 deletion, 16.13.11 deletion and 8q24.21-q24.3 gain identified by array. The FISH results confirmed CNAs in most cases while CNA was not confirmed in one patient. Moreover, the FISH data analysis showed that the CNAs were found in both cells without inv (16) and cells harbouring the inv (16) rearrangement. In the final study, indirect immunofluorescence (IF) was used to determine the ki67 staining patterns in 8 stimulated and unstimulated peripheral blood samples and k562 cell lines. The results showed a high percentage of ki67 positive staining in the stimulated samples in comparison with unstimulated samples, which showed a low percentage of ki67 positive staining. In addition, a high percentage of proliferating cells were detected in the k562 cell line. ImmunoFISH was performed on five different patient samples and leukaemia cell lines using specific probes in the regions of interest to detect the chromosomal abnormalities and using the ki67 antibody to assess the proliferation state of the cells. The results showed that the proliferation state of the cells carrying chromosomal abnormalities in two patients was higher than the proliferation state of the cells carrying abnormalities in three patients; in other words, most of the cells carrying abnormalities were proliferating in two cases and non-proliferating in three cases.

Atherosclerosis and post-angioplasty neointimal proliferation, which are leading causes of cardiovascular morbidity and mortality, develop as a result of chronic or acute vascular injury producing inflammatory and proliferative responses in the vessel wall. Glucocorticoids, the stress hormones produced by the adrenal cortex, have anti-inflammatory and anti-proliferative characteristics and can also influence systemic cardiovascular risk factors. The systemic levels of these hormones are controlled by the hypothalamic pituitary adrenal axis. However, there is also a tissue-specific pre-receptor regulation of these hormones by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD); type 1 regenerates active glucocorticoids within the cells and type 2 inactivates glucocorticoids. Whilst it has been shown that the inhibition of 11β-HSD1 has favourable effect on cardiovascular risk factors and the inhibition of 11β-HSD2 results in hypertension; the effect of these enzymes on vascular lesion development is not known. The work described in this thesis tested the hypothesis that 11β-HSD1 inhibition reduces vascular lesion development due to improvement in cardiovascular risk factors, whereas 11β-HSD2 inhibition leads to adverse vascular remodelling. Apolipoprotein-E deficient (ApoE-/-) mice fed on western diet were used to study atherosclerosis, whereas neointimal proliferation was investigated using a well-established mouse model of wire-angioplasty. Vascular lesions were assessed using novel imaging and standard histological techniques. 11β-HSD1 inhibition reduced the size of atherosclerotic lesions and improved markers of plaque stability with a reduction in lipid content and increase in collagen content of the plaques. This was associated with a reduction in weight gain and blood pressure but without any effect on lipid profile. 11β-HSD1 inhibition did not produce any significant effect on neointimal proliferation in C57Bl/6J mice. However in ApoE-/- mice, 11β-HSD1 inhibition reduced neointimal proliferation with corresponding increase in size of patent lumen and with an associated reduction in macrophage content of neointimal lesions. 11β-HSD2 deletion produced an outward remodelling in un-injured vessels but there was no effect on neointimal proliferation after wire-angioplasty. Administration of a selective mineralocorticoid antagonist, eplerenone, reduced neointimal lesions significantly but to a similar degree in both C57Bl/6J and 11β-HSD2-/- mice, associated with a significant reduction in macrophage content of lesions but without any effect on blood pressure. Data in this thesis highlight the potential therapeutic application of 11β-HSD1 inhibition in reducing the size and vulnerability of atherosclerotic plaques and also reduction in neointimal proliferation (and hence post-angioplasty restenosis) in high risk patients with „metabolic syndrome‟ phenotype. The results also indicate that 11β-HSD2 has a limited, if any, role to play in the development of neointimal lesions.