A Mechanistic Investigation of the Relationship Between Extramural Vascular Invasion (EMVI) and CpG Island Methylator Phenotype (CIMP) in Rectal Cancer

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer death worldwide. Each year, one million people will develop CRC, and 40-50% will die within five years. Furthermore, rectal and distal sigmoid cancers are known to present at a later stage and have a poorer prognosis than other colonic cancers. Rectal cancers that demonstrate pathological extramural vascular invasion (EMVI-positive) are known to have a poorer prognosis than those that do not (EMIV-negative), and EMVI has is acknowledged as an important risk factor for systemic recurrence, local recurrence and death. Additionally, EMVI status influences the need for pre- and post-operative chemoradiation (CRT); which may influence survival outcomes. Aberrant DNA methylation is emerging as a carcinogenic mechanism and potential biomarker in colorectal cancer. This study investigates the association between hypermethylation and EMVI in vivo and in vitro. Firstly, the in vivo associations between hypermethylation, EMVI, and clinical and histopathological outcomes are examined. Secondly, an investigation of the effects of demethylation on invasive colorectal cell lines in vitro aims to illuminate the genetic and cellular mechanisms that underlie methylation-dependent pathological cellular behaviour. Finally, highlighted biologic mechanisms are investigated in vivo to discover if there is an association with EMVI and survival outcomes. By these means the axis of association between hypermethylation, EMVI, and clinical outcomes is investigated. The investigation is conducted within the framework of consensus molecular subtyping in colorectal cancer, and in concordance with current methodologies of assessing DNA methylation status. The primary findings demonstrate that EMVI is associated with hypermethylation in vivo, but that there is no direct correlation between hypermethylation and disease-free (DFS) or overall survival (OS). In vitro, demethylation of hypermethylated colorectal cancer cells, by means of established demethylating agent 5-azacytidine and putative demethylator RRx-001, reduces their propensity to migrate and invade. Demethylation in vitro is also associated with changes in the expression of the metalloproteinases involved in the metabolism of the basement membrane and the epithelial-to-mesenchymal transition and tumour metastasis, notably MMP2 and TIMP2. Changes in expression were confirmed at transcriptomal and proteomic levels in response to demethylation. In vivo, MMP2 expression was shown to be statistically significantly associated with EMVI, DFS, and OS, and was also independently predictive of EMVI, raising the possibility that it could act as a diagnostic and predictive biomarker in rectal cancers. These findings indicate a mechanistic association between hypermethylation and EMVI, mediated by methylation-dependent expression of metalloproteinases. Metalloproteinase expression, specifically MMP2, may act as a potential biomarker for EMVI and correlates to survival outcomes in rectal cancer

Investigating the functional role of SMOC-1 in zebrafish

True anophthalmia is the most severe congenital eye malformation. With absence of the eye, optic nerve, chiasm and optic tracts. Identifying the genes that cause genetic true anophthalmia should improve our understanding of the critical processes required for development of the eye. Recessive loss-of-function mutations in SMOC1 have been identified as the cause of Ophthalmo-acromelic syndrome (OAS), a multisystem disorder which has true anophthalmia as a prominent feature with characteristic limb and facial malformations. In order to establish the function of SMOC1 in development I used the zebrafish as a model organism to support a link between SMOC-1 and BMP signalling. As a first step I characterised the genomic structure of zebrafish smoc1 gene. I was able to correct an error in the zebrafish genome (Zv8) that annotated zsmoc1 as two fragmented and rearranged orthologous loci. However, using RTPCR I could show that there is in fact a single intact zsmoc1 transcript. In addition, I was able to identify an un-annotated 5’ coding exon using 5' RACE which showed that the full open reading frame includes a signalling peptide. RT-PCR was also used to identify several novel zsmoc1 splice isoforms. To explore the link between zsmoc1 and bmp signalling I used injection of antisense morpholino oligonucleotide and capped mRNA to examine the effects of loss-of-function and overexpression respectively of smoc1 and genes functioning in the bmp signalling pathway. The resulting embryos were analysed using morphometric analysis (Kishimoto scale), a quantitative assay of dorsalisation/ventralisation and live imaging of reporter transgenic fish. I developed a quantitative RT-PCR assay for expression of dorsal (otx2 and runx3) and ventral (eve1 and gata2) marker genes. I established a reliable system for live imaging of zebrafish development between 8 hpf and 24 hpf. By combining this system with fluorescent transgenic reporters marking the eye field (rx3:gfp reporter) and BMP-signaling (BRE:gfp reporter) I was able to accurately quantitate the effect of smoc1 depletion on eye size and SMAD1/5/8 signalling in the eye. These results support the predictions from the Drosophila homologue Pent that zsmoc1 functions as an antagonist of bmp signalling. Finally, I describe my attempt to produce a zebrafish model for OAS using genome editing technology. I designed, produced and validated transcription activator like effectors nucleases (TALENs) targeted to the zsmoc1 open reading frame using the Voytas Goldengate method. I designed and optimised a novel strategy to demonstrate targeted cutting activity for in vitro validation. Following injections of the in vitro validated TALEN into zebrafish embryos I used Ion Torrent sequencing to assess the in vivo activity of the engineered TALEN pairs. Unfortunately these TALENs were not able to cut the targeted locus in vivo

Craft Sciences

The field of ‘Craft Sciences’ refers to research conducted across and within different craft subjects and academic contexts. This anthology aims to expose the breadth of topics, source material, methods, perspectives, and results that reside in this field, and to explore what unites the research in such diverse contexts as, for example, the arts, conservation, or vocational craft education. The common thread between each of the chapters in the present book is the augmented attention given to methods—the craft research methods—and to the relationship between the field of inquiry and the field of practice. A common feature is that practice plays an instrumental role in the research found within the chapters, and that the researchers in this publication are also practitioners. The authors are researchers but they are also potters, waiters, carpenters, gardeners, textile artists, boat builders, smiths, building conservators, painting restorers, furniture designers, illustrators, and media designers. The researchers contribute from different research fields, like craft education, meal sciences, and conservation crafts, and from particular craft subjects, like boat-building and weaving. The main contribution of this book is that it collects together a number of related case studies and presents a reflection on concepts, perspectives, and methods in the general fields of craft research from the point of view of craft practitioners. It adds to the existing academic discussion of crafts through its wider acknowledgement of craftsmanship and extends its borders and its discourse outside the arts and crafts context. This book provides a platform from which to develop context-appropriate research strategies and to associate with the Craft Sciences beyond the borders of faculties and disciplines

Environmental, genetic and epigenetic contributors to the risk of bipolar disorder

Bipolar disorder (BD) is a complex mental illness for which both environmental and biological (genetic and epigenetic) factors play an aetiological role. This thesis aimed to find factors behind risk of psychopathology, using a multi-site cohort with parents or siblings with BD (high-risk; HR) and offspring of healthy control parents. My aim was to explore the trajectory towards development of BD and related psychopathology, guided by the hypothesis that DNA methylation (DNAm), a key epigenetic process, may provide a molecular link of relevant gene-environment interactions. Firstly, association of stressful life events and family environment with clinical status was investigated. Next, blood-derived DNAm of a subset with available genotype data (n=415) was used to investigate epigenetic signals. Epigenome-wide association studies were used to identify differentially methylated CpG sites based on familial risk and emergent clinical status, and polygenic risk. Finally, methylation-derived markers, indexing C-reactive protein (CRPm) and neutrophil-to-lymphocyte ratio (NLRm), were used to investigate the influence of inflammatory processes in the development of psychopathology. Stressful life events and an unbalanced family environment increase risk of emergent psychopathology, providing targets for early intervention. Nominally differentially methylated CpG sites that were related to family history, clinical transition and polygenic risk showed enrichment in neurological pathways and functions. Family environment influenced DNAm signatures of clinical transition, and methylation profile scores showed partial prediction of clinical transition in independent validation. The influence of polygenic risk scores for BD revealed wide-ranging nominal effects on DNAm signatures and an epigenome-wide significant signal at mitochondrial gene VARS2, within the major histocompatibility complex (MHC) region. The relationship between DNAm signatures and polygenic background was independently validated. While the role of inflammation on psychopathology was not strong, CRPm could potentially predict psychopathology in HR individuals, and a trend of higher NLRm and CRPm was evident in HR with suicidal ideation. My findings showed that epigenetic processes are impacted by both familial background and genetic predisposition, may reflect underlying environmental exposures, and influence emergent psychopathology, providing insights into biological dysregulation leading to BD

Putting reaction-diffusion systems into port-Hamiltonian framework

Reaction-diffusion systems model the evolution of the constituents distributed in space under the influence of chemical reactions and diffusion [6], [10]. These systems arise naturally in chemistry [5], but can also be used to model dynamical processes beyond the realm of chemistry such as biology, ecology, geology, and physics. In this paper, by adopting the viewpoint of port-controlled Hamiltonian systems [7] we cast reaction-diffusion systems into the portHamiltonian framework. Aside from offering conceptually a clear geometric interpretation formalized by a Stokes-Dirac structure [8], a port-Hamiltonian perspective allows to treat these dissipative systems as interconnected and thus makes their analysis, both quantitative and qualitative, more accessible from a modern dynamical systems and control theory point of view. This modeling approach permits us to draw immediately some conclusions regarding passivity and stability of reaction-diffusion systems. It is well-known that adding diffusion to the reaction system can generate behaviors absent in the ode case. This primarily pertains to the problem of diffusion-driven instability which constitutes the basis of Turing’s mechanism for pattern formation [11], [5]. Here the treatment of reaction-diffusion systems as dissipative distributed portHamiltonian systems could prove to be instrumental in supply of the results on absorbing sets, the existence of the maximal attractor and stability analysis. Furthermore, by adopting a discrete differential geometrybased approach [9] and discretizing the reaction-diffusion system in port-Hamiltonian form, apart from preserving a geometric structure, a compartmental model analogous to the standard one [1], [2] is obtaine