Myeloproliferative neoplasms (MPNs) are a heterogeneous group of haematological stem cell malignancies characterised by proliferation of one or more cells of the myeloid lineage. The molecular investigation of MPN was revolutionized in 2005 by the finding that approximately 95% of cases with polycythaemia vera (PV) and 50-60% of cases of essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are characterised by a single acquired mutation, JAK2 V617F. My study has focused on four principal areas: (i) Involvement of V617F in other myeloid disorders. After developing sensitive methods to detect and quantify V617F, this mutation was identified in 17% of cases of atypical chronic myeloid leukaemia (17/99) as well as other atypical MPN, thus demonstrating that it was more widely involved in myeloid disorders that initially thought. Homozygosity of V617F was shown to have arisen by acquired uniparental disomy (UPD) and examination of two cases with V617F plus either KIT D816V or BCR-ABL demonstrated that the mutations had arisen in independent clones. (ii) In vitro assays to predict imatinib sensitivity. Haemopoietic colony and liquid cultures were used to determine if peripheral blood or bone marrow cells from atypical MPN cases (n=200) were sensitive to imatinib. Of those that responded in one or both cultures (n=185) some had known abnormalities of PDGFRA or PDGFRB, but a significant minority proved negative for all molecular tests suggesting the presence of uncharacterised imatinib-sensitive mutations. (iii) V617F as a marker of response to therapy. JAK2 V617F was used as a molecular marker to monitor the response of PV patients (n=21) to therapy with imatinib and interferon-α. Neither therapy eradicated V617F but there was a modest reduction in %V617F which correlated with haematological response. By contrast, in those patients that did not respond (n=13) the %V617F marginally increased. (iv) Genetic predisposition to MPN. Whilst investigating the possible contribution of JAK2 single nucleotide polymorphisms to the phenotypic diversity associated with V617F, marked skewing of alleles associated with the mutation was observed. Further investigation revealed that V617Fassociated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (PV, n=192, P=2.9x10-16; ET, n=78, P=8.2x10-9 and MF, n=41, P=8.0x10-5). Furthermore, allele-specific PCR demonstrated that V617F specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of V617F associated MPNs (OR=3.7; 95% CI 3.1-4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation.

Huntington's Disease (HD) is one of many neurodegenerative diseases that are associated with protein misfolding, aggregation and oxidative stress. While several changes in the redox homeostasis have been shown to occur in HD animal models and HD brains, the formal relationships between intracellular protein misfolding that occurs in HD, redox dysregulation and cellular toxicity are unknown. Therefore, several cellular models of intracellular polyglutamine (polyQ) protein misfolding were established for mechanistic studies. Various in vitro transient and stable cell expression systems expressing an N-terminal fragment of huntingtin (htt) (httExon 1, httEx1) with/or without a polyQ expansion and fused to fluorescent proteins were characterized. Mutant httEx1 (mhttEx1) constructs expressed in both neuronal and non-neuronal cell lines produced early polyQ aggregates and intracellular inclusion bodies (IBs) followed by cell toxicity that increased over time in time-course experiments. Using oxidation-sensitive probes, reactive oxygen species (ROS) were measured in polyQ-expressing cells using single, live-cell imaging analysis by confocal microscopy or population assays in order to explore the relationship between polyQ aggregation, ROS production and cellular toxicity. This study highlighted an early increase in ROS due to the expression of aggregation-prone mhttEx1 in both transient and stable cellular systems that coincided with polyQ aggregation, but preceded cell death. Suppression of ROS and toxicity was achieved by two antioxidant compounds (L-NAC and Trolox). Moreover, the use of MitoQ (Coenzyme Q10 covalently attached to triphenylphosphonium cation (TPP+)) at nanomolar concentrations abrogated the increased ROS due to mhttEx1 suggesting a mitochondrial origin of ROS. Given that molecular chaperones regulate the folding/misfolding of proteins and are involved in the regulation of the cellular redox homeostasis, the role of the redoxactivatable chaperone DJ-1 in HD was investigated. Protein expression analysis in HD cell models, a rodent model of HD and human HD brain samples showed an up-regulation of DJ-1 protein expression compared to control samples. Oxidation of DJ-1 was also elevated in the human HD cortex. To test for a functional role of DJ-1 elevation and oxidation in HD, DJ-1 was overexpressed with wild-type or mhttEx1 in cell lines and mouse primary astrocytes. Overexpression of DJ-1 accelerated mhttEx1 aggregation and toxicity both of which could be suppressed by exposure of cells to mild oxidants suggesting that DJ-1, when redox-activated to a chaperone, modulates polyQ aggregation and toxicity. This hypothesis was tested by overexpression of mhttEx1 with a DJ-1 mutant lacking a critical redox activatable cysteine (Cys106). The C106S-DJ-1 mutant lost its ability to reduce polyQ aggregation and toxicity under oxidising conditions upon co-expression with mhttEx1 suggesting that DJ-1 indeed functions as a modulator of polyQ misfolding and toxicity. Together this work suggests that ROS may be produced during polyQ aggregation and is involved in cellular toxicity. This study also shows that DJ-1 regulates both, polyQ aggregation and toxicity in cell models and given the increased DJ-1 expression in vitro and in vivo (human HD), this protein could be a potential target for HD therapy.

Recent evidence suggests that stem cells are important for cancer metastasis and that the epithelial-to-mesenchymal transition also involves a transition toward stemness. Current thinking suggests that lgr5, a 7-transmembrane spanning G-protein, also marks a certain population of stem cells capable of regenerating an intestinal crypt and that the specialised immune secretory paneth cell, is important for maintenance of the stem cell niche. We implicate fascin in regulating the balance of lgr5 stem cells during acute intestinal inflammation and in regenerating intestinal tissue. Fascin is an actin bundling protein that drives the assembly of filopodia through the cross-linking of actin filaments into straight bundles. Conserved from amoebas to man, fascin was originally purified from extracts of sea urchin oocytes and coelomocytes and later found in Drosophila as the singed gene product. It is involved in the invasion and metastasis of multiple epithelial cancer types through stabilisation of actin in invadopodia, finger like protrusions used by cancer cells to invade into and degrade the extra-cellular matrix. Fascin, whilst normally low or absent from epithelia, localises to the leading edges of migratory cells and is over-expressed in many cancers of the same epithelial origin including lung, colorectal, pancreatic and liver. Fascin has also recently been shown to increase during inflammatory bowel disease (IBD) conditions such as diverticulitis, Crohn’s disease and ulcerative colitis. In this thesis I have investigated the role of fascin in murine models of IBD and have demonstrated that fascin is required for the haematopoietic production of leucocytes, in response to inflammation and that the loss of fascin, in the presence of high Wnt levels, results in enhanced proliferation of intestinal epithelial cells. One of the serious consequences of IBD is the increased lifetime risk of the patient developing an intestinal malignancy secondary to the disease. The exact mechanism underlying the increase in malignancies has not yet been fully established, however it is postulated that chronic inflammation and the effect this has on the major molecular pathways involved in carcinogenesis underlies the transformation from benign to malignant disease. Highest fascin expression has been shown in the dysplastic, pre-malignant cells in human IBD tissues indicating an important role of fascin in the transformation of benign to malignant cells. In this thesis, I have demonstrated that loss of fascin impairs tumour initiation in inflammatory driven and spontaneous intestinal tumourigenesis models, which is likely, in part, to be as a consequence of reduced leucocytes, in particular neutrophils, which may be CXCL2 mediated.

Os métodos radiométricos (Rb-Sr e Sm-Nd) têm sido aplicados, com sucesso, em rochas sedimentares visando a obtenção de idades deposicionais e informações sobre proveniência. Em muitos casos, apesar dos resultados geologicamente significativos, ainda persistem dúvidas em relação a extensão e interpretação dos dados obtidos. Isto ocorre porque as rochas sedimentares resultam da mistura de fragmentos detríticos de diversas proveniências com diferentes sistemas isotópicos, conseqüentemente, já contendo um registro isotópico da rocha fonte. No caso do método Rb-Sr, aplicado em rochas sedimentares de granulometria fina, pode-se obter idades absolutas para o evento deposicional. Isto é viável desde que este registro isotópico proveniente das rochas fonte seja apagado no momento da deposição, ou seja, deve ocorrer a homogeneização isotópica do Sr no ambiente sedimentar. Para tanto, devem ser observados alguns pré-requisitos em relação a amostragem, granulometria, ambiente deposicional e composição mineralógica das amostras, entre outros. No entanto, ainda existem questionamentos em relação a esta metodologia especialmente quanto à ocorrência e à extensão do processo de homogeneização isotópica do Sr em ambiente sedimentar.(Continua0 O método Sm-Nd, apesar de ser uma técnica relativamente nova quando aplicada a rochas sedimentares, tem se tornado uma ferramenta fundamental para auxílio na determinação de proveniência de rochas sedimentares. As maiores dificuldades estão na correta interpretação dos valores obtidos e sua associação com um ambiente sedimentar, ou seja, de baixa temperatura. Neste trabalho, foram aplicados os métodos radiométricos Rb-Sr e Sm-Nd em amostras de rochas sedimentares coletadas em diferentes contextos geológicos da Bacia do Paraná. Foram tentativamente testados os diferentes parâmetros que atuam no sentido da homogeneização isotópica do Sr e do comportamento do Nd. Os resultados obtidos permitiram aprimorar a metodologia radiométrica Rb-Sr e Sm-Nd quando aplicada em rochas sedimentares, bem como obter resultados sobre a idade deposicional de diferentes unidades sedimentares e, por vezes, sua proveniência.

The success of forensic investigation very often depends on the establishment of the correct timeline of events. In the investigation of fatalities, this depends greatly on the estimation of the time since death of the victim. Current methods lead to inaccurate results and depend greatly on the experience of the investigator. Pathologists estimate the time since death based on visual inspection of the bodies as well as body temperature measurement. Only very short post-mortem intervals (PMIs) can be evaluated with some degree of certainty. This investigation used untargeted proteomic and metabolomic approaches to identify potential molecular markers (proteins, metabolites) which could help to quantify post-mortem changes and aid PMI estimation. Animal models were used in the initial stages of the project. Aged beef meat (stored at 4°C for 13 days) and rat muscle samples (intact cadavers stored at ambient temperature for 3 days) were sampled at 24 h time intervals. In the final stages of the project, human tissue samples were collected at the Forensic Anthropology Centre at Texas State University (San Marcos, Texas). Muscle samples were collected at various times post-mortem from 6 different subjects over the period of two weeks. For the proteomics experiment, 0.5g of tissue was homogenized in extraction buffer consisting of urea, thiourea and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). Protein separation was carried out using two-dimensional gel electrophoresis. Protein identification was performed using liquid chromatography-tandem mass spectrometry. For the metabolomics experiment, 0.5g of tissue was homogenized in chloroform/methanol/water solution. The extracted samples were analysed using liquid chromatography-mass spectrometry as well as gas chromatography – mass spectrometry. The investigation allowed the identification of potential biomarker candidates. The proteins of interest varied between the sampled mammals. However, myosin and actin appear as promising candidates for all three species. The metabolomics experiments yielded a large number of possible biomarker candidates. Both liquid and gas chromatography approaches were successfully applied, pointing towards various compounds. Proteogenic amino acids were identified as main compounds of interest in all species using both methods. The study has shown that both proteomic and metabolomic approaches can be successfully applied in forensic medical science and can help to find PMI markers. Using the untargeted approach gives the advantage of looking at a whole range of detected molecules and choosing the most appropriate ones for the task. Furthermore, the combination of these two approaches gives a deeper insight into the post-mortem biological processes. The biomarker candidates proposed in this study require further validation in a larger cohort of subjects.

In the early days of PKU, micronutrient deficiencies, undernutrition and growth failure were common features of patients with the condition. This was mainly due to the limited availability of engineered special low protein foods (SLPF) and PKU protein substitutes to use. Nowadays, SLPF foods and micronutrient-enriched PKU protein substitutes have become widely available and are free on prescription in most countries. These SLPF are high in carbohydrates and often in fat content, have a higher glycaemic index and provide more energy per weight compared with the protein-containing equivalent normal foods. Advancement in the PKU management, including dietary practices, led to nutritional problems that had never been reported before, but have become more frequent in the recent years. Overweight and obesity, rather than undernutrition, have become increasingly reported in patients with PKU, with some studies suggesting higher prevalence in females than males. Data on body composition in patients with PKU are inconsistent with some studies showing that patients with PKU have higher FM and lower FFM compared to healthy controls. This suggests that for a given body weight and height, patients with PKU might be fatter and look bigger. However, there is very little research looking at the determinants of nutrient status, body composition and obesity in patients with PKU. Hence, among the aims of this thesis was to investigate the impact of a PKU SLPF-based meal on appetite ratings, gut appetite hormones, thermic effect of feeding (TEF) and fat oxidation (Chapter 2). Twenty-three healthy adults (mean ± SD age: 24.3 ± 5.1 years; BMI: 22.4 ± 2.5 kg/m2) participated in a randomised crossover study. Each participant conducted two (PKU and Control) experimental trials which involved consumption of a PKU SLPF-based meal and protein substitute drink or an isocaloric and weight matched ordinary meal and protein-enriched milk drink. Appetite, metabolic rate, fat oxidation measurements and blood collections were conducted for the duration of 300 minutes. On completion of the measurements, an ad libitum buffet dinner was served. Responses of appetite ratings, plasma concentrations of GLP-1 and PYY (P > 0.05, trial effect, two-way ANOVA) and energy intake during ad libitum buffet dinner (P > 0.05, paired t-test) were not significantly different between the two trials. The TEF (PKU, 10.2 ±1.5%; Control, 13.2 ± 1.0%) and the total amount of fat oxidised (PKU, 18.90 ± 1.10 g; Control, 22.10 ± 1.10 g) were significantly (P < 0.05, paired t-tests) lower in the PKU than in the Control trial. The differences in TEF and fat oxidation were significant (P < 0.05, paired t-tests) for the post-meal period. Therefore, from this first study we concluded that consumption of a meal composed of SLPF has no detrimental impact on appetite and appetite hormones but produces a lower TEF and postprandial fat oxidation than an ordinary meal. We hypothesised that these metabolic alterations may contribute to the increased prevalence of obesity reported in patients with PKU on contemporary dietary management. In the second experimental chapter, we tested the hypothesis generated from the study above and measured TEF, fasting and postprandial fat and CHO oxidation in 13 patients with PKU and 10 healthy controls of similar age and BMI. Participants in the PKU group were provided an SLPF-based meal while those in the Control group were provided an isocaloric normal meal. It was found that TEF, and postprandial fat and CHO oxidation were not signifcantly different between the PKU and the Control groups. In addition, this study compared body composition characteristics (measured by Deuterium Oxide dilution technique) between PKU patients and healthy controls and revealed that differences in body composition are not significant between the two groups despite a tendency of PKU patients having higher percentage of body fatness (P=0.08). However, data generated from this study should be interpreted with caution and requires confirmation from studies with larger sample size. Micronutrient imbalance has been noted in patients with PKU despite their high provision through the PKU protein substitutes. Recent studies showed high blood levels of vitamin B12 and folate, but simultaneously deficient plasma levels of selenium and zinc in PKU patients prescribed with micronutrient-enriched PKU protein substitutes. Factors associated with micronutrient imbalance have rarely been studied in the literature. Therefore, the last chapter of this thesis aimed to evaluate the micronutrient status of children with PKU and explore factors associated with micronutrient imbalances and deficiencies. This was analysis of a large clinical dataset with serial measurements obtained from PKU children (≤16 years) attending the metabolic medicine clinic at the Glasgow Royal Hospital for Sick Children between 1990 and 2013. The study included 81 patients who provided a total of 512 blood samples for their routine annual micronutrient screening. Data on blood micronutrient measurements was available for vitamins A, B12, D, E, serum folate, and erythrocyte folate and the trace elements copper, selenium, zinc and serum ferritin as a biomarker of iron stores. Status of vitamin B12, E, and serum and erythrocyte folate measurements were above the normal range (NR) in 27%, 54%, 46% and 35% of the blood samples, respectively. However, 44% of selenium and 14% of zinc measurements were below the NR. Moreover, when we compared results with those from the UK National Dietary and Nutritional Survey, selenium and zinc deficiencies were specific to PKU condition and not a reflection of the epidemiology in the general UK population. In our PKU sample, poor metabolic control, PKU severity, and low adherence to PKU protein substitutes predicted low selenium status; while deficient zinc status was solely predicted by low adherence to PKU protein substitutes. Yet, these predictors, collectively, explained a small (5.8 – 8.8 %) variation in the status of selenium and zinc in this group of patients. Selenium and zinc deficiencies are common in PKU patients despite high levels of other nutrients including vitamin B12, E and folate. The findings of this study suggest that selenium and zinc deficiencies reported in patients with PKU may be attributed to other factors which we were unable to measure in this retrospective study, such as low bioavailability of these nutrients from the artificial PKU protein substitutes.

Os métodos radiométricos (Rb-Sr e Sm-Nd) têm sido aplicados, com sucesso, em rochas sedimentares visando a obtenção de idades deposicionais e informações sobre proveniência. Em muitos casos, apesar dos resultados geologicamente significativos, ainda persistem dúvidas em relação a extensão e interpretação dos dados obtidos. Isto ocorre porque as rochas sedimentares resultam da mistura de fragmentos detríticos de diversas proveniências com diferentes sistemas isotópicos, conseqüentemente, já contendo um registro isotópico da rocha fonte. No caso do método Rb-Sr, aplicado em rochas sedimentares de granulometria fina, pode-se obter idades absolutas para o evento deposicional. Isto é viável desde que este registro isotópico proveniente das rochas fonte seja apagado no momento da deposição, ou seja, deve ocorrer a homogeneização isotópica do Sr no ambiente sedimentar. Para tanto, devem ser observados alguns pré-requisitos em relação a amostragem, granulometria, ambiente deposicional e composição mineralógica das amostras, entre outros. No entanto, ainda existem questionamentos em relação a esta metodologia especialmente quanto à ocorrência e à extensão do processo de homogeneização isotópica do Sr em ambiente sedimentar.(Continua0 O método Sm-Nd, apesar de ser uma técnica relativamente nova quando aplicada a rochas sedimentares, tem se tornado uma ferramenta fundamental para auxílio na determinação de proveniência de rochas sedimentares. As maiores dificuldades estão na correta interpretação dos valores obtidos e sua associação com um ambiente sedimentar, ou seja, de baixa temperatura. Neste trabalho, foram aplicados os métodos radiométricos Rb-Sr e Sm-Nd em amostras de rochas sedimentares coletadas em diferentes contextos geológicos da Bacia do Paraná. Foram tentativamente testados os diferentes parâmetros que atuam no sentido da homogeneização isotópica do Sr e do comportamento do Nd. Os resultados obtidos permitiram aprimorar a metodologia radiométrica Rb-Sr e Sm-Nd quando aplicada em rochas sedimentares, bem como obter resultados sobre a idade deposicional de diferentes unidades sedimentares e, por vezes, sua proveniência.

Introduction: Malignant Melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Breslow thickness remains the best predictor of metastasis and Sentinel Lymph Node Biopsy is the only method of detecting nodal spread in clinically node negative patients. Surgery is the only effective therapy. Angiogenesis – the growth of new vessels from pre-existing vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. Anti- angiogenic isoforms of VEGF have been demonstrated previously to be protective with regard to metastasis. The aims of this thesis were to determine whether VEGF expression within the tumour may allow prediction of the nodal status. Furthermore another aim was to determine whether via the “Seed and Soil” theory, by examination of angiogenic and lymphangiogenic profiles of the tumour and node can we determine that the tumour may control the microenvironment around the Sentinel Node? Finally, as a cohort of false negative patients emerged with a higher mortality rate than their true negative and true positive patient cohort counterparts, could any further patterns be established by performing the same experiments on these patients? Methods: Archived human tumour and corresponding Sentinel Node samples were used and immunohistochemistry was used to investigate the role of pro and anti angiogenic isoforms of VEGF, VEGF-C, LYVE-1 and CD31 within these patients. Results: VEGF-C expression was significantly increased in the intranodal component of positive Sentinel Lymph Nodes (p < 0.01 Bonferroni). This increased expression appeared to be independent of tumoural influences and no strong evidence for the “Seed and Soil” theory was proved. A significantly higher number of lymphatic vessel counts were identified within node negative patients (p < 0.05 ANOVA). No further significant findings were defined on examination of the false negative cohort of patients. Conclusions: This study has shown that positive Sentinel Lymph Nodes exhibit high levels of intranodal VEGF-C. This expression does not appear to be related to tumoural influences. It would therefore appear that VEGF-C expression within Sentinel Nodes warrants further investigation and may aid diagnosis of spread or represent a target to slow or even prevent the onset of metastasis.

Os métodos radiométricos (Rb-Sr e Sm-Nd) têm sido aplicados, com sucesso, em rochas sedimentares visando a obtenção de idades deposicionais e informações sobre proveniência. Em muitos casos, apesar dos resultados geologicamente significativos, ainda persistem dúvidas em relação a extensão e interpretação dos dados obtidos. Isto ocorre porque as rochas sedimentares resultam da mistura de fragmentos detríticos de diversas proveniências com diferentes sistemas isotópicos, conseqüentemente, já contendo um registro isotópico da rocha fonte. No caso do método Rb-Sr, aplicado em rochas sedimentares de granulometria fina, pode-se obter idades absolutas para o evento deposicional. Isto é viável desde que este registro isotópico proveniente das rochas fonte seja apagado no momento da deposição, ou seja, deve ocorrer a homogeneização isotópica do Sr no ambiente sedimentar. Para tanto, devem ser observados alguns pré-requisitos em relação a amostragem, granulometria, ambiente deposicional e composição mineralógica das amostras, entre outros. No entanto, ainda existem questionamentos em relação a esta metodologia especialmente quanto à ocorrência e à extensão do processo de homogeneização isotópica do Sr em ambiente sedimentar.(Continua0 O método Sm-Nd, apesar de ser uma técnica relativamente nova quando aplicada a rochas sedimentares, tem se tornado uma ferramenta fundamental para auxílio na determinação de proveniência de rochas sedimentares. As maiores dificuldades estão na correta interpretação dos valores obtidos e sua associação com um ambiente sedimentar, ou seja, de baixa temperatura. Neste trabalho, foram aplicados os métodos radiométricos Rb-Sr e Sm-Nd em amostras de rochas sedimentares coletadas em diferentes contextos geológicos da Bacia do Paraná. Foram tentativamente testados os diferentes parâmetros que atuam no sentido da homogeneização isotópica do Sr e do comportamento do Nd. Os resultados obtidos permitiram aprimorar a metodologia radiométrica Rb-Sr e Sm-Nd quando aplicada em rochas sedimentares, bem como obter resultados sobre a idade deposicional de diferentes unidades sedimentares e, por vezes, sua proveniência.

Development, tissue renewal and longevity of multi-cellular organisms require the ability to switch between a proliferative state and quiescence, a reversible arrest from the cell cycle. The balance of quiescence and proliferation underlies the fundamental feature of generating and maintaining the appropriate number of cells, which is essential for tissue architecture, regeneration, and function. Disruption of quiescence and proliferation balance leads to hypo- or hyper-proliferative diseases. To date, the regulatory mechanism of proliferation has been well established, while cellular quiescence has remained a phenotypic description without a clearly defined molecular control mechanism. Simply, quiescence has long been considered a passive counterpart to proliferation. However, recent findings have revealed that quiescence is an actively maintained state exhibiting a unique gene expression pattern. While quiescence has been traditionally considered as a state (namely G0) outside of the cell cycle, it is in fact a collection of heterogeneous states. In studies conducted in the 70's and 80's using fibroblasts and lymphocytes, it has been observed that the longer the cells were kept under quiescence inducing conditions such as contact inhibition, the deeper the cells moved into quiescence. Deep quiescent cells are still able to reenter the cell cycle upon growth stimulation but they exhibit a longer pre-DNA synthesis phase [1-4]. Shallow quiescent state has also been recently reported in muscle and neural stem cells termed GAlert and "prime" quiescent state, respectively. Heterogeneous quiescent depth entails that cells vary in their sensitivity to growth signals, representing an important yet underappreciated layer of complexity in cell growth control. The cellular mechanisms that control the depth of quiescence remains elusive. In my thesis work, I first investigate the strengths of serum stimulation required for cells to exit deep and shallow quiescence as a determinant of quiescence depth. Through model simulations and experimental measurements, I further demonstrate that various components of the Rb-E2F pathway control quiescence depth with varying efficacy. The Rb-E2F pathway interacts with diverse cellular pathways that respond to environmental signals to jointly modulate quiescence depth. Given that certain circadian clock genes (e.g., Cry) affect key components in the Rb-E2F pathway, I tested the effect of Cry activity on quiescence depth. I found that increased Cry activity resulted in deeper quiescence, contrary to our anticipation based on the literature. Next, we constructed a library of mathematical models that represent possible interactions between Cry and the Rb-E2F pathway. We computationally searched this model library for links that could explain the experimental observations. The modeling search suggested that Cry upregulation may lead to increased expression of cyclin dependent kinase inhibitor (e.g., p21), which in turn drives cells into deeper quiescence. This model prediction was confirmed by my follow-up experiments. Collectively, my thesis work establishes an integrated modeling and experimental framework that will help us to further investigate diverse cellular mechanisms controlling the heterogeneous quiescence depth.