AML1 long splice isoform regulates transcription of many haematopoietic specific genes and may act as both transcriptional activator and repressor. The AML1 short isoform which lacks the transactivation domain can bind DNA but is not capable of activating transcription; suggesting that it acts in a dominant-negative manner. Thus, if the long AML1 isoform induces differentiation of haematopoietic progenitors, the short isoform may promote their self-renewal. This hypothesis may be tested using an engineered ES cell differentiation system overexpressing individual AML1 isoforms. Therefore we aimed here to generate ES cell lines overexpressing individual AML1 isoforms constitutively or in a regulatable fashion in order to test their biological functions. Both, long and short AML1 isoforms demonstrated cytotoxic effect when overexpressed constitutively in ES cells. In order to overcome the toxicity problem, these isoforms have been expressed conditionally in an inducible fashion using the tetracycline gene expression system. Different experimental designs were tried to conditionally overexpress the isoforms in ES cells. Finally, the AML1 isoforms were targeted into the HPRT homing site of Ainv15 ES cells which harbour both regulatory and responsive elements of the tetracycline gene expression system. Since AML1 isoforms were linked to a fluorescent EGFP reporter the expression of AML1 could be readily monitored following induction with doxycycline.  Upon doxycycline induction reduction in size and number of undifferentiated colonies was observed that has not been associated with obvious apoptotic events. AML1 induction has also been initiated during the putative haemangioblast differentiation stage. However, no obvious deviation in clonogenic haematopoietic activity has been observed in induced and non-induced differentiating embryo bodies.

No description available.

Cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway were prepared to analyze changes in nuclear morphology during apoptotic execution. When isolated interphase nuclei were exposed to S/M extracts prepared from morphologically normal cells in the "committed stage" stage of the apoptotic pathway, they became condensed and the DNA was cleaved. These changes were dependent on caspase activity. In contrast, when the same nuclei were exposed to E/X extracts, prepared from morphologically apoptotic cells, they underwent apoptotic changes in a caspase-independent manner. These results suggest that nuclear disassembly is driven largely by factors activated downstream of the caspases at the onset of apoptotic execution. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40, can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors. However, as morphological changes occur even when CAD activity is inhibited, CAD cannot be the sole factor triggered by caspases. Indeed, in this study it was found that DNA topoisomerase IIa (topoIIa), which is essential both for chromosome condensation and segregation in mitosis, also functions during apoptotic execution. Simultaneous inhibition of topo IIa plus either caspases or CAD completely abolished apoptotic chromatin condensation. Furthermore, <i>in vitro </i>experiments demonstrated that CAD binds to topo IIa. This study presented the first evidence that activities such as CAD and topo II that function downstream of caspases in apoptosis are largely responsible for nuclear disassembly. Furthermore, it is demonstrated that CAD and topo II which are located in the nucleus in healthy cells, have interactions but work in parallel during chromatin condensation.

Despite the fundamental significance of desiccation in determining the distributions and activities of living organisms, there is virtually no insight as to the state of the cytoplasm of an air-dried, or even a wet, cell. In bacterial cells that have been subjected to air-drying the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (VQ and the environmental water (vapor) potential (Ψwv)) may be achieved very rapidly. In the air-dried state some bacteria survive only for seconds, others can tolerate desiccation for thousands, perhaps for millions, of years. The means by which certain cells, the anhydrobiotes, overcome and then tolerate acute water deficit remains one of the most intractable problems in cell biology. One such anhydrobiote, the cyanobacterium Nostoc commune, is cosmopolitan, its colonies form visually-conspicuous and abundant growths in situ, and it constitutes an ecologically-significant component of terrestial nitrogen-fixing communities. The cyanobacteria are phylogenetically-significant organisms that provide model systems for the study of a broad range of problems in cell biology. The studies described in this thesis established the molecular ecology and cell biology of Nostoc commune, and they provide a chronicle of the development of this microorganism as the prokaryotic model for the anhydrobiotic cell. In the design of experiments to investigate this problem the bias was, and remains, this: to understand desiccation tolerance, understand an organism that tolerates desiccation. The thesis documents an investigation into the consequences of acute cell-water deficit and the cellular basis for desiccation tolerance. An eclectic approach has been adopted to study desiccation tolerance and it includes the application of techniques of cell biology, biochemistry, microbiology, molecular biology, structural biology and biophysics.

Deciduous tooth formation begins before birth and ends after birth. This makes it more difficult to construct a continuous developmental chronology for deciduous teeth than for permanent teeth. The discovery of the neonatal line in enamel and confirmation that it marks birth, allowed the expansion of deciduous dental chronologies, which until this time had been largely based on qualitative descriptions. The aim of this study was to use the daily incremental record in deciduous enamel to document rates of enamel formation and to use these data to produce regression equations that describe the average rates of deciduous enamel formation for each tooth type. These formulae can then be applied to all deciduous teeth even when daily increments are not visible, in order to estimate crown formation times and other events during crown development, as well as to determine the age at death where enamel formation has ceased prior to completion. In permanent teeth, rates of enamel formation vary between 2.5μm per day at the EDJ to 6.5μm per day at the enamel surface. Seventy deciduous ground sections were examined and it was established that the daily rates in deciduous enamel varied less, with regional weighted means for all tooth types ranging from 2.85μm per day at the EDJ to 3.40μm per day at the enamel surface with extreme outliers of 2.07 to 4.97μm per day. The average daily incremental growth rate of enamel in deciduous teeth was calculated for each tooth type, the weighted mean of the apposition rate over both aspects (labial/buccal and lingual) and over all three regions (cervical, lateral and occlusal) for all tooth types was 3.23μm per day. A key finding of this study was that there is a marked reduction in the enamel formation rate in the zone immediately following the neonatal line or following other accentuated striae assumed to be associated with stressful events. A catch-up phase usually followed these events, during which the previous rates recovered. These data provide clear evidence of enamel hypoplasia associated with both the birth process and other events that cause stress in perinatal life.

Weibel-Palade Bodies (WPBs) are the regulated storage organelles of endothelial cells that play an important role in haemostasis and the initiation of inflammation through release of their major content protein, von Willebrand Factor (VWF), and other factors present in/on the organelle. Their large size and novel shape – a 1–5 μm long cigar-shape – raises a unique problem as to how they form at the trans-Golgi network (TGN). In this thesis I have examined the way in which these organelles form at and subsequently scission from the TGN, and possible factors involved in their regulated release in response to extracellular stimulus. I have shown that the formation of WPBs at the TGN, which is critically dependent on the presence of clathrin and AP-1, does not appear to follow the standard model of formation of a spherical clathrin-coated vesicle. Rather, the organelle appears to form through maturation of the TGN using clathrin as a scaffold. Consistent with this model, the classic candidate molecule for vesicle scission from a donor membrane, dynamin, does not play a role in the budding of the WPB from the TGN in Human Umbilical Vein Endothelial Cells (HUVECs). However, in HEK293 cells, dynamin appears crucial for scission of heterologously expressed WPBs – an example of different cells using different machinery to form the same organelle. During formation, and also following scission, a variety of factors are recruited to, or help regulate, the maturing organelle. One pair of such factors, the small GTPases Rab10 and Rab8a, play a role in controlling WPB exocytosis in response to endothelial activation, since their removal by siRNA results in a significant reduction in VWF release.

The endothelial-specific secretory organelles called Weibel-Palade Bodies (WPBs) play an important role in haemostasis and in the initiation of the inflammatory response. Activation of the endothelium induces fusion of WPBs at the plasma membrane releasing the pro-thrombotic cargo protein von Willebrand Factor (VWF) and the leukocyte receptor P-selectin, which is involved in the early stages of the inflammatory cascade. In addition, WPBs contain the ubiquitously expressed tetraspanin CD63, which is recruited to the mature organelles in an AP-3 dependent manner. Although CD63 is a long established component of WPBs, the functional significance of this protein within WPB was unknown. This thesis primarily concerns the role of CD63 in inflammation and haemostasis, attempting to determine its function. Using siRNA to knockdown CD63 in Human Umbilical Vein Endothelial Cells (HUVECs), no phenotype was observed in the haemostatic functions nor biogenesis maturation or exocytosis of WPBs. However, CD63 depletion resulted in a failure of P-selectin dependent recruitment of leukocytes to endothelial monolayers. CD63-deficient mice also displayed a reduction in leukocyte recruitment and a decrease in leukocyte extravasation using a peritonitis model. High-resolution scanning electron microscopy and proximity-ligation assays revealed CD63 co-clusters with P-selectin on the surface of activated endothelial cells and that CD63 acts to cluster the receptor, retaining it at the plasma membrane. In addition, scanning electron microscopy of the activated endothelium was carried out to study the structure of VWF exit sites and string formation in static conditions and under shear stress, to recreate physiological conditions. The results reveal new insights into the behaviour of VWF strings and the mechanism of WPB exocytosis at the membrane, involving the actin cytoskeleton.

The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs can also regulate signalling downstream of other families of receptors and are now known to have a number of non-receptor substrates and binding partners. Here I identify RhoAGTP, Raf1 and ERK2 as novel binding partners of GRK2 and report a previously unsuspected function for this kinase. GRK2 acts as a RhoA-activated scaffold protein for the ERK MAP kinase cascade downstream of the epidermal growth factor (EGF) receptor. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoAGTP binding to the catalytic domain of the kinase, however, while RhoAGTP binding is common to all of the ubiquitously expressed GRKs, the ability to act as a RhoA-regulated Raf/MEK/ERK scaffold is specific to GRK2. GRK2 over-expression in HEK-293 cells potentiates EGF-induced ERK activation in a Rho-dependent fashion. Conversely, depleting GRK2 expression by RNAi reveals that GRK2 is required for EGF-induced thymidine incorporation in vascular smooth muscle cells (VSMCs). Rho-dependent ERK MAP kinase scaffolding by GRK2 may therefore have an important role in the vasculature, where increased levels of GRK2 and RhoA have been associated with hypertension.

The circadian clock is an endogenous time-keeping mechanism that allows an organism to coordinate its biology with the 24 hour variations in its external environment. Epidemiological studies have linked clock disruption to an increase incidence of cancer, in particular breast malignancy. On a molecular level, clock components have been shown to regulate cell cycle gene expression and its progression in a number of models. This thesis set out to further dissect the link between these two important systems. Within the zebrafish cell-line, PAC2, mitosis was demonstrated to be under circadian control via clock regulation of the cell cycle mediator, Cyclin B1. Techniques used were then translated into a human cell-line model to study species specific clock function, with particular reference to breast epithelial tissue. Glucocorticoids, putative clock synchronisation agents in vivo, were observed to induce cellular clock synchronisation in HEK 293 cells and the benign breast epithelial cell-line MCF10A. Clock disruption inhibited cell growth. Study of the breast epithelial cell cycle mutant, MDA-MB-231, demonstrated a functional clock, revealing no reciprocal regulation. In contrast, decreased expression levels of clock gene and putative tumour suppressor, Per1, were observed within the malignant breast epithelial cell-line, MCF7, leading to greatly disrupted clock function and circadian independent cell growth. Unlike the zebrafish model, no intracellular clock regulation of cell cycle genes expression or function was observed, expression being preferentially modified by homeral circadian regulators such as glucocorticoids and melatonin. This also contradicts mammalian in vivo studies, leading to the hypothesis that the clock and cell cycle maybe uncoupled in immortalised cell cultures. In conclusion this study has demonstrated that clock regulation of the cell cycle in mammalian system is a multifactorial process and that disruption of this system leads to changes in the character of the cell cycle within the host tissue. Further work must explore this relationship in an in vivo setting.

Senescence, like apoptosis, functions to remove damaged cells from the cell cycle and thereby prevents the accumulation of multiple genetic changes that drive tumourigenesis. In contrast to apoptosis – also commonly referred to as programmed cell-death – senescence is characterised by an irreversible cell-cycle arrest. A number of stimuli have been reported to induce senescence including oncogenic stimuli, e.g. oncogenic RAS–RAF signalling; telomere shortening; culture stress; oxidative stress and DNA damaging agents. However, many of the senescence signals generated by these stimuli remain poorly defined. Oncogenes are drivers of tumourigenesis and therefore a complete understanding of the senescence signals generated by oncogenes will be important for advancing our knowledge of how tumours evade senescence and for the rational design of prosenescence cancer therapies. Three proposed signals include DNA replicational stress, oxidative DNA damage from an increase in intracellular reactive oxygen species (ROS) and p38 stress-activated protein kinase (SAPK) signalling. In this thesis, data are presented that support that an oncogenic RAF-induced senescent cell-cycle arrest can occur in rat Schwann cells without an increase in ROS or a DNA damage response (DDR) as well as p38 SAPK signalling, and thereby suggest that the arrest is induced by an alternative senescence signal. Furthermore, data are presented that show that low oxygen does not prevent oncogenic RAS-induced senescence in rodent or human cells and thereby question the role of ROS in oncogene-induced senescence. Senescence by definition is an irreversible cell-cycle arrest. However, the stability of the cell-cycle arrest in the absence of a continuous oncogenic signal has been relatively unexplored. This is an important question as the answer is also likely to impact the rational design of pro-senescence cancer therapies. Analyses of rat Schwann cells and human fibroblasts using an inducible RAF protein construct that enables reversible RAF signalling show that an oncogenic RAF-induced senescent arrest is inherently more stable in human cells than rodent cells but that the arrest is ultimately reversible in both species.