Obesity and associated comorbidities such as diabetes are currently major global health problems. It has been shown that individual sites or depots of fat have different properties and effects on whole body physiology. Of particular interest are depots that display thermogenic characteristics, known as brown or beige adipose tissue, which may be able to increase energy expenditure and clear excess glucose and lipids from the circulation. The aim of this thesis was to compare adipose tissue depots in terms of their capacity for thermogenesis in early life using sheep as an animal model and browning response to chronic adrenergic stress in humans with pheochromocytoma. Epicardial and paracardial adipose tissues were focused on in the sheep study as they have been described as thermogenic in some adult humans but these findings have been inconclusive. The characterisation of these depots and their capacity for thermogenesis in early life has not been established, especially in comparison to other known depots of white and brown adipose tissue. My results show that epicardial and paracardial adipose tissue have increased thermogenic gene expression and uncoupling protein 1 in comparison to omental white adipose tissue suggesting they may play a role in thermogenesis in early life. These depots undergo remodelling over the first 28 days changing from brown to white adipose tissue. In sheep, this adipose tissue transition occurs without evidence of apoptosis and which may suggest other cellular mechanisms are responsible for this process. The browning capacity of different fat depots is important for targeting therapeutic interventions. As a model of catecholamine excess, samples of human periadrenal and subcutaneous adipose tissue were collected from patients with pheochromocytoma as well as patients undergoing adrenalectomy for benign tumours (control group). The data suggests periadrenal adipose tissue can undergo browning in some patients with pheochromocytoma. These patients had the highest plasma catecholamine concentrations suggesting a possible threshold is required to elicit browning. Subcutaneous adipose tissue did not display signs of browning suggesting that this depot does not respond to chronic adrenergic stress. It may therefore have a lack of browning capacity compared to visceral depots. In conclusion, both studies suggest differences between adipose tissue depots in thermogenic capacity both in development in early life and capacity for browning in later life.

Articular cartilage in synovial joints can become damaged due to mechanical loading, trauma or wear and tear. The initiation and progression of damage in cartilage may lead to degenerative changes of the joint. However, links between mechanical loading and the initiation/progression of damage in cartilage remain poorly understood. In this thesis, the damaging effects of loading frequencies representative of normal (1 Hz), above normal (10Hz) and rapid heel-strikes (100Hz) on cartilage/cartilage-on-bone were assessed and compared to test the hypothesis that failure can be influenced by frequency. Bovine cartilage was used as a model for human cartilage. Materials testing machines were used to apply sinusoidally varying loads at different frequencies and altered maximum forces under different loading types. A metal indenter was used to apply cyclic loading on cartilage-on-bone specimens to produce failure on the surface of cartilage-on-bone specimens in compression. Fatigue failure of cartilage-on-bone specimens were determined using cyclic three-point bending. Propagation of an initial crack across the area of cartilage specimens with respect to increasing number of loading cycles were measured and compared under tension. The results from this thesis indicated that failure increases significantly (p < 0.05) in cartilage-on-bone specimens with increasing the loading frequency under compression and bending. Strain experienced by the cartilage specimens at higher frequency, e.g. 100 Hz, caused a greater crack growth under tension. The results from this work have many potential implications in the early onset of osteoarthritis. This is because rapid heel-strike rise times have been implicated in the early onset of osteoarthritis.

The prevalence of muscle dysfunction in elderly populations represents a significant burden on healthcare due to the increased risk of injury, and difficulty in maintaining activities of daily living. This thesis describes the application of advanced computational techniques designed to “learn” the structure of molecular networks to understanding human skeletal muscle ageing. Using this approach we have been able to discover a link between protein translation and age-dependent metabolic reprogramming. Experimental validation using the haploinsufficient eukaryotic initiation factor 6 (eIF6) mouse confirmed this important hypothesis and revealed a substantial molecular reprogramming. The role of eIF6 in skeletal muscle and myoblasts was further investigated, revealing potential up and down-stream signalling mechanisms. The process of angiogenesis is an important step in morphogenesis including systems as diverse as muscle regeneration and tumour growth. This thesis presents the first temporal model of transcriptional alterations in tumour and the surrounding stroma during vascularisation. The application of reverse engineering approaches that are able to integrate perturbation data lead to the hypothesis that modulation of the pro-inflammatory cytokine IL1α may be an important upstream event in angiogenesis.

This thesis investigates the effect of various physical and chemical surface modification methods on the permeation of topically applied pharmaceutical compounds through poly(dimethylsiloxane) (PDMS), a polymer frequently employed as a model barrier in in vitro skin permeation studies. Such studies are essential for safety, risk assessment, and quality control purposes, in addition to assisting in the design and development of efficacious topically applied medicines. The commercial availability, legal status, ease of handling, and the reproducibility of the permeation data associated with polymeric skin mimics renders them an attractive alternative to biological tissue. However, over-predictions of percutaneous absorption observed following the use of such membranes are a significant disadvantage when attempting to obtain quantitative toxicological data. Accordingly, the aims of the work presented in this thesis were to both reduce the permeability of PDMS to pharmaceutical compounds, and to increase correlation between permeation data obtained using the synthetic substitute and data obtained similarly using suitable biological tissue. Primarily, the potential of an air plasma pre-treatment to produce a lamellae-type structure in PDMS, endeavouring to more accurately model the architectural, physical, and chemical properties of the human stratum corneum, was investigated. Reductions in the permeability coefficient of up to 54.4 % were observed, rendering the modified system promising. Correlation analysis revealed an increase in correlation between the data collected using the modified synthetic substitute (R 2 = 0.86) and a selfcollated library of literature-derived epidermal tissue permeability data, relating to eighteen compounds and spanning a range of typical penetrants, compared to similar analysis using data obtained using the native substitute ( R 2 = 0.75), suggesting an increase in the predictive capability. It was hypothesised that an N2 plasma treatment may provide suitable surface functional groups on the PDMS substrate, namely amine groups, for the covalent attachment of biomolecules via an N,N'- dicylohexylcarbodiimide (DCC) coupling reaction, enabling the production of a skin mimic displaying enhanced biorelevance. Therefore, the effect of an N2 plasma pre-treatment on the permeation of a subset of the eighteen compounds investigated. It was found that the N2 plasma pre-treatment was advantageous in terms of offering a greater reduction in permeability, since longer treatment times could be employed i.e. reductions of up to 61.8 % were observed. However, significant surface oxidation was still observed, with only a marginal increase in nitrogen containing functionalities compared with the air plasma analogue i.e. 0.31 %. Furthermore, the treatment did not offer any additional increase in correlation between epidermal-derived data than previously observed. Further chemical methods of biomolecule attachment were pursued for use in the development of a lipidproteinaceous bilayer model, initiated in both cases by surface amination using an alkoxysilane. This was followed by a DCC coupling to an amino acid in the former approach, and use of a glutaraldehyde III linker molecule to attach the same amino acid, namely lysine, in the latter approach. In either case, no further reductions in the permeation of the pharmaceutical compounds tested were observed, with respect to that through plasma treated PDMS. In summary, the air plasma treatment of PDMS was found to be a promising approach to simultaneously reducing the permeability of a silicone skin mimic and increasing correlation with data obtained in similar studies employing biological tissue. Further, the covalent coupling of biomolecules to the surface of PDMS following surface amine group generation, via both plasma and wet chemical methods, appeared not to compromise the integrity the PDMS membranes relating to such applications, rendering the techniques compatible with the production of biorelevant semi-synthetic skin mimics.

The character of the functional output of a motor unit within skeletal muscle has been linked experimentally to the proteins found in the sarcomere, the smallest contractile unit of muscle fibre. Current mathematical models focus on either individual chemical reactions or the bulk properties of muscle with limited reference to the internal processes and structures within the muscle. Without an understanding of those internal properties, the normal function of muscle cannot be simulated and consequently muscular diseases and their treatments cannot be accurately modelled. In this project, a mathematical model has been developed which relates the chemomechanical cycle of individual events (crossbridges) to the transfer of mechanical energy through an actin filament, myosin cofilament and, by incorporating the protein titin, the mechanical properties of the interconnecting proteins in a section of sarcomere. Evaluation and parameterisation of the model were made by comparison with in vitro test data from the published literature at the level of a single crossbridge and single filaments. At the single filament level, the model was evaluated against two conditions: a low load high displacement (concentric contraction) and a high load low displacement (isometric contraction). In isometric loading the peak force level per unit length of actin filament was higher than that observed in vitro, the difference being attributable to the greater compliance in the substrate used in vitro to hold the myosin fragments (~37pN compared to ~12pN). The mean number of concurrent crossbridges was consistent between the model and in vitro data. Under low load the model demonstrated filament movement at speeds comparable to those measured in in vitro motility studies, although longer filaments in the model were required than those in vitro to reach the higher speeds (7μm vs. 2μm for ~8μm/s). By making the pre-lever reaction duration of the crossbridge cycle strain dependent it was possible to obtain long reaction cycles in low load scenarios comparable to those observed for fragments in solution while generating the actin filament speeds observed in vitro. It was necessary to have a distribution of attachment times across the filament in order to generate and maintain filament movement in the model; the variation being governed by the tension distribution in the filament. By applying a passive loading as generated by the titin protein the filaments moved more rapidly, with an increased contribution from each crossbridge to filament movement. Initial results indicate examination of the strain dependency of the post-lever reaction duration may modify filament speeds and will increase the proportion of each crossbridge movement that contributes to the actin filament propulsion (increase crossbridge efficiency). Examination of a selection of the model’s parameters gave an initial evaluation of how the model could be ‘tuned’ to change the number, reaction state and distribution in time of crossbridges to achieve changes in filament contraction speed, isometric force generation and the efficiency with which crossbridges are used; noting that one desired output may conflict with another. The interaction of the passive components in the structure of the sarcomere with the strain dependent reaction cycle at each crossbridge demonstrated the potential limitations of scaling and averaging localised events without consideration of the passive structures present in the fibre and muscle bulk. The model provides a means to examine the mechanisms and parameters of the sarcomere’s function and how those parameters may be adjusted to achieve different output characteristics. The model provides a foundation for the emulation of muscle fibres and a motor unit in health and disease.

Thesis--University of Chicago, 1916. / Bibliography: p. vii-ix.

Thesis--University of Chicago, 1916. / Bibliography: p. vii-ix.

The amniotic membrane, well known scaffolding tissue, which widely uses and benefits of having anti-inflammatory, anti-microbial, anti-fibrosis, anti-scarring with low immunogenicity and reasonable mechanical properties. Amniotic Membrane Transplantation (AMT) is an established treatment modality, which favourably influence ocular surface re-epithelisation and prevents angiogenesis, thus promoting healing and minimising scarring. It is also used at several other sites of the body. Despite its widespread use, key elements of the membrane and its precise mechanism(s) of action remain to be elucidated. Unfortunately, over the years conflicting clinical reports have suggested variations in the efficacy of AM utility. Conventional methods for amnion preparation do not acknowledge the presence of the SL. My project is a continuation of previous PhD completed in the department, which mentioned the Spongy Layer as one of the important layers in Amniotic Membrane, which had not been look for in any previous work and Dr A Hopkinson, the author of previously mentioned PhD, accidentally discovered possibility to separate that layer from the amnion, it had been decided to take a close look at the layer and investigate properties. Researchers at the University of Nottingham have developed and improved techniques of manufacturing clinical grade the amnion and they have identified the SL as a substance that is variably present in conventional amnion. They have developed techniques to entirely isolate the SL, which allows comprehensive characterisation of its composition and biological properties. The project originally designed to investigate all layers of Amniotic Membrane separately in comparison with amniotic membrane which is completely free from Spongy Layer (SL detached) as well as Spongy Layer attached to the amniotic membrane (classically used layer) and identify a layer which is richest in proteins and growth factors. So, three different samples were investigated: 1) Isolated Spongy Layer; 2) Isolated Amniotic Membrane; 3) Amniotic Membrane with Spongy Layer attached (classical layer, which well- known used). I used technique developed in the department. This technique allows separating the SL without unnecessary mechanical tissue disturbance and isolated SL was used to investigate comprehensive characterisation of the composition and biological properties. Our technique of removing SL is simple and could be easily adapted. The SL imbibes water well and significantly expands, which makes it thick and easy to pull using forceps or using blunt edge of the scalpel blade to push the SL from the amnion surface without mechanical interruption. Investigation of origin of the Spongy Layer during gestation period done through intensive literature search and came to conclusion that the Spongy Layer developed from the extraembryonic endoderm. It is well known that the SL acts as a barrier between vascular amnion and avascular chorion. Also, question was about similarities and differences of embryological origin of the Wharton Jelly and the Spongy Layer. The present in which of TGFb1 (immunofluorescent staining) could be result of cross link during the embryological development. As previously, reported by Dr A Hopkinson et al in 2006, that the Spongy Layer’s biochemical composition is containing TGF-b1, EGF and HGF. The structure of SL is reported to be composed of Collagen types I-VIII, the SL contains high level of hyaluronan, which is a major carbohydrate component of the ECM. To extract proteins from the SL, a few different techniques were used, first of all the tissue weighted, freeze dried and lyophilised in buffers, which were different and depended on the experiment planned. After, proteins were analysed through the Searchlight protein array analysis, 2-D protein quantitation, the Bradford (Commassie staining) assay, the mass spectrometry, had been discovered that the SL contains angiogenic factors, biomarkers, cell adhesions factors, cytokine proteins, growth factors, metalloproteinases, chemokine proteins, neurotrophic factors and cellular components. Experiments show that the level of those factors and proteins in fresh AM, the Transplant Ready Amniotic Membrane (TRAM) and the SL shows that significant amount of proteins was simply washed out during preparation from the TRAM, however the SL is holding those proteins in significant level probably due to imbibing while absorbing water. The important discovery of this project was the cytotoxic effect of the SL and its antimicrobial properties. Some fractions of the SL with high molecular weight proteins show apoptotic activity to corneal keratofibroblasts by necrosis rather than apoptosis. However, cells occurred apoptosis after treatment with lower molecular weight proteins. As preliminary data shows the SL to be cytotoxic, this could lead to some understanding of different outcome in the Amniotic Membrane transplantation (AMT). However, this area needs further investigation. Well known antimicrobial properties of the amniotic membrane were established only in the TRAM and in the samples of the SL which were prepared in the same way as TRAM (washed in gentamicin), however samples of the SL which were not washed in gentamicin did not show antimicrobial properties. One of the next steps of investigation properties of Spongy Layer was the measurement of the thickness of the layer in normal physiological situation, during gestation. As the SL is imbibing the water quickly, in vitro it was difficult to measure “real” thickness of the membrane; this gave an idea to measure it in vivo. To answer the question of “real” (physiological) thickness of the Spongy Layer, different methods were used, however the Ultrasound technique was our method of interest, as it gave the possibility to see the layer during gestation without any interruption and see changes in thickness prior to delivery. These measurements were done in the Fetal Maternal Medicine Department by very experienced sonographer. The Spongy Layer has a variable thickness in three different areas (cervical part, mid region and apical part of the uterus) the SL regions had been divided accordingly. The Spongy Layer has a variable thickness depending on the anatomical location. The difference of the SL thickness had been measured in vivo (ultrasound technic – described below) and in vitro (this work published by JJ Gicquel) the compared results matched. The major problem of the project was the separation of proteins and to break hyaluronic chains to extract clean proteins. In my project, I used two different techniques to separate proteins according the molecular weight of proteins presented in the sample. 1) Revers Phase Solid Phase Extraction (RP-SPE) isolation of proteins; and 2) Soluble Protein Fractionation using Vivaspin columns The second technique in my hands was more successful and I decided to use this for all my samples. To minimise the possibility of sample variations from the point of sample preparation of the SL and the AM itself, I used the same technique for all experiments and combined samples. Nevertheless, my samples were not 100% clear due to different protein structure and shape. This layer therefore has the potential to be exploited clinically for the treatment of several indications. However, before it can be employed, the layer requires further investigation to determine characterise the content of potential factors. In addition, as the spongy layer is predominantly composed of mucin and proteoglycans resulting in a gelatinous/viscous substance, a processing procedure must be developed to either modify the substance in a usable format, or to extract the beneficial factors, for clinical use. Being able to demonstrate the isolated SL and its derivatives can be exploited as potential therapeutic agents in the treatment of many ocular surface disorders, would have significant translational potential. / The control of inflammation caused by disease (e.g. Ocular cicatricial pemphigoid) and any injury (e.g. Chemical burns) and the limitation of scarring and vascularisation would preserve sight or allow successful secondary intervention such as corneal drafting, which otherwise has a high risk of failure in such situations, the potential for preventing visual impairment and promoting quality of life in all age groups in therefore immense. My work proved that the SL is a separate layer and is having a vast number of different factors in a significantly high amount compare to amnion itself.

Orientadores: Fausto Berzin, Eduardo Grossmann / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T13:02:50Z (GMT). No. of bitstreams: 1 Zeilmann_PatriciaPereira_M.pdf: 1075202 bytes, checksum: 0a785029a41f3d911baf96f7d87ca1e1 (MD5) Previous issue date: 2010 / Resumo: O presente trabalho empregou o exame de ressonância magnética nuclear do músculo temporal para verificar se existia, ou não, diferenças quanto ao gênero e lado bem como na tentativa de diferenciá-lo de um músculo denominado esfenomandibular. Foram avaliados 20 voluntários, 10 do gênero feminino e 10 do masculino, assintomáticos para cefaléia, disfunção temporomandibular, parafunção oclusal, dor articular e/ou muscular durante atividades funcionais e/ou dor muscular ao acordar, com idades entre 18 e 46 anos. Os voluntários foram submetidos a uma anamnese, a um exame clínico da cavidade bucal, das articulações temporomandibulares, dos músculos da região e dos nervos cranianos, seguido de um exame de ressonância magnética nuclear. As imagens foram realizadas em cortes sagitais, coronais e axiais. Os dados obtidos foram registrados em uma ficha clínica. Posteriormente foi realizada uma análise descritiva dos dados. O músculo temporal apresentou duas partes distintas, uma profunda e outra superficial, sendo que a profunda mostrou-se sempre maior. O volume do músculo esquerdo apresentou-se maior que o direito e a profundidade do direito apresentou-se maior que a do esquerdo, ambos independentes do gênero. No masculino observou-se que tal músculo apresentava maior volume e profundidade que o feminino. Dentro das condições desse estudo, concluiu-se que o exame de ressonância magnética nuclear possibilitou identificar a região estudada como sendo músculo temporal e não músculo esfenomandibular / Abstract: This study used the nuclear magnetic resonance of the temporalis muscle to check if there was or not gender and side differences and in an attempt to differentiate it from a muscle called sphenomandibularis. We evaluated 20 volunteers, 10 females and 10 males, with no symptoms of headache, temporomandibular disorders, occlusal parafunction, joint pain and / or muscle during functional activities and / or muscle pain on waking, aged between 18 and 46 years. The volunteers underwent a medical history, a clinical examination of the buccal cavity, temporomandibular joints, muscles of the region and the cranial nerves, followed by an examination of nuclear magnetic resonance. The images were taken in sagittal, coronal and axial slices. The data were recorded in a clinical record. It was later performed a descriptive analysis of data. The temporalis muscle had two distinct parts, one deep and one superficial and the deep was always greatest. The volume of muscle left was larger than the right and the depth of the right was higher than the left, both independent of gender. In male was observed that this muscle has greater volume and depth than females. Under the conditions of this study, it was concluded that the examination of nuclear magnetic resonance enabled identified this region as being the temporalis muscle and not sphenomandibularis muscle / Mestrado / Anatomia / Mestre em Biologia Buco-Dental

The neuromodulator adenosine is involved in both physiological and pathological activity, such as sleep, epilepsy and stroke. However, the complex processes underlying the release, transport and clearance of adenosine from the extracellular space and their interactions are still poorly quantified. In this thesis I develop the �rst detailed model of the dynamics of adenosine in neural tissue, including intracellular and extracellular metabolism, using parameters taken from an extensive search of the literature. This approach also identifies physiological and metabolic parameters that have yet to be experimentally measured. The model provides estimates of the range of influence of adenosine, the distance where the extracellular concentration is greater than that required for half of the maximum inhibition by the dominant type of adenosine receptors in the cortex, and suggests that under physiological conditions the adenosine signal will be highly localised. The model predicts that adenosine concentration profiles are primarily determined by diffusion and that neuronal transport and metabolism are the dominant clearance mechanisms. The model can be used with either experimental or endogenous sources of adenosine, and I apply it to the bath application of adenosine to a tissue slice, (a method used extensively to study the e�ect of adenosine on synaptic transmission). The model is used to predict the effective dose response curve of bath applied adenosine and to compare the effects of transporter blockers. I then turn to the modelling of biosensors, which are used extensively to measure the concentration of various analytes in tissue, including adenosine. Biosensors are often calibrated in a flow injection system with a known concentration of the analyte. Mathematical and computational models are used to compare the response characteristics of biosensors in this free environment with the tortuous environment in which they are used. An estimated correction factor is obtained together with the sensitivity of this factor to the characteristics of the biosensor. This work provides a framework to move from qualitative studies of changes of adenosine in the brain to quantitative analysis of the spatio-temporal dynamics of adenosine signalling and its in uence on networks of neurons.