Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Chlorine Contribution to Quantitative Structure and Activity Relationship Models of Disinfection By-Products' Quantum Chemical Descriptors and Toxicities

Quantitative Structure-Activity Relationship (QSAR) has been applied extensively in predicting toxicity of Disinfection By-Products (DBPs) in drinking water. Among many toxicological properties, acute and chronic toxicities of DBPs have been widely used in health risk assessment of DBPs. These toxicities are correlated with molecular properties, which are usually correlated with molecular descriptors. The primary goals of this thesis are: 1) to investigate the effects of molecular descriptors (e.g., chlorine number) on molecular properties such as energy of the lowest unoccupied molecular orbital (ELUMO) via QSAR modelling and analysis; 2) to validate the models by using internal and external cross-validation techniques; 3) to quantify the model uncertainties through Taylor and Monte Carlo Simulation. One of the very important ways to predict molecular properties such as ELUMO is using QSAR analysis. In this study, number of chlorine (NCl) and number of carbon (NC) as well as energy of the highest occupied molecular orbital (EHOMO) are used as molecular descriptors. There are typically three approaches used in QSAR model development: 1) Linear or Multi-linear Regression (MLR); 2) Partial Least Squares (PLS); and 3) Principle Component Regression (PCR). In QSAR analysis, a very critical step is model validation after QSAR models are established and before applying them to toxicity prediction. The DBPs to be studied include five chemical classes: chlorinated alkanes, alkenes, and aromatics. In addition, validated QSARs are developed to describe the toxicity of selected groups (i.e., chloro-alkane and aromatic compounds with a nitro- or cyano group) of DBP chemicals to three types of organisms (e.g., Fish, T. pyriformis, and P.pyosphoreum) based on experimental toxicity data from the literature. The results show that: 1) QSAR models to predict molecular property built by MLR, PLS or PCR can be used either to select valid data points or to eliminate outliers; 2) The Leave-One-Out Cross-Validation procedure by itself is not enough to give a reliable representation of the predictive ability of the QSAR models, however, Leave-Many-Out/K-fold cross-validation and external validation can be applied together to achieve more reliable results; 3) ELUMO are shown to correlate highly with the NCl for several classes of DBPs; and 4) According to uncertainty analysis using Taylor method, the uncertainty of QSAR models is contributed mostly from NCl for all DBP classes.

Porous Materials with Tunable Structure and Mechanical Properties via Templated Layer-by-Layer Assembly

The deposition of stiff and strong coatings onto porous templates offers a novel strategy for fabricating macroscale materials with controlled architectures at the micro- and nanoscale. Here, layer-by-layer assembly is utilized to fabricate nanocomposite-coated foams with highly customizable properties by depositing polymer–nanoclay coatings onto open-cell foam templates. The compressive mechanical behavior of these materials evolves in a predictable manner that is qualitatively captured by scaling laws for the mechanical properties of cellular materials. The observed and predicted properties span a remarkable range of density-stiffness space, extending from regions of very soft elastomer foams to very stiff, lightweight honeycomb and lattice materials.

An effective three-dimensional ordered mesoporous CuCo2O4 as electrocatalyst for Li-O2 batteries

Three-dimensional ordered mesoporous (3DOM) CuCo₂O₄ materials have been synthesized via a hard template and used as bifunctional electrocatalysts for rechargeable Li-O₂ batteries. The characterization of the catalyst by X-ray diffractometry and transmission electron microscopy confirms the formation of a single-phase, 3-dimensional, ordered mesoporous CuCo₂O₄ structure. The as-prepared CuCo₂O₄ nanoparticles possess a high specific surface area of 97.1 m² g^{- 1} and a spinel crystalline structure. Cyclic voltammetry demonstrates that mesoporous CuCo₂O₄ catalyst enhances the kinetics for either oxygen reduction reaction (ORR) or oxygen evolution reaction (OER). The Li-O₂ battery utilizing 3DOM CuCo₂O₄ shows a higher specific capacity of 7456 mAh g^{- 1} than that with pure Ketjen black (KB). Moreover, the CuCo₂O₄-based electrode enables much enhanced cyclability with a 610 mV smaller discharge-recharge voltage gap than that of the carbon-only cathode at a current rate of 100 mA g^{- 1}. Such excellent catalytic performance of CuCo₂O₄ could be associated with its larger surface area and 3D ordered mesoporous structure. The excellent electrochemical performances coupled with its facile and cost-effective way will render the 3D mesoporous CuCo₂O₄ nanostructures as attractive electrode materials for promising application in Li-O₂ batteries.

Benchmarking a modified version of the civ3 nonrelativistic atomic-structure code within Na-like-tungsten R -matrix calculations

In this work we explore the validity of employing a modified version of the nonrelativistic structure code civ3 for heavy, highly charged systems, using Na-like tungsten as a simple benchmark. Consequently, we present radiative and subsequent collisional atomic data compared with corresponding results from a fully relativistic structure and collisional model. Our motivation for this line of study is to benchmark civ3 against the relativistic grasp0 structure code. This is an important study as civ3 wave functions in nonrelativistic R-matrix calculations are computationally less expensive than their Dirac counterparts. There are very few existing data for the W LXIV ion in the literature with which we can compare except for an incomplete set of energy levels available from the NIST database. The overall accuracy of the present results is thus determined by the comparison between the civ3 and grasp0 structure codes alongside collisional atomic data computed by the R-matrix Breit-Pauli and Dirac codes. It is found that the electron-impact collision strengths and effective collision strengths computed by these differing methods are in good general agreement for the majority of the transitions considered, across a broad range of electron temperatures.

Functional alkali tantalate 2D structures for microelectronics and related applications

Alkali tantalates and niobates, including K(Ta / Nb)O3, Li(Ta / Nb)O3 and Na(Ta / Nb)O3, are a very promising ferroic family of lead-free compounds with perovskite-like structures. Their versatile properties make them potentially interesting for current and future application in microelectronics, photocatalysis, energy and biomedics. Among them potassium tantalate, KTaO3 (KTO), has been raising interest as an alternative for the well-known strontium titanate, SrTiO3 (STO). KTO is a perovskite oxide with a quantum paraelectric behaviour when electrically stimulated and a highly polarizable lattice, giving opportunity to tailor its properties via external or internal stimuli. However problems related with the fabrication of either bulk or 2D nanostructures makes KTO not yet a viable alternative to STO. Within this context and to contribute scientifically to the leverage tantalate based compounds applications, the main goals of this thesis are: i) to produce and characterise thin films of alkali tantalates by chemical solution deposition on rigid Si based substrates, at reduced temperatures to be compatible with Si technology, ii) to fulfil scientific knowledge gaps in these relevant functional materials related to their energetics and ii) to exploit alternative applications for alkali tantalates, as photocatalysis. In what concerns the synthesis attention was given to the understanding of the phase formation in potassium tantalate synthesized via distinct routes, to control the crystallization of desired perovskite structure and to avoid low temperature pyrochlore or K-deficient phases. The phase formation process in alkali tantalates is far from being deeply analysed, as in the case of Pb-containing perovskites, therefore the work was initially focused on the process-phase relationship to identify the driving forces responsible to regulate the synthesis. Comparison of phase formation paths in conventional solid-state reaction and sol-gel method was conducted. The structural analyses revealed that intermediate pyrochlore K2Ta2O6 structure is not formed at any stage of the reaction using conventional solid-state reaction. On the other hand in the solution based processes, as alkoxide-based route, the crystallization of the perovskite occurs through the intermediate pyrochlore phase; at low temperatures pyrochlore is dominant and it is transformed to perovskite at >800 °C. The kinetic analysis carried out by using Johnson-MehlAvrami-Kolmogorow model and quantitative X-ray diffraction (XRD) demonstrated that in sol-gel derived powders the crystallization occurs in two stages: i) at early stage of the reaction dominated by primary nucleation, the mechanism is phase-boundary controlled, and ii) at the second stage the low value of Avrami exponent, n ~ 0.3, does not follow any reported category, thus not permitting an easy identification of the mechanism. Then, in collaboration with Prof. Alexandra Navrotsky group from the University of California at Davis (USA), thermodynamic studies were conducted, using high temperature oxide melt solution calorimetry. The enthalpies of formation of three structures: pyrochlore, perovskite and tetragonal tungsten bronze K6Ta10.8O30 (TTB) were calculated. The enthalpies of formation from corresponding oxides, ∆Hfox, for KTaO3, KTa2.2O6 and K6Ta10.8O30 are -203.63 ± 2.84 kJ/mol, - 358.02 ± 3.74 kJ/mol, and -1252.34 ± 10.10 kJ/mol, respectively, whereas from elements, ∆Hfel, for KTaO3, KTa2.2O6 and K6Ta10.8O30 are -1408.96 ± 3.73 kJ/mol, -2790.82 ± 6.06 kJ/mol, and -13393.04 ± 31.15 kJ/mol, respectively. The possible decomposition reactions of K-deficient KTa2.2O6 pyrochlore to KTaO3 perovskite and Ta2O5 (reaction 1) or to TTB K6Ta10.8O30 and Ta2O5 (reaction 2) were proposed, and the enthalpies were calculated to be 308.79 ± 4.41 kJ/mol and 895.79 ± 8.64 kJ/mol for reaction 1 and reaction 2, respectively. The reactions are strongly endothermic, indicating that these decompositions are energetically unfavourable, since it is unlikely that any entropy term could override such a large positive enthalpy. The energetic studies prove that pyrochlore is energetically more stable phase than perovskite at low temperature. Thus, the local order of the amorphous precipitates drives the crystallization into the most favourable structure that is the pyrochlore one with similar local organization; the distance between nearest neighbours in the amorphous or short-range ordered phase is very close to that in pyrochlore. Taking into account the stoichiometric deviation in KTO system, the selection of the most appropriate fabrication / deposition technique in thin films technology is a key issue, especially concerning complex ferroelectric oxides. Chemical solution deposition has been widely reported as a processing method to growth KTO thin films, but classical alkoxide route allows to crystallize perovskite phase at temperatures >800 °C, while the temperature endurance of platinized Si wafers is ~700 °C. Therefore, alternative diol-based routes, with distinct potassium carboxylate precursors, was developed aiming to stabilize the precursor solution, to avoid using toxic solvents and to decrease the crystallization temperature of the perovskite phase. Studies on powders revealed that in the case of KTOac (solution based on potassium acetate), a mixture of perovskite and pyrochlore phases is detected at temperature as low as 450 °C, and gradual transformation into monophasic perovskite structure occurs as temperature increases up to 750 °C, however the desired monophasic KTaO3 perovskite phase is not achieved. In the case of KTOacac (solution with potassium acetylacetonate), a broad peak is detected at temperatures <650 °C, characteristic of amorphous structures, while at higher temperatures diffraction lines from pyrochlore and perovskite phases are visible and a monophasic perovskite KTaO3 is formed at >700 °C. Infrared analysis indicated that the differences are due to a strong deformation of the carbonate-based structures upon heating. A series of thin films of alkali tantalates were spin-coated onto Si-based substrates using diol-based routes. Interestingly, monophasic perovskite KTaO3 films deposited using KTOacac solution were obtained at temperature as low as 650 °C; films were annealed in rapid thermal furnace in oxygen atmosphere for 5 min with heating rate 30 °C/sec. Other compositions of the tantalum based system as LiTaO3 (LTO) and NaTaO3 (NTO), were successfully derived as well, onto Si substrates at 650 °C as well. The ferroelectric character of LTO at room temperature was proved. Some of dielectric properties of KTO could not be measured in parallel capacitor configuration due to either substrate-film or filmelectrode interfaces. Thus, further studies have to be conducted to overcome this issue. Application-oriented studies have also been conducted; two case studies: i) photocatalytic activity of alkali tantalates and niobates for decomposition of pollutant, and ii) bioactivity of alkali tantalate ferroelectric films as functional coatings for bone regeneration. Much attention has been recently paid to develop new type of photocatalytic materials, and tantalum and niobium oxide based compositions have demonstrated to be active photocatalysts for water splitting due to high potential of the conduction bands. Thus, various powders of alkali tantalates and niobates families were tested as catalysts for methylene blue degradation. Results showed promising activities for some of the tested compounds, and KNbO3 is the most active among them, reaching over 50 % degradation of the dye after 7 h under UVA exposure. However further modifications of powders can improve the performance. In the context of bone regeneration, it is important to have platforms that with appropriate stimuli can support the attachment and direct the growth, proliferation and differentiation of the cells. In lieu of this here we exploited an alternative strategy for bone implants or repairs, based on charged mediating signals for bone regeneration. This strategy includes coating metallic 316L-type stainless steel (316L-SST) substrates with charged, functionalized via electrical charging or UV-light irradiation, ferroelectric LiTaO3 layers. It was demonstrated that the formation of surface calcium phosphates and protein adsorption is considerably enhanced for 316L-SST functionalized ferroelectric coatings. Our approach can be viewed as a set of guidelines for the development of platforms electrically functionalized that can stimulate tissue regeneration promoting direct integration of the implant in the host tissue by bone ingrowth and, hence contributing ultimately to reduce implant failure.

SOCIAL STRUCTURE OF ASIAN ELEPHANTS (ELEPHAS MAXIMUS) IN SRI LANKA

Asian elephants (Elephas maximus) are critically endangered and live in fragmented populations spread across 13 countries. Yet in comparison to the African savannah elephant (Loxodonta africana), relatively little is known about the social structure of wild Asian elephants because the species is mostly found in low visibility habitat. A better understanding of Asian elephant social structure is critical to mitigate human-elephant conflicts that arise due to increasing human encroachments into elephant habitats. In this dissertation, I examined the social structure of Asian elephants at three sites: Yala, Udawalawe, and Minneriya National Parks in Sri Lanka, where the presence of large open areas and high elephant densities are conducive to behavioral observations. First, I found that the size of groups observed at georeferenced locations was affected by forage availability and distance to water, and the effects of these environmental factors on group size depended on site. Second, I discovered that while populations at different sites differed in the prevalence of weak associations among individuals, a core social structure of individuals sharing strong bonds and organized into highly independent clusters was present across sites. Finally, I showed that the core social structure preserved across sites was typically composed of adult females associating with each other and with other age-sex classes. In addition, I showed that females are social at all life stages, whereas males gradually transition from living in a group to a more solitary lifestyle. Taking into consideration these elements of Asian elephant social structure will help conservation biologists develop effective management strategies that account for both human needs and the socio-ecology of the elephants.

Effect of soil structure on temporal and spatial dynamics of bacteria

Soil is a complex heterogeneous system comprising of highly variable and dynamic micro-habitats that have significant impacts on the growth and activity of resident microbiota. A question addressed in this research is how soil structure affects the temporal dynamics and spatial distribution of bacteria. Using repacked microcosms, the effect of bulk-density, aggregate sizes and water content on growth and distribution of introduced Pseudomonas fluorescens and Bacillus subtilis bacteria was determined. Soil bulk-density and aggregate sizes were altered to manipulate the characteristics of the pore volume where bacteria reside and through which distribution of solutes and nutrients is controlled. X-ray CT was used to characterise the pore geometry of repacked soil microcosms. Soil porosity, connectivity and soil-pore interface area declined with increasing bulk-density. In samples that differ in pore geometry, its effect on growth and extent of spread of introduced bacteria was investigated. The growth rate of bacteria reduced with increasing bulk-density, consistent with a significant difference in pore geometry. To measure the ability of bacteria to spread thorough soil, placement experiments were developed. Bacteria were capable of spreading several cm’s through soil. The extent of spread of bacteria was faster and further in soil with larger and better connected pore volumes. To study the spatial distribution in detail, a methodology was developed where a combination of X-ray microtopography, to characterize the soil structure, and fluorescence microscopy, to visualize and quantify bacteria in soil sections was used. The influence of pore characteristics on distribution of bacteria was analysed at macro- and microscales. Soil porosity, connectivity and soil-pore interface influenced bacterial distribution only at the macroscale. The method developed was applied to investigate the effect of soil pore characteristics on the extent of spread of bacteria introduced locally towards a C source in soil. Soil-pore interface influenced spread of bacteria and colonization, therefore higher bacterial densities were found in soil with higher pore volumes. Therefore the results in this showed that pore geometry affects the growth and spread of bacteria in soil. The method developed showed showed how thin sectioning technique can be combined with 3D X-ray CT to visualize bacterial colonization of a 3D pore volume. This novel combination of methods is a significant step towards a full mechanistic understanding of microbial dynamics in structured soils.

Iron mineral structure, reactivity, and isotopic composition in a South Pacific Gyre ferromanganese nodule over 4 Ma

Deep-sea ferromanganese nodules accumulate trace elements from seawater and underlying sediment porewaters during the growth of concentric mineral layers over millions of years. These trace elements have the potential to record past ocean geochemical conditions. The goal of this study was to determine whether Fe mineral alteration occurs and how the speciation of trace elements responds to alteration over ∼3.7 Ma of marine ferromanganese nodule (MFN) formation, a timeline constrained by estimates from 9Be/10Be concentrations in the nodule material. We determined Fe-bearing phases and Fe isotope composition in a South Pacific Gyre (SPG) nodule. Specifically, the distribution patterns and speciation of trace element uptake by these Fe phases were investigated. The time interval covered by the growth of our sample of the nodule was derived from 9Be/10Be accelerator mass spectrometry (AMS). The composition and distribution of major and trace elements were mapped at various spatial scales, using micro-X-ray fluorescence (μXRF), electron microprobe analysis (EMPA), and inductively coupled plasma mass spectrometry (ICP-MS). Fe phases were characterized by micro-extended X-ray absorption fine structure (μEXAFS) spectroscopy and micro-X-ray diffraction (μXRD). Speciation of Ti and V, associated with Fe, was measured using micro-X-ray absorption near edge structure (μXANES) spectroscopy. Iron isotope composition (δ56/54Fe) in subsamples of 1-3 mm increments along the radius of the nodule was determined with multiple-collector ICP-MS (MC-ICP-MS). The SPG nodule formed through primarily hydrogeneous inputs at a rate of 4.0 ± 0.4 mm/Ma. The nodule exhibited a high diversity of Fe mineral phases: feroxyhite (δ-FeOOH), goethite (α-FeOOH), lepidocrocite (γ-FeOOH), and poorly ordered ferrihydrite-like phases. These findings provide evidence that Fe oxyhydroxides within the nodule undergo alteration to more stable phases over millions of years. Trace Ti and V were spatially correlated with Fe and found to be adsorbed to Fe-bearing minerals. Ti/Fe and V/Fe ratios, and Ti and V speciation, did not vary along the nodule radius. The δ56/54Fe values, when averaged over sample increments representing 0.25 to 0.75 Ma, were homogeneous within uncertainty along the nodule radius, at -0.12 ± 0.07 ‰ (2sd, n=10). Our results indicate that the Fe isotope composition of the nodule remained constant during nodule growth and that mineral alteration did not affect the primary Fe isotope composition of the nodule. Furthermore, the average δ56/54Fe value of -0.12 ‰ we find is consistent with Fe sourced from continental eolian particles (dust). Despite mineral alteration, the trace element partitioning of Ti and V, and Fe isotope composition, do not appear to change within the sensitivity of our measurements. These findings suggest that Fe oxyhydroxides within hydrogenetic ferromanganese nodules are out of geochemical contact with seawater once they are covered by subsequent concentric mineral layers. Even though Fe-bearing minerals are altered, trace element ratios, speciation and Fe isotope composition are preserved within the nodule.

Environmental factors affecting maerl bed structure in Brittany (France)

This study used a large spatial scale approach in order to better quantify the relationships between maerl bed structure and a selection of potentially forcing physical factors. Data on maerl bed structure and morpho-sedimentary characteristics were obtained from recent oceanographic surveys using underwater video recording and grab sampling. Considering the difficulties in carrying out real-time monitoring of highly variable hydrodynamic and physicochemical factors, these were generated by three-dimensional numerical models with high spatial and temporal resolution. The BIOENV procedure indicated that variation in the percentage cover of thalli can best be explained (correlation = 0.76) by a combination of annual mean salinity, annual mean nitrate concentration and annual mean current velocity, while the variation in the proportion of living thalli can best be explained (correlation = 0.47) by a combination of depth and mud content. Linear relationships showed that the percentage cover of maerl thalli was positively correlated with nitrate concentration (R2 = 0.78, P < 0.01) and negatively correlated with salinity (R2 = 0.81, P < 0.01), suggesting a strong effect of estuarine discharge on maerl bed structure, and also negatively correlated with current velocity (R2 = 0.81, P < 0.01). When maerl beds were deeper than 10 m, the proportion of living thalli was always below 30% but when they were shallower than 10 m, it varied between 4 and 100%, and was negatively correlated with mud content (R2 = 0.53, P < 0.01). On the other hand, when mud content was below 10%, the proportion of living thalli showed a negative correlation with depth (R2 = 0.84, P < 0.01). This large spatial scale explanation of maerl bed heterogeneity provides a realistic physical characterization of these ecologically interesting benthic habitats and usable findings for their conservation and management.

Gestural communication of music structure during solo classical piano performance

The production and perception of music is a multimodal activity involving auditory, visual and conceptual processing, integrating these with prior knowledge and environmental experience. Musicians utilise expressive physical nuances to highlight salient features of the score. The question arises within the literature as to whether performers’ non-technical, non-sound-producing movements may be communicatively meaningful and convey important structural information to audience members and co-performers. In the light of previous performance research (Vines et al., 2006, Wanderley, 2002, Davidson, 1993), and considering findings within co-speech gestural research and auditory and audio-visual neuroscience, this thesis examines the nature of those movements not directly necessary for the production of sound, and their particular influence on audience perception. Within the current research 3D performance analysis is conducted using the Vicon 12- camera system and Nexus data-processing software. Performance gestures are identified as repeated patterns of motion relating to music structure, which not only express phrasing and structural hierarchy but are consistently and accurately interpreted as such by a perceiving audience. Gestural characteristics are analysed across performers and performance style using two Chopin preludes selected for their diverse yet comparable structures (Opus 28:7 and 6). Effects on perceptual judgements of presentation modes (visual-only, auditory-only, audiovisual, full- and point-light) and viewing conditions are explored. This thesis argues that while performance style is highly idiosyncratic, piano performers reliably generate structural gestures through repeated patterns of upper-body movement. The shapes and locations of phrasing motions are identified particular to the sample of performers investigated. Findings demonstrate that despite the personalised nature of the gestures, performers use increased velocity of movements to emphasise musical structure and that observers accurately and consistently locate phrasing junctures where these patterns and variation in motion magnitude, shape and velocity occur. By viewing performance motions in polar (spherical) rather than cartesian coordinate space it is possible to get mathematically closer to the movement generated by each of the nine performers, revealing distinct patterns of motion relating to phrasing structures, regardless of intended performance style. These patterns are highly individualised both to each performer and performed piece. Instantaneous velocity analysis indicates a right-directed bias of performance motion variation at salient structural features within individual performances. Perceptual analyses demonstrate that audience members are able to accurately and effectively detect phrasing structure from performance motion alone. This ability persists even for degraded point-light performances, where all extraneous environmental information has been removed. The relative contributions of audio, visual and audiovisual judgements demonstrate that the visual component of a performance does positively impact on the over- all accuracy of phrasing judgements, indicating that receivers are most effective in their recognition of structural segmentations when they can both see and hear a performance. Observers appear to make use of a rapid online judgement heuristics, adjusting response processes quickly to adapt and perform accurately across multiple modes of presentation and performance style. In line with existent theories within the literature, it is proposed that this processing ability may be related to cognitive and perceptual interpretation of syntax within gestural communication during social interaction and speech. Findings of this research may have future impact on performance pedagogy, computational analysis and performance research, as well as potentially influencing future investigations of the cognitive aspects of musical and gestural understanding.

STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

Changes in the community structure of urban and rural forest patches in Baltimore from 1998 to 2015

Urban forests are often highly fragmented with many exotic species. Altered disturbance regimes and environmental pollutants influence urban forest vegetation. One of the best ways to understand the impacts of land-use on forest composition is through long-term research. In 1998, the Baltimore Ecosystem Study established eight forest plots to investigate the impacts of urbanization on natural ecosystems. Four plots were located in urban forest patches and four were located in rural forests. In 2015, I revisited these plots to measure abundances and quantify change in forest composition, diversity, and structure. Sapling, shrub, and seedling abundance were reduced in the rural plots. Alpha diversity and turnover was lower in the rural plots. Beta diversity was reduced in the rural plots. The structure of the urban plots was mostly unchanged, except for a highly reduced sapling layer. Beta diversity in the urban plots was consistent across surveys due to high species turnover.

Gross and microscopic observations on the lingual structure of the franciscana (Pontoporia blainvillei-gervais and d'orbigny, 1844)

In most anatomical studies developed with mammals, the tongue is described as highly differentiated among different species. However, studies on the tongue of aquatic mammals are still limited as compared to those on terrestrial mammals. The aim of this study was to describe the tongue morphology of the Franciscana dolphin (Pontoporia blainvillei) using macroscopic observations, light, and scanning electron microscopy. Microscopically, the dorsal surface was covered by a keratinized stratified epithelium. Salivary gland acini were found on the middle and caudal third of the tongue. The dorsal surface was totally covered by filiform papillae with a connective tissue core and a connective tissue structure round in shape in the middle and caudal regions. Microsc. Res. Tech. 75:737742, 2012. (C) 2012 Wiley Periodicals, Inc.

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A(2) (sPLA2) from Both tops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the a-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of 5PLA2 and have a direct effect on the Ca2+-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this 5PLA2. (C) 2011 Elsevier Ltd. All rights reserved.