Integrative genomic profiling reveals conserved genetic mechanisms for tumorigenesis in common entities of non-Hodgkin's lymphoma

Recent developments in genomic technologies have resulted in increased understanding of pathogenic mechanisms and emphasized the importance of central survival pathways. Here, we use a novel bioinformatic based integrative genomic profiling approach to elucidate conserved mechanisms of lymphomagenesis in the three commonest non-Hodgkin's lymphoma (NHL) entities: diffuse large B-cell lymphoma, follicular lymphoma, and B-cell chronic lymphocytic leukemia. By integrating genome-wide DNA copy number analysis and transcriptome profiling of tumor cohorts, we identified genetic lesions present in each entity and highlighted their likely target genes. This revealed a significant enrichment of components of both the apoptosis pathway and the mitogen activated protein kinase pathway, including amplification of the MAP3K12 locus in all three entities, within the set of genes targeted by genetic alterations in these diseases. Furthermore, amplification of 12p13.33 was identified in all three entities and found to target the FOXM1 oncogene. Amplification of FOXM1 was subsequently found to be associated with an increased MYC oncogenic signaling signature, and siRNA-mediated knock-down of FOXM1 resulted in decreased MYC expression and induced G2 arrest. Together, these findings underscore genetic alteration of the MAPK and apoptosis pathways, and genetic amplification of FOXM1 as conserved mechanisms of lymphomagenesis in common NHL entities. Integrative genomic profiling identifies common central survival mechanisms and highlights them as attractive targets for directed therapy.

Molecular mechanisms of migraine

Migraine is a common complex disorder that affects a large portion of the population and thus incurs a substantial economic burden on society. The disorder is characterized by recurrent headaches that are unilateral and usually accompanied by nausea, vomiting, photophobia, and phonophobia. The range of clinical characteristics is broad and there is evidence of comorbidity with other neurological diseases, complicating both the diagnosis and management of the disorder. Although the class of drugs known as the triptans (serotonin 5-HT1B/1D agonists) has been shown to be effective in treating a significant number of patients with migraine, treatment may in the future be further enhanced by identifying drugs that selectively target molecular mechanisms causing susceptibility to the disease. Genetically, migraine is a complex familial disorder in which the severity and susceptibility of individuals is most likely governed by several genes that may be different among families. Identification of the genomic variants involved in genetic predisposition to migraine should facilitate the development of more effective diagnostic and therapeutic applications. Genetic profiling, combined with our knowledge of therapeutic response to drugs, should enable the development of specific, individually-tailored treatment.

The function and mechanisms of action of ghrelin and obestatin in ovarian cancer

In this study, we have demonstrated that the preproghrelin derived hormones, ghrelin and obestatin, may play a role in ovarian cancer. Ghrelin and obestatin stimulated an increase in cell migration in ovarian cancer cell lines and may play a role in cancer progression. Ovarian cancer is the leading cause of death among gynaecological cancers and is the sixth most common cause of cancer-related deaths in women in developed countries. As ovarian cancer is difficult to diagnose at a low tumour grade, two thirds of ovarian cancers are not diagnosed until the late stages of cancer development resulting in a poor prognosis for the patient. As a result, current treatment methods are limited and not ideal. There is an urgent need for improved diagnostic markers, as well better therapeutic approaches and adjunctive therapies for this disease. Ghrelin has a number of important physiological effects, including roles in appetite regulation and the stimulation of growth hormone release. It is also involved in regulating the immune, cardiovascular and reproductive systems and regulates sleep, memory and anxiety, and energy metabolism. Over the last decade, the ghrelin axis, (which includes the hormones ghrelin and obestatin and their receptors), has been implicated in the pathogenesis of many human diseases and it may t may also play an important role in the development of cancer. Ghrelin is a 28 amino acid peptide hormone that exists in two forms. Acyl ghrelin (usually referred to as ghrelin), has a unique n-octanoic acid post-translational modification (which is catalysed by ghrelin O-acyltransferase, GOAT), and desacyl ghrelin, which is a non-octanoylated form. Octanoylated ghrelin acts through the growth hormone secretagogue receptor type 1a (GHSR1a). GHSR1b, an alternatively spliced isoform of GHSR, is C-terminally truncated and does not bind ghrelin. Ghrelin has been implicated in the pathophysiology of a number of diseases Obestatin is a 23 amino acid, C-terminally amidated peptide which is derived from preproghrelin. Although GPR39 was originally thought to be the obestatin receptor this has been disproven, and its receptor remains unknown. Obestatin may have as diverse range of roles as ghrelin. Obestatin improves memory, inhibits thirst and anxiety, increases pancreatic juice secretion and has cardioprotective effects. Obestatin also has been shown to regulate cell proliferation, differentiation and apoptosis in some cell types. Prior to this study, little was known regarding the functions and mechanisms of action ghrelin and obestatin in ovarian cancer. In this study it was demonstrated that the full length ghrelin, GHSR1b and GOAT mRNA transcripts were expressed in all of the ovarian-derived cell lines examined (SKOV3, OV-MZ-6 and hOSE 17.1), however, these cell lines did not express GHSR1a. Ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for ghrelin, obestatin, and GOAT, but not GHSR1a, or GHSR1b. No correlations between cancer grade and the level of expression of these transcripts were observed. This study demonstrated for the first time that both ghrelin and obestatin increase cell migration in ovarian cancer cell lines. Treatment with ghrelin (for 72 hours) significantly increased cell migration in the SKOV3 and OV-MZ-6 ovarian cancer cell lines. Ghrelin (100 nM) stimulated cell migration in the SKOV3 (2.64 +/- 1.08 fold, p <0.05) and OV-MZ-6 (1.65 +/- 0.31 fold, p <0.05) ovarian cancer cell lines, but not in the representative normal cell line hOSE 17.1. This increase in migration was not accompanied by an increase in cell invasion through Matrigel. In contrast to other cancer types, ghrelin had no effect on proliferation. Ghrelin treatment (10nM) significantly decreased attachment of the SKOV3 ovarian cancer cell line to collagen IV (24.7 +/- 10.0 %, p <0.05), however, there were no changes in attachment to the other extracellular matrix molecules (ECM) tested (fibronectin, vitronectin and collagen I), and there were no changes in attachment to any of the ECM molecules in the OV-MZ-6 or hOSE 17.1 cell lines. It is, therefore, unclear if ghrelin plays a role in cell attachment in ovarian cancer. As ghrelin has previously been demonstrated to signal through the ERK1/2 pathway in cancer, we investigated ERK1/2 signalling in ovarian cancer cell lines. In the SKOV3 ovarian cancer cell line, a reduction in ERK1/2 phosphorylation (0.58 fold +/- 0.23, p <0.05) in response to 100 nM ghrelin treatment was observed, while no significant change in ERK1/2 signalling was seen in the OV-MZ-6 cell line with treatment. This suggests that this pathway is unlikely to be involved in mediating the increased migration seen in the ovarian cancer cell lines with ghrelin treatment. In this study ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for obestatin, however, no correlation between cancer grade and level of obestatin transcript expression was observed. In the ovarian-derived cell lines studied (SKOV3, OV-MZ-6 and hOSE 17.1) it was demonstrated that the full length preproghrelin mRNA transcripts were expressed in all cell lines, suggesting they have the ability to produce mature obestatin. This is the first study to demonstrate that obestatin stimulates cell migration and cell invasion. Obestatin induced a significant increase in migration in the SKOV3 ovarian cancer cell line with 10 nM (2.80 +/- 0.52 fold, p <0.05) and 100 nM treatments (3.12 +/- 0.68 fold, p <0.05) and in the OV-MZ-6 cancer cell line with 10 nM (2.04 +/- 0.10 fold, p <0.01) and 100 nM treatments (2.00 +/- 0.37 fold, p <0.05). Obestatin treatment did no affect cell migration in the hOSE 17.1normal ovarian epithelial cell line. Obestatin treatment (100 nM) also stimulated a significant increase in cell invasion in the OV-MZ-6 ovarian cancer cell line (1.45 fold +/- 0.13, p <0.05) and in the hOSE17.1 normal ovarian cell line cells (1.40 fold +/- 0.04 and 1.55 fold +/- 0.05 respectively, p <0.01) with 10 nM and 100 nM treatments. Obestatin treatment did not stimulate cell invasion in the SKOV3 ovarian cancer cell line. This lack of obestatin-stimulated invasion in the SKOV3 cell line may be a cell line specific result. In this study, obestatin did not stimulate cell proliferation in the ovarian cell lines and it has previously been shown to have no effect on cell proliferation in the BON-1 pancreatic neuroendocrine and GC rat somatotroph tumour cell lines. In contrast, obestatin has been shown to affect cell proliferation in gastric and thyroid cancer cell lines, and in some normal cell lines. Obestatin also had no effect on attachment of any of the cell lines to any of the ECM components tested (fibronectin, vitronectin, collagen I and collagen IV). The mechanism of action of obestatin was investigated further using a two dimensional-difference in gel electrophoresis (2D-DIGE) proteomic approach. After treatment with obestating (0, 10 and 100 nM), SKOV3 ovarian cancer and hOSE 17.1 normal ovarian cell lines were collected and 2D-DIGE analysis and mass spectrometry were performed to identify proteins that were differentially expressed in response to treatment. Twenty-six differentially expressed proteins were identified and analysed using Ingenuity Pathway Analysis (IPA). This linked 16 of these proteins in a network. The analysis suggested that the ERK1/2 MAPK pathway was a major mediator of obestatin action. ERK1/2 has previously been shown to be associated with obestatin-stimulated cell proliferation and with the anti-apoptotic effects of obestatin. Activation of the ERK1/2 signalling pathway by obestatin was, therefore, investigated in the SKOV3 and OV-MZ-6 ovarian cancer cell lines using anti-active antibodies and Western immunoblots. Obestatin treatment significantly decreased ERK1/2 phosphorylation at higher obestatin concentrations in both the SKOV3 (100 nM and 1000 nM) and OV-MZ-6 (1000 nM) cell lines compared to the untreated controls. Currently, very little is known about obestatin signalling in cancer. This thesis has demonstrated for the first time that the ghrelin axis may play a role in ovarian cancer migration. Ghrelin and obestatin increased cell migration in ovarian cancer cell lines, indicating that they may be a useful target for therapies that reduce ovarian cancer progression. Further studies investigating the role of the ghrelin axis using in vivo ovarian cancer metastasis models are warranted.

Building project competence : developing dynamic capabilities with knowledge governance mechanisms

This research investigates the development of project competence, and particularly, three related dynamic capabilities (shifting, adapting, leveraging) that contribute to project competence development. In doing so, we make use of the emerging literature on knowledge governance and theorize how knowledge governance mechanisms can move the organization towards desired knowledge-based goals. A multiple-case study comprising 23 cases advances our understanding of the elements that trigger, enable, hamper, and drive shifting, leveraging and adapting. Finally, the paper offers a tentative framework of dynamic capability building promoting project competence development.

Polyalanine repeat polymorphism in RUNX2 is associated with site-specific fracture in post-menopausal females

Runt related transcription factor 2 (RUNX2) is a key regulator of osteoblast differentiation. Several variations within RUNX2 have been found to be associated with significant changes in BMD, which is a major risk factor for fracture. In this study we report that an 18bp deletion within the polyalanine tract (17A>11A) of RUNX2 is significantly associated with fracture. Carriers of the 11A allele were found to be nearly twice as likely to have sustained fracture. Within the fracture category, there was a significant tendency of 11A carriers to present with fractures of bones of intramembranous origin compared to bones of endochondral origin (p=0.005). In a population of random subjects, the 11A allele was associated with decreased levels of serum collagen cross links (CTx, p=0.01), suggesting decreased bone turnover. The transactivation function of the 11A allele was quantitatively decreased. Interestingly, we found no effect of the 11A allele on BMD at multiple skeletal sites, although these were not the sites where a relationship with fracture was most evident. These findings suggest that the 11A allele is a biologically relevant polymorphism that influences serum CTx and confers enhanced fracture risk in a site-selective manner related to intramembranous bone ossification.

Mathematical modelling of soft callus formation in early murine bone repair

Fracture healing is a complicated coupling of many processes. Yet despite the apparent complexity, fracture repair is usually effective. There is, however, no comprehensive mathematical model addressing the multiple interactions of cells, cytokines and oxygen that includes extra-cellular matrix production and that results in the formation of the early stage soft callus. This thesis develops a one dimensional continuum transport model in the context of early fracture healing. Although fracture healing is a complex interplay of many local factors, critical components are identified and used to construct an hypothesis about regulation of the evolution of early callus formation. Multiple cell lines, cellular differentiation, oxygen levels and cytokine concentrations are examined as factors affecting this model of early bone repair. The model presumes diffusive and chemotactic cell migration mechanisms. It is proposed that the initial signalling regime and oxygen availability arising as consequences of bone fracture, are sufficient to determine the quantity and quality of early soft callus formation. Readily available software and purpose written algorithms have been used to obtain numerical solutions representative of various initial conditions. These numerical distributions of cellular populations reflect available histology obtained from murine osteotomies. The behaviour of the numerical system in response to differing initial conditions can be described by alternative in vivo healing pathways. An experimental basis, as illustrated in murine fracture histology, has been utilised to validate the mathematical model outcomes. The model developed in this thesis has potential for future extension, to incorporate processes leading to woven bone deposition, while maintaining the characteristics that regulate early callus formation.

Sintering and densification mechanisms of Y2BaCuO5 pellets

The sintering and densification of Y2BaCuO5 (Y-211) pellets made from powders with different characteristics have been investigated in the temperature range 1000-1140°C. A pellet made from powder containing Ba-rich secondary phases shows very early liquid-assisted sintering and densification and clear evidence of exaggerated grain growth. The melting of BaCuO2 and YBa2Cu3O7-δ (Y-123) secondary phases increases the rate of densification of Y-211 pellets made from other powders at temperatures above 1025-1030°C. All the liquid produced by the melting of the latter phases recrystallizes as intergranular layers of Y-123. These intergranular layers account for the darker appearance and for measurable electrical conductivities at room temperature of the pellets sintered at the higher temperatures. The development of exaggerated grain growth within a uniform fine-grained matrix opens the possibility of using controlled secondary recrystallization to obtain large single domains of Y-211, provided that the trapping of porosity can be avoided or minimized. © 1999 Elsevier Science S.A.

Improving in vivo models of fracture fixation associated osteomyelitis

This project’s aim was to create new experimental models in small animals for the investigation of infections related to bone fracture fixation implants. Animal models are essential in orthopaedic trauma research and this study evaluated new implants and surgical techniques designed to improve standardisation in these experiments, and ultimately to minimise the number of animals needed in future work. This study developed and assessed procedures using plates and inter-locked nails to stabilise fractures in rabbit thigh bones. Fracture healing was examined with mechanical testing and histology. The results of this work contribute to improvements in future small animal infection experiments.

Local plastic mechanisms in thin steel plates under in-plane compression

Thin-walled steel plates subjected to in-plane compression develop two types of local plastic mechanism, namely the roof-shaped mechanism and the so-called flip-disc mechanism, but the intriguing question of why two mechanisms should develop was not answered until recently. It was considered that the location of first yield point shifted from the centre of the plate to the midpoint of the longitudinal edge depending on the b/t ratio, imperfection level, and yield stress of steel, which then decided the type of mechanism. This paper has verified this hypothesis using analysis and laboratory experiments. An elastic analysis using Galerkin's method to solve Marguerre's equations was first used to determine the first yield point, based on which the local plastic mechanism/imperfection tolerance tables have been developed which give the type of mechanism as a function of b/t ratio, imperfection level and yield stress of steel. Laboratory experiments of thin-walled columns verified the imperfection tolerance tables and thus indirectly the hypothesis. Elastic and rigid-plastic curves were them used to predict the effect on the ultimate load due to the change of mechanism. A finite element analysis of selected cases also confirmed the results from simple analyses and experiments.

A novel iterative method for simulating patient-specific optimal deformation and fit of fracture fixation plates

This paper proposes a new iterative method to achieve an optimally fitting plate for preoperative planning purposes. The proposed method involves integration of four commercially available software tools, Matlab, Rapidform2006, SolidWorks and ANSYS, each performing specific tasks to obtain a plate shape that fits optimally for an individual tibia and is mechanically safe. A typical challenge when crossing multiple platforms is to ensure correct data transfer. We present an example of the implementation of the proposed method to demonstrate successful data transfer between the four platforms and the feasibility of the method.

Comparative study of failure mechanisms in steel and concrete members strengthened with CFRP composites

Over the last decade advanced composite materials, like carbon fibre reinforced polymer (CFRP), have increasingly been used in civil engineering infrastructure. The benefits of advanced composites are rapidly becoming evident. This paper focuses on the comparative performance of steel and concrete members retrofitted by carbon fibre reinforced polymers. The objective of this work is a systematic assessment and evaluation of the performance of CFRP for both the concrete and steel members available in the technical literature. Existing empirical and analytical models were studied. Comparison is made with respect to failure mode, bond characteristics, fatigue behaviour, durability, corrosion, load carrying capacity and force transfer. It is concluded that empirical expressions for the concrete-CFRP composite are not readily suited for direct use in the steel-CFRP composite. This paper identifies some of the major issues that need further investigation.

Multiscale investigation of microfilament networks disruption in living cells

Actin is the most abundantly distributed protein in living cells which plays critical roles in the cell interior force generation and transmission. The fracture mechanism of microfilament networks, whose principle component is actin, would provide insights which can contribute to the understandings of self-protective characters of cytoskeleton. In this study, molecular simulations are conducted to investigate the molecular mechanisms of disruption of microfilament networks from the viewpoint of biophysics. By employing a coarse-grained (CG) model of actin filament networks, we focused on the ultimate strength and crack growth mode of microfilament networks that have dependency on the crack length. It can be found that, the fracture mechanism of microfilament network has dependency on the structural properties of microfilament networks. The structure flaws marginally change the strength of microfilament networks which would explain the self-protective characters of cytoskeleton.

Multiple types of data are required to identify the mechanisms influencing the spatial expansion of melanoma cell colonies

Background The expansion of cell colonies is driven by a delicate balance of several mechanisms including cell motility, cell-to-cell adhesion and cell proliferation. New approaches that can be used to independently identify and quantify the role of each mechanism will help us understand how each mechanism contributes to the expansion process. Standard mathematical modelling approaches to describe such cell colony expansion typically neglect cell-to-cell adhesion, despite the fact that cell-to-cell adhesion is thought to play an important role. Results We use a combined experimental and mathematical modelling approach to determine the cell diffusivity, D, cell-to-cell adhesion strength, q, and cell proliferation rate, ?, in an expanding colony of MM127 melanoma cells. Using a circular barrier assay, we extract several types of experimental data and use a mathematical model to independently estimate D, q and ?. In our first set of experiments, we suppress cell proliferation and analyse three different types of data to estimate D and q. We find that standard types of data, such as the area enclosed by the leading edge of the expanding colony and more detailed cell density profiles throughout the expanding colony, does not provide sufficient information to uniquely identify D and q. We find that additional data relating to the degree of cell-to-cell clustering is required to provide independent estimates of q, and in turn D. In our second set of experiments, where proliferation is not suppressed, we use data describing temporal changes in cell density to determine the cell proliferation rate. In summary, we find that our experiments are best described using the range D = 161 - 243 ?m2 hour-1, q = 0.3 - 0.5 (low to moderate strength) and ? = 0.0305 - 0.0398 hour-1, and with these parameters we can accurately predict the temporal variations in the spatial extent and cell density profile throughout the expanding melanoma cell colony. Conclusions Our systematic approach to identify the cell diffusivity, cell-to-cell adhesion strength and cell proliferation rate highlights the importance of integrating multiple types of data to accurately quantify the factors influencing the spatial expansion of melanoma cell colonies.