The evaluation of the remodeling of soft biomaterial implants often involves surgical removal of the implant for subsequent histological assessment. This approach is very resource intensive, often destructive, and imposes practical limitations on how effectively these materials can be evaluated. Magnetic resonance imaging (MRI) has the potential to non-invasively monitor the remodeling of collagen sponges, specifically the biodegradation, cellular infiltration, extracellular matrix deposition and angiogenesis within the sponge. This project involves the development of an in vivo model system for the evaluation of collagen-sponge remodeling using MRI and conventional histological techniques. Collagen sponges made using insoluble bovine collagen, and subjected various crosslinking treatments, were implanted subcutaneously into rats. Changes in water T2 relaxations times, water apparent diffusion coefficients (ADC), and MRI contrast agent uptake/washout were collected using spin-echo and diffusion-weighted MRI pulse sequences. These measurements were compared with histological assessments of sponge remodeling. Regions of differential cellularity were distinguished using calculated T2 maps and confirmed by histology. Calculated ADC maps corroborated these results and showed a decreasing trend with increased tissue in-growth. Results from MRI-contrast-agent studies were consistent with the development of angiogenesis within the sponge over time. The MRI approach allows for longitudinal studies that significantly reduce the resources required to evaluate these materials as well as improves the quality of the statistical information obtained from these studies.

The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

Blood vessels are dynamic structures whose properties are continuously adapted by resident vascular cells. Existing mechanobiological models tend to ignore regulatory signalling and cell population dynamics, both key determinants of arterial growth and remodelling (G&R). In this D.Phil., a combined theoretical, experimental and computational approach is used to formulate, refine and implement a novel model of the arterial wall that includes vascular mechanics, microstructure, biochemical metabolism and signalling, and cell phenotype and population dynamics. A mathematical chemo-mechano-biological (CMB) model is formulated by coupling a biomechanical model of the arterial wall as a cylindrical nonlinear elastic membrane to a system of biologically-informed evolution laws governing fibroblast cell-mediated, transforming growth factor (TGF)-β-regulated collagen metabolism. Model simulation of inflammatory aneurysm development suggests that increasing TGF-β levels promotes a cell-driven profibrotic response leading to aneurysm stabilisation, illustrating the model's ability to couple chemo-biological processes to tissue-level mechanical evolution. To inform the theoretical framework experimentally, a recent mouse model of post-developmental disruption of medial smooth muscle TGF-β signalling is for the first time subjected to hypertension, and characterised by biaxial mechanical testing and (immuno)histological staining. Increased adventitial TGF-β levels following perturbation are associated with strong profibrotic responses (increased cellularity, collagen deposition, thicker walls) altering tissue mechanics (lower biaxial stress, higher structural stiffness). Simulation of realistic arterial geometries is enabled by coupling the 1D CMB model to a three-dimensional structural solver. Heterogeneous spatial distributions of mechanical, microstructural and chemo-biological variables determining the evolution of complex saccular aneurysm geometries can be simulated with this 3D implementation. A novel chemo-mechano-biological model of vascular cell dynamics and regulatory signalling governing arterial G&R is formulated, informed by specifically-generated experimental data, and implemented in an advanced 3D computational framework. This will allow for virtual investigation of therapies acting on chemo-biological agents of arterial G&R, with potential benefits for vascular disease patients.

Inspired by biological membrane shaping in the cell through means of curvature-inducing proteins, we investigate the interplay between membrane curvature and the distribution and movement of shape-inducing objects which are free to move as a consequence of the underlying shape. We initially study the self-assembly of a filament, taken as a proxy for the cross-section of a biomembrane, which is primarily driven by the chemical kinetics of attaching proteins and find that, under certain mechanical stiffness regimes of the attaching proteins, pattern formation occurs. Regions of high and low protein concentration form before spatially uniform filament shapes are obtained by means of protein adhesion and movement governed by diffusion and local curvature-seeking. However, noting that the curvature-mediated protein movement on membranes has been biologically observed to be long-range, we next study the self-assembly of embedded inclusions on a membrane as a result of the underlying geometry. We first derive an interaction law for the shape-mediated interaction of inclusions which break symmetry and find that there is a finite equilibrium distance to which the inclusions will aggregate. We derive corresponding equations of motion which describe this curvature-mediated aggregation mechanism and, using this framework, we investigate some of the properties of these self-assembled configurations, including their energy, stability, and their collective elastic behavior. Lastly, we consider the interaction energies of embedded inclusions on a periodic domain and determine that this mechanism may explain computational results of how proteins form rings to promote tubulation on cylindrical membranes.

Osteoarthritis (OA) is the most common joint disease in human populations with onset and severity influenced by mechanical loading, aging effects, genetics, anatomy, and body mass. Despite major advancements in knowledge, the aetiopathogenesis of OA is complex and still poorly understood. Lack of standardization in methods to quantify skeletal OA make it difficult to study the effects of interacting explanatory variables on arthritic response, and prevents comparison of results between bioarchaeological studies. Joint changes of OA as a function of both the natural aging process and of mechanical stress can make an individual appear older than their chronological age, potentially impacting current methods to derive accurate skeletal age at death estimates, particularly in load-bearing regions. This project addressed these issues through three studies, using a large skeletal sample of modern Europeans for which sex, age, and occupation were available. The first study used principal component analysis (PCA) as a standardized procedure to compute aggregate scores for joint complexes and a systemic measure of OA in each region of the lumbar spine, pelvis, and knee. The second study analyzed the composite scores with a multiple regression model to determine the relative contribution of three predictors: age, activity, and body size, and their effect on skeletal expression of OA in each region. Body size (stature and mass) was calculated from postcranial skeletal measurements; torsional strength (J) of the femoral midshaft was calculated from three-dimensional surface models, size standardized and used as a proxy for measure of activity. The third study considered the effect of OA severity on the validity and reliability of three methods to estimate age at death from load-bearing joints of the os coxa: the pubic symphysis, auricular surface, and acetabulum. The study was designed to determine whether OA in adults acts as a potential limitation or benefit in deriving accurate skeletal age at death estimates from pelvic joint morphology that will contribute to standardized methods in establishing physiological degeneration of the skeleton due to aging. Body size and activity factors did not contribute significantly to OA pathology outside of the age-related expression in either of the lumbar vertebrae or knee regions, and only demonstrated a weak association at pelvic joints. Differences in adult patterns of age are reflected in joint arthritic changes of the os coxa and OA severity has an effect on the accuracy of age estimates from the pelvis; those with OA consistently aging faster in all three joint areas. This influence is most significant for young individuals at the auricular surface and pubic symphysis, over-aging at both. Oldest persons with little arthritic patterning at the acetabulum were under-aged, but accuracy of the age estimate improved as OA severity increased. Systemic measures of OA determined through PCA as an indicator of age, appear useful to identify the very old, but may also help to distinguish between systemic age-related stresses and localized biomechanical effects. Interpreting OA as evidence for old age, measures of habitual activity, and larger body mass should be exercised with caution in skeletal populations. / Graduate / 2018-04-18 / 0327 / 0339 / 0571 / calce.stephanie@gmail.com

Le spermatozoïde des animaux à reproduction sexuée est une cellule extrêmement spécialisée, dont la chromatine très particulière est le siège de nombreux remodelages tant lors de la gamétogenèse que lors de la formation du zygote. Chez D. melanogaster comme chez les mammifères, lors de la spermiogenèse, les histones qui condensent l'ADN sont remplacées par des petites protéines basiques spécifiques du noyau spermatique : les protamines. Cette architecture est stabilisée par des liaisons disulfures. Lors de la fécondation, ces protéines sont éliminées du noyau paternel, qui réincorporent des histones pour former une chromatine fonctionnelle. Toutefois, les mécanismes régissant la mise en place et l'enlèvement des ponts disulfures et des protamines sont inconnus chez la Drosophile.Au cours de ma thèse, j'ai démontré l'importance de deux thiorédoxines sexuelles pour la reproduction.D'une part, j'ai pu montrer que DHD, qui est une thiorédoxine strictement maternelle, est essentielle à l'éviction des protamines de la chromatine paternelle lors de la fécondation. Sans cette protéine essentielle, la décondensation du noyau mâle n'a pas lieu, les protamines ne sont pas enlevées et le développement zygotique ne peut pas avoir lieu. Cette thiorédoxine est directement responsable de la réduction des liaisons disulfures qui stabilisent la chromatine spermatique.D'autre part, j'ai démontré que TrxT, une thiorédoxine exclusivement testiculaire, est nécessaire au bon déroulement de la spermiogenèse. Sans cette protéine, les spermatides subissent des dommages à l'ADN et sont éliminées.Ce travail met en évidence les rôles essentiels des thiorédoxines sexuelles pour la reproduction / In animal sexual reproduction, spermatozoon is a very specialized cell. Its very peculiar chromatin is remodeled both during spermiogenesis and fertilization. During mammalian and drosophilian spermiogenesis, histones involved in DNA condensation are replaced with sperm specific small nuclear basic proteins : the protamines. This sperm specific architecture is stabilized by disulfide bonds. At fertilization,protamines are removed from the male nucleus and maternally-provided histones are incorporated to form a functional paternal chromatin. However, the mecanisms involved in the incorporation and the removal of protamines of their disulfide bonds are unknown in Drosophila.During my PhD, I demonstrated that two sexual thioredoxins are important for spermiogenesis and fertilization in D. melanogaster. In one hand, I showed that DHD, a female specific thioredoxin, is essential for protamine eviction at fertilization. Without this major protein, male nucleus does not decondense, protamines are not removed from sperm chromatin and zygotic development does not occur. Besides, I demonstrated that DHD is directly responsible for the reduction of the disufide bonds which stabilize sperm chromatin.On the other hand, I showed that TrxT, a testis-specific thioredoxin, is needed for spermiogenesis. Without this protein, DNA damages appear on spermatid nuclei, and those spermatozoon are then eliminated during spermatogenesis.This work highlights that drosophilian sex-specific thioredoxins are essential for sexual reproduction success

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Angiotensin II signals cardiovascular disease through metalloproteinases including MMP-2, MMP-7 and ADAM-17/TACE. We hypothesized that these metalloproteinases regulate each other at the transcriptional level. Further, MMP-2, being a major gelatinase in cardiac and vascular tissue, could mediate angiotensin II-induced cardiovascular disease. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography and qRT-PCR analysis of hypertrophy marker genes) and fibrosis (by collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. Angiotensin II induced hypertension, cardiac hypertrophy and fibrosis which correlated with an upregulation of MMP-2. Downregulation of MMP-2 by pharmacological inhibition and RNA interference attenuated hypertension but not cardiac hypertrophy or fibrosis. Downregulation of MMP-7 or ADAM-17/TACE by RNA interference attenuated angiotensin II-induced MMP-2 upregulation as well as hypertension, cardiac hypertrophy and fibrosis. We conclude that MMP-2 selectively mediates angiotensin II-induced hypertension under the transcriptional control of MMP-7 and ADAM-17/TACE.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.