Dissertations / Theses on the topic 'Dynamical structure functions'

Dynamical structure functions were developed as a partial structure representation of linear time-invariant systems to be used in the reconstruction of biological networks. Dynamical structure functions contain more information about structure than a system's transfer function, while requiring less a priori information for reconstruction than the complete computational structure associated with the state space realization. Early sufficient conditions for network reconstruction with dynamical structure functions severely restricted the possible applications of the reconstruction process to networks where each input independently controls a measured state. The first contribution of this thesis is to extend the previously established sufficient conditions to incorporate both necessary and sufficient conditions for reconstruction. These new conditions allow for the reconstruction of a larger number of networks, even networks where independent control of measured states is not possible. The second contribution of this thesis is to extend the robust reconstruction algorithm to all reconstructible networks. This extension is important because it allows for the reconstruction of networks from real data, where noise is present in the measurements of the system. The third contribution of this thesis is a Matlab toolbox that implements the robust reconstruction algorithm discussed above. The Matlab toolbox takes in input-output data from simulations or real-life perturbation experiments and returns the proposed Boolean structure of the network. The final contribution of this thesis is to increase the applicability of dynamical structure functions to more than just biological networks by applying our reconstruction method to wireless communication networks. The reconstruction of wireless networks produces a dynamic interference map that can be used to improve network performance or interpret changes of link rates in terms of changes in network structure, enabling novel anomaly detection and security schemes.

The signal structure is a partial structure representation for dynamic systems. It characterizes the causal relationship between manifest variables and is depicted in a weighted graph, where the weights are dynamic operators. Earlier work has defined signal structure for linear time-invariant systems through dynamical structure function. This thesis focuses on the search for the signal structure of nonlinear systems and proves that the signal structure reduces to the linear definition when the systems are linear. Specifically, this work: (1) Defines the complete computational structure for nonlinear systems. (2) Provides a process to find the complete computational structure given a state space model. (3) Defines the signal structure for dynamic systems in general. (4) Provides a process to find the signal structure for a class of dynamic systems from their complete computational structure.

Linear time-invariant (LTI) dynamic networks are described by their dynamical structure function, and generally, they have many possible state space realizations. This work characterizes the necessary and sufficient conditions on a state transformation that preserves the dynamical structure function, thereby generating the entire set of realizations of a given order for a specific dynamic network.

In this work we study the interesting physics of the rare earths, and the microscopic state after ultrafast magnetization dynamics in iron. Moreover, this work covers the development, examination and application of several methods used in solid state physics. The first and the last part are related to strongly correlated electrons. The second part is related to the field of ultrafast magnetization dynamics. In the first part we apply density functional theory plus dynamical mean field theory within the Hubbard I approximation to describe the interesting physics of the rare-earth metals. These elements are characterized by the localized nature of the 4f electrons and the itinerant character of the other valence electrons. We calculate a wide range of properties of the rare-earth metals and find a good correspondence with experimental data. We argue that this theory can be the basis of future investigations addressing rare-earth based materials in general. In the second part of this thesis we develop a model, based on statistical arguments, to predict the microscopic state after ultrafast magnetization dynamics in iron. We predict that the microscopic state after ultrafast demagnetization is qualitatively different from the state after ultrafast increase of magnetization. This prediction is supported by previously published spectra obtained in magneto-optical experiments. Our model makes it possible to compare the measured data to results that are calculated from microscopic properties. We also investigate the relation between the magnetic asymmetry and the magnetization. In the last part of this work we examine several methods of analytic continuation that are used in many-body physics to obtain physical quantities on real energies from either imaginary time or Matsubara frequency data. In particular, we improve the Padé approximant method of analytic continuation. We compare the reliability and performance of this and other methods for both one and two-particle Green's functions. We also investigate the advantages of implementing a method of analytic continuation based on stochastic sampling on a graphics processing unit (GPU).

Network reconstruction is the process of recovering a unique structured representation of some dynamic system using input-output data and some additional knowledge about the structure of the system. Many network reconstruction algorithms have been proposed in recent years, most dealing with the reconstruction of strictly proper networks (i.e., networks that require delays in all dynamics between measured variables). However, no reconstruction technique presently exists capable of recovering both the structure and dynamics of networks where links are proper (delays in dynamics are not required) and not necessarily strictly proper.The ultimate objective of this dissertation is to develop algorithms capable of reconstructing proper networks, and this objective will be addressed in three parts. The first part lays the foundation for the theory of mathematical representations of proper networks, including an exposition on when such networks are well-posed (i.e., physically realizable). The second part studies the notions of abstractions of a network, which are other networks that preserve certain properties of the original network but contain less structural information. As such, abstractions require less a priori information to reconstruct from data than the original network, which allows previously-unsolvable problems to become solvable. The third part addresses our original objective and presents reconstruction algorithms to recover proper networks in both the time domain and in the frequency domain.

One of the most powerful properties of mathematical systems theory is the fact that interconnecting systems yields composites that are themselves systems. This property allows for the engineering of complex systems by aggregating simpler systems into intricate patterns. We call these interconnection patterns the "structure" of the system. Similarly, this property also enables the understanding of complex systems by decomposing them into simpler parts. We likewise call the relationship between these parts the "structure" of the system. At first glance, these may appear to represent identical views of structure of a system. However, further investigation invites the question: are these two notions of structure of a system the same? This dissertation answers this question by developing a theory of dynamical structure. The work begins be distinguishing notions of structure from their associated mathematical representations, or models, of a system. Focusing on linear time invariant (LTI) systems, the key technical contributions begin by extending the definition of the dynamical structure function to all LTI systems and proving essential invariance properties as well as extending necessary and sufficient conditions for the reconstruction of the dynamical structure function from data. Given these extensions, we then develop a framework for analyzing the structures associated with different representations of the same system and use this framework to show that interconnection (or subsystem) structures are not necessarily the same as decomposition (or signal) structures. We also show necessary and sufficient conditions for the reconstruction of the interconnection (or subsystem) structure for a class of systems. In addition to theoretical contributions, this work also makes key contributions to specific applications. In particular, network reconstruction algorithms are developed that extend the applicability of existing methods to general LTI systems while improving the computational complexity. Also, a passive reconstruction method was developed that enables reconstruction without actively probing the system. Finally, the structural theory developed here is used to analyze the vulnerability of a system to simultaneous attacks (coordinated or uncoordinated), enabling a novel approach to the security of cyber-physical-human systems.

In this thesis, computer simulations are used to study the properties of new colloidal systems with structured interactions. These are pair interactions that include both attraction and repulsion. Structured colloids differ from conventional colloids in which the interactions between particles are either strictly attractive or strictly repulsive. It is anticipated that these novel interactions will give rise to new microscopic structure and dynamics and therefore new material properties. Three classes of structured interactions are considered: radially structured interactions with an energetic barrier to pair association, Janus surface patterns with two hemispheres of different surface charge, and striped surface patterns. New models are developed to capture the structured interactions of these novel colloid systems. Dynamical computer simulations of these models are performed to quantify the effects of structured interactions on colloid properties. The results show that structured interactions can lead to unexpected particle ordering and novel dynamics. For Janus and striped particles, the particle order can be captured with simpler isotropic coarse-grained models. This relates the static properties of these new colloids to conventional isotropically attractive colloids (e.g. depletion attracting colloids). In contrast, Janus and striped particles are found to have substantially slower dynamics than isotropically attractive colloids. This is explained by the observation of longer-duration reversible bonds between pairs of structured particles. Dynamical mapping methods are explored to relates the dynamics of these structured colloids to isotropically attractive colloids. These methods could also facilitate future nonequilibrium simulation of structured colloids with computationally efficient coarse-grained models.

Protein dynamics are critical to the structure and function of proteins. However, due to the complexity they inherently bring to the protein design problem, dynamics historically have not been considered in computational protein design (CPD). Herein, we present meta-MSD, a new CPD methodology for the design of protein dynamics. We applied our methodology to the design of a novel mode of conformational exchange in Streptococcal protein G domain B1, producing dynamic variants we termed DANCERs. Predictions were validated by NMR characterization of selected DANCERs, confirming that our meta-MSD framework is suitable for the computational design of protein dynamics. We then performed a thorough NMR characterization of the sequence determinants of dynamics in one DANCER, isolating two mutations responsible for the novel dynamics this protein exhibits. The first, A34F, is responsible for destabilizing the highly stable native Gβ1 conformation, allowing the protein to sample other conformational states. The second, V39L mediates subtle interactions that stabilize the designed conformational trajectory in the context of the A34F mutation. Together, these results highlight the role of protein plasticity in the development of dynamics and the need for highly accurate computational tools to approach similar design problems. Finally, we present an NMR-based characterization of structural dynamics in a family of related red fluorescent proteins (RFPs) and pinpoint regions of the RFP structure where dynamics correlate to RFP brightness. This overview of the RFP dynamics-function relationship will be used in future projects to perform a computation design of functional dynamics in RFPs.

The aim of this study is to investigate means of efficiently assessing the effects of distributed structural modification on the dynamic properties of a complex structure. The helicopter structure is normally designed to avoid resonance at the main rotor rotational frequency. However, very often military helicopters have to be modified (such as to carry a different weapon system or an additional fuel tank) to fulfill operational requirements. Any modification to a helicopter structure has the potential of changing its resonance frequencies and mode shapes. The dynamic properties of the modified structure can be determined by experimental testing or numerical simulation, both of which are complex, expensive and time-consuming. Assuming that the original dynamic characteristics are already established and that the modification is a relatively simple attachment such as beam or plate modification, the modified dynamic properties may be determined numerically without solving the equations of motion of the full-modified structure. The frequency response functions (FRFs) of the modified structure can be computed by coupling the original FRFs and a delta dynamic stiffness matrix for the modification introduced. The validity of this approach is investigated by applying it to several cases, 1) 1D structure with structural modification but no change in the number of degree of freedom (DOFs). A simply supported beam with double thickness in the middle section is treated as an example for this case; 2) 1D structure with additional DOFs. A cantilever beam to which a smaller beam is attached is treated as an example for this case, 3) 2D structure with a reduction in DOFs. A four-edge-clamped plate with a cut-out in the centre is treated as an example for this case; and 4) 3D structure with additional DOFs. A box frame with a plate attached to it as structural modification with additional DOFs and combination of different structures. The original FRFs were obtained numerically and experimentally except for the first case. The delta dynamic stiffness matrix was determined numerically by modelling the part of the modified structure including the modifying structure and part of the original structure at the same location. The FRFs of the modified structure were then computed. Good agreement is obtained by comparing the results to the FRFs of the modified structure determined experimentally as well as by numerical modelling of the complete modified structure.

Matrix models are formulated to study the dynamics of the structured populations. We consider closed populations, that is, without migration, and populations with migration. The effects of specific patterns of migration, whether with constant or time-dependent terms, are explored within the context of how they manifest in model output, such as population size. Time functions, commonly known as relative sensitivities, are employed to rank the parameters of the models from most to least influential in the population size or abundance of individuals per group

La résistance antimicrobienne est devenue un problème majeur de santé publique. L’usage parfois abusif d’antibiotiques conduit à la sélection et à la propagation mondiale de mécanismes de résistance. Grâce à leur efficacité clinique et faible toxicité, les β-lactamines sont les antibiotiques les plus prescrits actuellement, et le mécanisme de résistance le plus répandu est l’expression de β-lactamases. Dans ces conditions, le développement de nouveaux traitements antimicrobiens, pour des cibles connues ou nouvelles, est essentiel. Plus particulièrement, le développement de nouveaux inhibiteurs des β-lactamases est très prometteur, permettant de continuer l’utilisation des antibiotiques existants. Les études biochimiques et structurales des nouvelles β-lactamases et leurs mutants synthétiques, par cristallographie aux rayons X ou par différentes techniques de modélisation moléculaire (modélisation par homologie, « docking », dynamique moléculaire, analyse du réseau des molécules d’eau) permettent une meilleure compréhension de ces enzymes. Dans ce contexte, nous avons caractérisé plusieurs β-lactamases du point de vue phénotypique, biochimique et structural.La β-lactamase CMY-136 contient une mutation inhabituelle, Y221H, par rapport à CMY-2, dans une position qui est très conservée parmi les enzymes de classe C. Les études cristallographiques et de modélisation moléculaire ont révélé des interactions stériques défavorables autour de la position mutée 221 qui peuvent affecter la conformation et la dynamique de la boucle Ω, et qui pourraient expliquer l’hydrolyse plus efficace des substrats volumineux et l’affinité plus faible de la plupart des substrats par rapport à la CMY-2.La structure cristallographique de la β-lactamase OXA-427, une nouvelle carbapénèmase de classe D, montre une Lys73 très peu carbamoylée, ce qui est très inhabituel pour cette classe d’enzyme, ainsi qu’un pont hydrophobe à proximité du site actif. Les simulations de dynamique moléculaire ont montré que la boucle β5-β6 est plus flexible et dans une conformation étendue. Ces résultats peuvent expliquer le profil d’hydrolyse unique observé expérimentalement pour cette enzyme.Les modifications dans la boucle β5-β6 de la β-lactamase OXA-48 (mutations d’alanines, délétions systematiques, remplacement par la boucle β5-β6 de la β-lactamase OXA-18) provoquent des modifications importantes dans le profil d’hydrolyse, avec une acquisition graduelle d’une activité cephalosporinase et une diminution de l’activité carbapénémase dans certains cas. Des études de cristallographie aux rayons X et modélisation moléculaire suggèrent que les différences de conformation et de flexibilité dans cette boucle et les régions adjacentes permettent une meilleure fixation des céphalosporines plus volumineuses, par rapport à l’OXA-48. L’analyse de la dynamique des molécules d’eau dans le site actif montre des changements qui sont potentiellement responsables de la diminution de l’activité par rapport aux carbapénèmes. En complément des études sur les mutants naturels, ces résultats confirment l’importance de la boucle β5-β6 pour la spécificité de substrat des enzymes de type OXA-48. La structure cristallographique du mutant 217ΔP de l’OXA-48 présente une conformation auto-inhibée inattendue, induite par la présence d’un ion nitrate, un inhibiteur auparavant inconnu des β-lactamases de classe D.La Beta-Lactamase DataBase(BLDB, http://bldb.eu) développée dans notre équipe est une ressource publique exhaustive contenant des données relatives aux β-lactamases, vérifiées et mises à jour régulièrement. Cette base de données contient tous les mutants naturels et synthétiques de β-lactamases, ainsi que toutes les structures 3D disponibles dans la PDB et la caractérisation phénotypique.Globalement, ces résultats représentent le prérequis pour une meilleure compréhension des relations structure-fonction des β-lactamases et pour le futur développement rationnel d’inhibiteurs pour ces enzymes
Antimicrobial resistance (AMR) has become a major threat to public health nowadays. The use and abuse of antibiotics is increasingly leading to selection and spread of resistance mechanisms worldwide, greatly compromising our capacity to treat infectious diseases. AMR might ultimately result in a future without effective antimicrobial therapy. Due to their safety and clinical efficacy, β-lactams are the most utilized antimicrobial therapy, and the most common resistance mechanism is the expression of β-lactamases. Therefore, the development of new antimicrobial drugs, for novel or already known targets, is of utmost importance. In particular, the development of novel inhibitors towards β-lactamases is also quite promising, as it would allow us to continue using the effective and safe antimicrobial drugs already available today. The biochemical and structural study of novel β-lactamases or synthetic mutants, through X-ray crystallography and various molecular modelling techniques (homology modelling, docking, molecular dynamics, water network analysis), can provide valuable information. In this context, we have characterized phenotypically, biochemically and structurally several β-lactamases.The CMY-136 β-lactamase possesses an unusual mutation, Y221H, as compared to CMY-2, in a position highly conserved among class C ß-lactamases. Crystallographic and molecular modelling experiments reveal a steric impediment around the mutated position 221 that may affect the conformation and dynamics of the Ω-loop, and therefore account for an increased turnover rate for bulky substrates and a decreased affinity for most substrates as compared to CMY-2.The crystal structure of the OXA-427, a novel class D carbapenemase, shows the Lys73 only partially carbamoylated, a very unusual characteristic for this class of β-lactamases, and an unexpected hydrophobic bridge in the vicinity of the active site. Moreover, molecular dynamics simulations revealed an extended and highly flexible β5-β6 loop. Altogether, these features may explain the unique hydrolytic profile determined experimentally for this enzyme.Modifications in the β5-β6 loop of the OXA-48 β-lactamase (alanine scanning, systematic deletions, replacement with the β5-β6 loop from OXA-18) result in profound changes in the hydrolytic profile, with gradual acquisition of cephalosporinase activity and decrease of carbapenemase activity in some cases. X-ray crystallography and molecular modelling studies suggest that the altered conformation and flexibility of this loop and of adjacent regions in these mutants may allow for the better accommodation of the bulkier cephalosporins, compared to OXA-48. Additionally, water dynamics analysis highlighted changes in the water network around and inside the active site cavity that may be responsible for the lower activity towards carbapenems. Together with studies on other naturally occurring mutants, results corroborate the relevance of the β5-β6 loop on the substrate profile of OXA-type enzymes. Crystal structure of the OXA-48 217ΔP mutant reveals an unexpected self-inhibited conformation induced by the presence of a nitrate ion, a previously unknown inhibitor of class D β-lactamases.Finally, the Beta-Lactamase DataBase (BLDB, http://bldb.eu) developed in our laboratory is a comprehensive, manually curated public resource providing up-to-date structural and functional information on β-lactamases. It contains all reported naturally-occurring β-lactamases and synthetic mutants, together with all available 3D structures from the PDB and the phenotypical characterization.Overall, these results constitute an essential foundation for a better understanding of the structure-function relationship of β-lactamases, which may prove crucial for the future rational development of β-lactamase inhibitors

Los receptores acoplados a proteínas G (GPCRs) son la superfamilia más grande y diversa de proteínas transmembrana en Eucariotas. Estos receptores transducen una gran variedad de señales exógenas y endógenas como fotones, hormonas o neurotransmisores para iniciar la respuesta biológica en el interior de la célula. Son, por lo tanto, muy interesantes como dianas farmacológicas. Esta Tesis Doctoral se centra en la comprensión de la estructura y función de los GPCRs, mediante el uso de técnicas de la química computacional como son el modelado por homología, el anclaje molecular y las simulaciones de dinámica molecular. En concreto, la tesis aborda los determinantes estructurales asociados al mecanismo de activación, la regulación por moduladores alostéricos, la oligomerización con otro GPCR o proteínas adicionales, asícomo el acoplamiento de transductores (proteínas G o arrestinas).
G protein-coupled receptors (GPCRs) are the largest and most diverse superfamily of transmembrane proteins in Eukaryotes. GPCRs transduce a huge variety of exogenous and endogenous signals such as photons, hormones or neurotransmitters to initiate biological responses in the cell interior. Therefore, they are very interesting therapeutic targets. This Doctoral Thesis focusses on the understanding of the structure and function of GPCRs, by applying computational chemistry techniques such as homology modelling, docking and molecular dynamics simulations. Particularly, the thesis addresses the structural determinants associated to the activation mechanism, the regulation by allosteric modulators, the oligomerization with other GPCR or additional proteins and the coupling to transducers (G proteins or arrestins).

Understanding the function of membrane proteins is crucial to elucidate the molecular mechanisms by which transmembrane signaling based physiological processes,i. e., the interactions of extracellular ligands with membrane-bound receptors, are regulated. In this work, synthetic transmembrane segments derived from the visual photoreceptor rhodopsin, the full length system rhodopsin and mutants of opsin are used to study physical processes that underlie the function of this prototypical class-A G-protein coupled Receptor. The dependency of membrane protein hydration and protein-lipid interactions on side chain charge neutralization is addressed by fluorescence spectroscopy on synthetic transmembrane segments in detergent and lipidic environment constituting transmembrane segments of rhodopsin in the membrane. Results from spectroscopic studies allow us to construct a structural and thermodynamical model of coupled protonation of the conserved ERY motif in transmembrane helix 3 of rhodopsin and of helix restructuring in the micro-domain formed at the protein/lipid water phase boundary. Furthermore, synthesized peptides and full length systems were studied by time resolved FTIR-Fluorescence Cross Correlation Hydration Modulation, a technique specifically developed for the purpose of this study, to achieve a full prospect of time-resolved hydration effects on lipidic and proteinogenic groups, as well as their interactions. Multi-spectral experiments and time-dependent analyses based on 2D correlation where established to analyze large data sets obtained from time-resolved FTIR difference spectra and simultaneous static fluorescence recordings. The data reveal that lipids play a mediating role in transmitting hydration to the subsequent membrane protein response followed by water penetration into the receptor structure or into the sub-headgroup region in single membrane-spanning peptides carrying the conserved proton uptake site (monitored by the fluorescence emission of hydrophobic buried tryptophan). Our results support the assumption of the critical role of the lipid/water interface in membrane protein function and they prove in particular the important influence of electrostatics, i. e., side chain charges at the phase boundary, and hydration on that function
Für die Aufklärung der molekularen Wirkungsweise von physiologischen, auf Signaltransduktion, d. h. dem Zusammenspiel von extrazellulären Reizen und membrangebundenen Rezeptoren, beruhenden Prozessen ist das Verständnis der Funktion von Membranproteinen unerlässlich. In dieser Arbeit werden von Rhodopsin abgleitete, synthetische transmembrane Segmentpeptide, Opsin-Mutanten und der vollständige Photorezeptor Rhodopsin untersucht, um die physikalischen Prozesse zu beleuchten, die der Funktionen dieses prototypischen Klasse-A G-Protein gekoppelten Rezeptors zugrunde liegen. Die Abhängigkeit der Membranprotein-Hydratation und der Lipid-Protein-Wechselwirkung von der Ladung einer Aminosäuren-Seitenkette wird erforscht. Hierzu werden synthetische, transmembrane Segmentpeptide in Lipid und Detergenz, als Modell transmembraner Segmente von Rhodopsin in der Membran mittels Fluoreszenzspektroskopie untersucht. Aus den erhaltenen Ergebnissen wird ein thermodynamisches und strukturelles Modell hergeleitet, welches die Kopplung der Protonierung des hochkonservierten ERY-Motivs in Transmembranhelix 3 von Rhodopsin an die Restrukturierung der Helix in der Mikroumgebung der Lipid-Wasser-Phasengrenze erklärt. Des Weiteren werden sowohl die Segementpeptide als auch die vollständigen Systeme Opsin und Rhodopsin mittels zeitaufgelöster FTIR-Fluoreszenz-Kreuzkorrelations-Hydratations-Modulation untersucht. Diese Technik wurde eigens zur Aufklärung von zeitabhängigen Hydratationseffekten auf Lipide und Proteine oder Peptide entwickelt. Dabei werden zeitaufgelöste FTIR Differenz-Spektren und gleichzeitig statische Fluoreszenzsignale aufgenommen und diese zeitabhängigen multispektralen Datensätze mittels 2D Korrelation analysiert. Die Auswertung der Experimente enthüllt einen sequentiellen Hydratationsprozess. Dieser beginnt mit der Bildung von Wasserstoffbrückenbindungen an der Carbonylgruppe des Lipids, gefolgt von Strukturänderungen der Transmembranproteine und abgeschlossen durch das Eindringen von Wasser in das Proteininnere. Letzteres wird nachgewiesen durch die Fluoreszenz von Tryptophan im hydrophoben Peptid- oder Proteininneren. Die Ergebnisse dieser Arbeit unterstreichen die Annahme, dass Lipid-Protein-Wechselwirkungen eine entscheidende Rolle in der Funktion von Membranproteinen spielen und das insbesondere Elektrostatik, in Form von Ladungen an der Phasengrenze, und die Hydratisierung einen kritischen Einfluss auf diese Funktion haben

In this thesis, I have used a combination of computational models such as mean field and spikingnetwork simulations to study various sub-circuits of basal ganglia. I first studied the striatum(chapter 2), which is the input nucleus of basal ganglia. The two types of Medium SpinyNeurons (MSNs), D1 and D2-MSNs, together constitute 98% of the neurons in striatum. Thecomputational models so far have treated striatum as a homogenous unit and D1 and D2 MSNs asinterchangeable subpopulations. This implied that a bias in a Go/No-Go decision is enforced viaexternal agents to the striatum (eg. cortico-striatal weights), thereby assigning it a passive role.New data shows that there is an inherent asymmetry in striatal circuits. In this work, I showedthat striatum due to its asymmetric connectivity acts as a decision transition threshold devicefor the incoming cortical input. This has significant implications on the function of striatum asan active participant in influencing the bias towards a Go/No-Go decision. The striatal decisiontransition threshold also gives mechanistic explanations for phenomena such as L-Dopa InducedDyskinesia (LID), DBS-induced impulsivity, etc. In chapter 3, I extend the mean field model toinclude all the nuclei of basal ganglia to specifically study the role of two new subpopulationsfound in GPe (Globus Pallidus Externa). Recent work shows that GPe, also earlier consideredto be a homogenous nucleus, has at least two subpopulations which are dichotomous in theiractivity with respect to the cortical Slow Wave (SWA) and beta activity. Since the data for thesesubpopulations are missing, a parameter search was performed for effective connectivities usingGenetic Algorithms (GA) to fit the available experimental data. One major result of this studyis that there are various parameter combinations that meet the criteria and hence the presenceof functional homologs of the basal ganglia network for both pathological (PD) and healthynetworks is a possibility. Classifying all these homologous networks into clusters using somehigh level features of PD shows a large variance, hinting at the variance observed among the PDpatients as well as their response to the therapeutic measures. In chapter 4, I collaborated on aproject to model the role of STN and GPe burstiness for pathological beta oscillations as seenduring PD. During PD, the burstiness in the firing patterns of GPe and STN neurons are shownto increase. We found that in the baseline state, without any bursty neurons in GPe and STN,the GPe-STN network can transition to an oscillatory state through modulating the firing ratesof STN and GPe neurons. Whereas when GPe neurons are systematically replaced by burstyneurons, we found that increase in GPe burstiness enforces oscillations. An optimal % of burstyneurons in STN destroys oscillations in the GPe-STN network. Hence burstiness in STN mayserve as a compensatory mechanism to destroy oscillations. We also propose that bursting inGPe-STN could serve as a mechanism to initiate and kill oscillations on short time scales, asseen in the healthy state. The GPe-STN network however loses the ability to kill oscillations inthe pathological state.

QC 20160509

Thesis advisor: Peter Clote
In addition to the well-characterized messenger RNA, transfer RNA and ribosomal RNA, many new classes of noncoding RNA(ncRNA) have been discovered in the past few years. ncRNA has been shown to play important roles in multiple regulation and development processes. The increasing needs for RNA structural analysis software provide great opportunities on computational biologists. In this thesis I present three highly non-trivial RNA parametric structural analysis algorithms: 1) RNAhairpin and RNAmultiloop, which calculate parition functions with respect to hairpin number, multiloop number and multiloop order, 2) RNAshapeEval, which is based upon partition function calculation with respect to a fixed abstract shape, and 3) RNAprofileZ, which calculates the expected partition function and ensemble free energy given an RNA position weight matrix.I also describe the application of these software in biological problems, including evaluating purine riboswitch aptamer full alignment sequences to adopt their consensus shape, building hairpin and multiloop profiles for certain Rfam families, tRNA and pseudoknotted RNA secondary structure predictions. These algorithms hold the promise to be useful in a broad range of biological problems such as structural motifs search, ncRNA gene finders, canonical and pseudoknotted secondary structure predictions
Thesis (MS) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Thesis advisor: Gabor J. Kalman
Mixtures of charged particles, where the components have different charge numbers (Z_A ), masses (m_A ) and densities (n_A ), with A = 1, 2 denoting the components, occur in Nature in a great variety. To be sure, even the simplest plasmas are necessarily multicomponent systems, consisting of negative and positive charges. This feature is, however, obscured within the centrally important and popular OCP (one component plasma) or jellium models, where the role of one of the components is reduced to providing a neutralizing background. When this background is inert, one is led to the Coulomb OCP model, while when the background is polarizable (such as an electron gas surrounding heavy particles), to a Yukawa OCP (YOCP), with a screened Yukawa potential replacing the Coulomb potential between the dynamically active particles. There are, however situations of physical importance, where the OCP description is inadequate and a genuine two component description of a plasma composed of two species is required. This Thesis focuses on the study of the dynamics of many-body systems consisting of two components of like charges (all the Z_A -s being of the same signature) in a neutralizing background. The methodology is based upon parallel attacks through theoretical analysis and Molecular Dynamics (MD) simulations, the latter yielding the capability of instant verification of the former. The investigation involves the study of the partial (i.e. species by species) structure functions S_AB (k, ω) and current-current correlation functions L_AB (k, ω). The Fluctuation–Dissipation Theorem (FDT) con- nects these quantities to the total and partial response functions χ_AB (k, ω) (matrices in species space), which are instrumental in the description of the collective mode excitations of the system. This analysis has revealed an entirely novel feature: both S_11 (k, ω) and S_22 (k, ω) exhibit very sharp and deep (several orders of magnitude) minima in the strongly coupled liquid phase at robust characteristic frequencies of the system, which are virtually coupling independent. The FDT then demands that these anti-resonances show up as well in the imaginary part of the partial density response function χ_AB (k, ω). Our theoretical analysis, based on the Quasi-Localized Charge Approximation (QLCA), has confirmed that this is indeed the case. These anti-resonant frequencies being related to the dissipative part of the response, require a physical description of the principal source of dissipation. This has been identified as the inter-species momentum transfer, governed by drag between the microscopic current fluctuations of the two species. The description of this effect was incorporatedv in the QLCA formalism, making it possible to derive a closed analytic representation of the fluctuation spectra in the frequency domain of interest and compare them with the results of the MD simulations. Other important novel concepts, such as the idea of coupling dependent effective mass, fast vs. slow sound, the mechanism of tran- sition from short-range to long-range interaction have been identified and analyzed. Furthermore, the investigation of the dynamics has led to the first comprehensive description of the mode structures of classical binary Coulomb and Yukawa mixtures at arbitrary coupling values, which has been a longstanding problem in statistical plasma physics. Focusing on the longitudinal excitations, we describe the transition from weak coupling (where one is acquainted with the RPA result yielding only the single plasmon mode in the Coulomb case or a single acoustic mode in the Yukawa case) to strong coupling, with a doublet of modes that arise from the complex rel- ative motion between the two components, as affected by the interaction with the background
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Physics

Research performed on the subject of dynamic soil-structure interaction (SS) concerning railway bridges is presented in this thesis with the focus on simply supported railway bridges supported by shallow foundations in soil strata on bedrock. The research aims to obtain insight into the SSI of high-speed railway bridges and to provide recommendations on how to model the soil-bridge system from a design perspective. A three-dimensional (3D) simply supported soil-bridge model was first developed and the effects from model assumptions made on the soil-foundation system was evaluated in a 3D setting (paper I). The soil-foundation system was then refined and a model assumptions study was performed in order to evaluate the effects of model assumptions on impedance functions, including the influence of the permanent load acting on the soil-foundation system (paper II). Finally, a study of the assembled soil-bridge system was performed in an extensive parametric study including a set of 2D bridge models in combination with a set of shallow foundations in soil strata on bedrock (paper III). A supplementary section related to paper III was also added in this thesis, showing the effects of the substructure mass. The model assumptions made when creating the soil-foundation model and the soil-bridge model can be very important and must be made with care. The permanent load acting on the soil-foundation systems of shallow foundations may alter the impedance functions significantly. The substructure mass may alter the behavior of the soil-bridge system depending on its magnitude, and neglecting it gives inaccurate results. The 3D effects of SSI do not cause high vibrations due to modes other than the first bending mode, and assuming a 2D bridge model is generally acceptable. The effects of SSI on the soil-bridge systems with shallow soil strata are largely dependent on the ratio between the natural frequency of the bridge and the fundamental frequency of the soil. Depending on the value of this ratio, the effect of including SSI in bridge models may contribute to the bridge obtaining a negligible, conservative, or non-conservative response, as compared to the bridge with the assumption of non-flexible supports.
Forskning i syfte att utröna effekten av dynamisk jord–struktur-interaktion (SSI)på järnvägsbroar presenteras i denna avhandling med huvudfokus på fritt upplagdabroar med stöd av plattgrundlagda fundament i jordar på fast berggrund. Forsknin-gen syftar till att ge förståelse för interaktionen mellan jord och järnvägsbroar samtatt ge rekommendationer på hur systemet kan modelleras ur ett designperspektiv.En tredimensionell (3D) fritt upplagd jord–bromodell utvecklades först och effek-terna av modellantaganden gjorda på jord–grundläggningssystemet utvärderadesi en 3D miljö (artikel I). Jord–grundläggningssystemet förfinades och en studiegenomfördes för att utvärdera effekterna av modellantaganden på impedansfunk-tioner, inklusive påverkan av den permanenta belastningen som verkar på jord–grundläggningssystemet (artikel II). Slutligen utfördes en omfattande parametriskstudie av det sammansatta jord–brosystemet där en uppsättning tvådimensionella(2D) bromodeller kombinerades med en uppsättning jordar (artikel III). Ett kom-pletterande avsnitt relaterat till artikel III lades till i denna avhandling som visareffekterna av massan av underbyggnaden på jord–brosystemet.De modellantaganden som görs vid skapandet av jord–grundläggningsmodeller ochjord–bromodeller kan vara mycket viktiga och bör utföras med varsamhet. Den per-manenta belastningen som verkar på jord–grundläggningssystemet kan väsentligtförändra impedansfunktionerna. Massan av underbyggnaden kan vidare ändra re-sponsen i jord–brosystemet, beroende på dess storlek, och att försumma den kan gefelaktiga resultat. De 3D effekterna av SSI orsakar inte höga vibrationer på grundav andra moder än den första böjmoden, och att anta en 2D bromodell är såledesgenerellt sett motiverat.Effekterna av SSI på jord–brosystemet i grunda jordar beror till stor del av kvotenmellan brons naturliga frekvens och jordens fundamentala frekvens. Beroende påvärdet på denna kvot kan effekten av att inkludera SSI i bromodeller bidra till attbron får en försumbar, konservativ, eller icke-konservativ respons, i jämförelse medbron med antagandet om fasta upplag.

QC 20200903

In this thesis several important proteins are investigated from a structural perspective. Some of the proteins are disease related while other have important but not completely characterised functions. The techniques used are general as demonstrated by applications on metabolic proteins (CYP21, CYP11B1, IAPP, ADH3), regulatory proteins (p53, GDNF) and a transporter protein (ANTR1). When the protein CYP21 (steroid 21-hydroxylase) is deficient it causes CAH (congenital adrenal hyperplasia). For this protein, there are about 60 known mutations with characterised clinical phenotypes. Using manual structural analysis we managed to explain the severity of all but one of the mutations. By observing the properties of these mutations we could perform good predictions on, at the time, not classified mutations. For the cancer suppressor protein p53, there are over thousand mutations with known activity. To be able to analyse such a large number of mutations we developed an automated method for evaluation of the mutation effect called PREDMUT. In this method we include twelve different prediction parameters including two energy parameters calculated using an energy minimization procedure. The method manages to differentiate severe mutations from non-severe mutations with 77% accuracy on all possible single base substitutions and with 88% on mutations found in breast cancer patients. The automated prediction was further applied to CYP11B1 (steroid 11-beta-hydroxylase), which in a similar way as CYP21 causes CAH when deficient. A generalized method applicable to any kind of globular protein was developed. The method was subsequently evaluated on nine additional proteins for which mutants were known with annotated disease phenotypes. This prediction achieved 84% accuracy on CYP11B1 and 81% accuracy in total on the evaluation proteins while leaving 8% as unclassified. By increasing the number of unclassified mutations the accuracy of the remaining mutations could be increased on the evaluation proteins and substantially increase the classification quality as measured by the Matthews correlation coefficient. Servers with predictions for all possible single based substitutions are provided for p53, CYP21 and CYP11B1. The amyloid formation of IAPP (islet amyloid polypeptide) is strongly connected to diabetes and has been studied using both molecular dynamics and Monte Carlo energy minimization. The effects of mutations on the amount and speed of amyloid formation were investigated using three approaches. Applying a consensus of the three methods on a number of interesting mutations, 94% of the mutations could be correctly classified as amyloid forming or not, evaluated with in vitro measurements. In the brain there are many proteins whose functions and interactions are largely unknown. GDNF (glial cell line-derived neurotrophic factor) and NCAM (neural cell adhesion molecule) are two such neuron connected proteins that are known to interact. The form of interaction was studied using protein--protein docking where a docking interface was found mediated by four oppositely charged residues in respective protein. This interface was subsequently confirmed by mutagenesis experiments. The NCAM dimer interface upon binding to the GDNF dimer was also mapped as well as an additional interacting protein, GFRα1, which was successfully added to the protein complex without any clashes. A large and well studied protein family is the alcohol dehydrogenase family, ADH. A class of this family is ADH3 (alcohol dehydrogenase class III) that has several known substrates and inhibitors. By using virtual screening we tried to characterize new ligands. As some ligands were already known we could incorporate this knowledge when the compound docking simulations were scored and thereby find two new substrates and two new inhibitors which were subsequently successfully tested in vitro. ANTR1 (anion transporter 1) is a membrane bound transporter important in the photosynthesis in plants. To be able to study the amino acid residues involved in inorganic phosphate transportation a homology model of the protein was created. Important residues were then mapped onto the structure using conservation analysis and we were in this way able to propose roles of amino acid residues involved in the transportation of inorganic phosphate. Key residues were subsequently mutated in vitro and a transportation process could be postulated. To conclude, we have used several molecular modelling techniques to find functional clues, interaction sites and new ligands. Furthermore, we have investigated the effect of muations on the function and structure of a multitude of disease related proteins.

Over the past few decades, dynamic solid mechanics has become a major field of interest in industrial applications involving crash simulation, impact problems, forging and many others to be named. These problems are typically nonlinear due to large deformations (or geometrical nonlinearity) and nonlinear constitutive relations (or material nonlinearity). For this reason, computer simulations for such problems are of practical importance. In these simulations, the Lagrangian formulation is typically used as it automatically satisfies the mass conservation law. Explicit numerical methods are considered to be efficient in these cases. Most of the numerical methods employed for such simulations are developed from the equation of motion (or momentum balance principle). The use of low-order elements in these numerical methods often exhibits the detrimental locking phenomena in the analysis of nearly incompressible applications, which produces an undesirable effect leading to inaccurate results. Situations of this type are usual in the solid dynamics analysis for rubber materials and metal forming processes. In metal plasticity, the plastic deformation is isochoric (or volume-preserving) whereas, the compressible part is due only to elastic deformation. Recently, a new mixed formulation has been established for explicit Lagrangian transient solid dynamics. This formulation, involving linear momentum, deformation gradient and total energy, results in first order hyperbolic system of equations. Such conservation-law formulation enables stresses to converge at the same rate as velocities and displacements. In addition, it ensures that low order elements can be used without volumetric locking and/or bending difficulty for nearly incompressible applications. The new mixed formulation itself shows a clear advantage over the classical displacement-based formulation, due to its simplicity in incorporating state-of-the-art shock capturing techniques. In this research, a curl-preserving cell centred finite volume computational methodology is presented for solving the first order hyperbolic system of conservation laws on quadrilateral cartesian grids. First, by assuming that the approximation to the unknown variables is constant within each cell. This will lead to discontinuities at cell edges which will motivate the use of a Riemann solver by introducing an upwind bias into the evaluation of the numerical flux function. Unfortunately, the accuracy is severely undermined by an excess of numerical dissipation. In order to alleviate this, it is vital to introduce a linear reconstruction procedure for enhancing the accuracy of the scheme. However the second-order spatial method does not prohibit spurious oscillation in the vicinity of sharp gradients. To circumvent this, a nonlinear slope limiter will then be introduced. It is now possible to evolve the semi-discrete evolutionary system of ordinary equations in time with the aid of the family of explicit Total Variation Diminishing Runge Kutta (TVD-RK) time marching schemes. Moreover, a correction procedure involving minimisation algorithm for conservation of the total angular momentum is presented. To this end, a number of interesting examples will be examined in order to demonstrate the robustness and general capabilities of the proposed approach.

Structural equation modeling (SEM) is often used to investigate effective connectivity in functional neuroimaging studies. Modeling effective connectivity refers to an approach in which a number of specific brain regions, called regions of interest (ROIs), are selected according to some prior knowledge about the regions, and directional (causal) relationships between them are hypothesized and tested. Existing methods for SEM, however, have serious limitations in terms of their computational capacity and the range of models that can be specified. To alleviate these difficulties, I propose a new method of SEM for analysis of effective connectivity, called Dynamic GSCA (Generalized Structured Component Analysis). This method is a component-based method that combines the original GSCA and a multivariate autoregressive model to account for the dynamic nature of data taken over time. Dynamic GSCA can accommodate more elaborate structural models that describe relationships among ROIs and is less prone to computational difficulties, such as improper solutions and the lack of model identification, than the conventional methods of SEM. To illustrate the use of the proposed method, results of empirical studies based on synthetic and real data are reported. Further extensions of Dynamic GSCA are also discussed, including higher order components, multi-sample comparison, multilevel analysis, and latent interactions.
La Modélisation par Équations Structurelles (MES) est souvent utilisée dans les études d'imagerie cérébrales fonctionnelles afin d'investiguer la connectivité effective. La modélisation de connectivité effective est une approche dans laquelle certaines régions cérébrales, appelées régions d'intérêts (RIs), sont spécifiquement sélectionnées à partir de connaissances établies sur ces régions, et des hypothèses sur les possibles liens directionnels (causals) entre les RIs sont formulées et testées. Par contre, les méthodes de MES existantes sont sérieusement limitées par leur capacité computationelle et le nombre et l'étendue des modèles qui peuvent être spécifiés. Afin d'adresser ces difficultés, je propose ici une nouvelle méthode de MES afin d'analyser la connectivité effective, appelée Analyse en Composantes Structurée Généralisée (ACSG) Dynamique. Cette méthode est une méthode basée sur les composantes, combinant la version originale des ACSGs et un modèle auto-régresseur multi-variable afin de tenir compte de la nature dynamique des données recueillies à différent temps. Les ACSG Dynamiques peuvent accommoder des modèles structurels plus complexes pour décrire les relations entre les RIs. De plus, comparé aux méthodes traditionnelles de MES, les ACSG Dynamiques sont moins susceptible de succomber aux difficultés computationelles, comme les solutions inappropriées et l'échec d'identification de modèle. Afin d'illustrer l'utilisation de la méthode proposée, des résultats d'études empiriques basées sur des données synthétiques et réelles sont présentées. Des extensions possibles des ACSG Dynamiques sont aussi discutées, incluant des composantes de plus haut niveau, la comparaison de plusieurs échantillons, l'analyse multi-niveau, et les interactions latentes.

Thesis (Ph.D)--Mechanical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Zhu, Cheng; Committee Member: Barry, Bridgette; Committee Member: Boyan, Barbara; Committee Member: McEver, Rodger; Committee Member: McIntire, Larry. Part of the SMARTech Electronic Thesis and Dissertation Collection.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
No presente trabalho se estuda um elemento finito subparamétrico que aproxima o campo de deslocamentos com funções spline, implementando um programa que pode ser utilizado para calculo estático, dinâmico e de instabilidade de estruturas compostas de placas, vigas de paredes finas, vigas caixão e em geral em elementos alongados (pontes e perfis metálicos). O grau de liberdade de rotação perpendicular ao plano do elemento é introduzido na formulação para possibilitar uma análise tridimensional. Apresenta-se um método que serve como base para determinar a constante de rigidez correspondente. Nos exemplos apresentados avalia-se a precisão obtida utilizando pouco número de divisões longitudinais do continuo, vantagem que justifica o uso desses elementos em estudos de pré-projeto ou otimização de estruturas. Comparam-se os resultados com soluções teóricas ou resultados de outros programas estruturais, permitindo apreciar as possibilidades e limitações da modelagem usando elementos finitos com funções spline. As diferenças observadas, que surgem principalmente em placas espessas, são explicadas pela aproximação da deformação de cisalhamento encontrada na literatura para os elementos utilizados na comparação. Mostra-se também exemplos de instabilidade analisados em três dimensões que permitem considerar diferentes condições de apoio e discutir os resultados de fórmulas conhecidas.
The present work presents a subparametric finite element model with spline displacement functions, implemented for static, dynamic and instability analysis of folded plates, thin-walled beams, box girders, and elongated structures such as bridges and structural shapes. A drilling degree of freedom (rotation about an axis perpendicular to the plane of the element) is introduced in the formulation to allow for three-dimensional analysis. A method for determining the corresponding contribution to the stiffness matrix is presented. The examples presented evaluate the accuracy obtained using a small number of longitudinal subdivisions of the continuum, convenient in the case of analyses for preliminary design and optimization. The results obtained are compared to theoretical solutions or results of standard structural analysis programs, allowing for an appraisal of the advantages and limitations of modeling with use of spline functions. The differences in the results, observed specially in the case of thick plates, are explained by the approximations for the shear strain in the elements used for comparison. From the examples it is possible to comment results of threedimensional modeling of instability problems with different boundary conditions.

Theory and small-scale experiments predict that biodiversity losses can decrease the magnitude and stability of ecosystem services such as production and nutrient cycling. Most of this research, however, has been isolated from spatial processes, such as dispersal and disturbance, which create and maintain diversity in nature. Since common anthropogenic drivers of biodiversity change, such as habitat fragmentation, species introductions, and climate change, are mediated by these understudied processes, it is unclear how environmental degradation will affect ecosystem services. This dissertation examines how diversity interacts with spatial processes to affect the magnitude and stability of ecosystem functions, using seagrass communities as a model system. Diverse communities were more resistant to colonization, but the order of species arrivals affected competition outcomes. as predicted, grazer metacommunities assembled from diverse species pools were more diverse at all scales, had larger grazer populations, and usually kept their primary food resource, epiphytic algae, at lower abundances than metacommunities assembled from smaller species pools. Counter to theory, increasing the number of mobile grazer species in these metacommunities increased spatial and temporal variability of producers and grazers. Effects of diversity on stability also differed qualitatively between patch and metacommunity scales. Moreover, allowing grazers to move among patches reduced diversity effects on production and modified relationships between grazer diversity and stability. Finally, dispersal significantly increased resistance to and recovery from a mimicked macroalgal bloom. However, diversity did not. None of the existing theories for biodiversity-ecosystem function relationships or consumer-resource metacommunity dynamics completely explained patterns observed in these experiments. Effects of diversity and dispersal on ecosystem functions were complex, but seemed to be influenced by habitat choice and synchronization of grazer and epiphyte dynamics among patches. Overall, these results emphasize the importance of incorporating spatial processes and trophic interactions into the study of biodiversity-ecosystem function relationships. This information is critical for conserving diversity and managing ecosystem services in light of the ongoing changes to regional species pools caused by anthropogenic disturbance.

The relationship between the protein motions, structure and catalytic activity is of contemporary interest in enzymology. Here, a broad array of kinetic techniques, site-directed mutagenesis, and methods involving isotopic labeling of substrates and proteins were used in studying thymidylate synthase (TSase). TSase catalyzes the de novo biosynthesis of the DNA building block thymidine, 2'-deoxythymidine 5'-monophosphate (dTMP) in most organisms and is medicinally important as an antibiotic and a chemotherapeutic drug target. The mechanism of TSase involves several steps including two major C-H bond activations: a rate limiting hydride transfer and a non-rate limiting proton transfer. Therefore, it is a good model system to study the dynamic and structural effects on different hydrogen transfers in the same catalytic cycle. Our experiments of a structurally identical but dynamically altered remote mutant of Escherichia Coli (ec)TSase, Y209W, showed that none of these two H-transfer steps under was altered significantly. Yet other kinetic steps were dramatically affected and reveal the importance of long-range dynamics of the enzymatic complex and its catalytic function in ecTSase. We also found that Mg2+ affects the hydride transfer although it binds to the surface of ecTSase, further indicating the important of long range dynamics across the protein. Furthermore, a comparative studies of light vs. heavy WT ecTSase suggest a direct coupling of the altered fast protein vibrations on the hydride transfer. Studies of an active site mutant of a conserved histidine suggest that it plays a major role in the hydride transfer step in ecTSase, supporting mechanisms that involve charge accumulation at carbonyl 4. The studies of an evolutionary divergent TSase from Bacillus subtilis that has a valine at the equivalent position suggest that evolutionary pressure led to extensive polymorphism across the protein, which compensate for the His→Val substitution at the active site. Altogether, these findings could assist in rational inhibitor design as leads to more specific and potent chemotherapeutic or antibiotic drugs in the future, and the design of biomimetic catalysts that include the cross-protein dynamics into consideration.

Doctor of Philosophy
Biology
Walter K. Dodds
Ari M. Jumpponen
Due to agricultural practices and urbanization, tallgrass prairie ecosystems have become threatened as < 5% of its historical coverage exists today. The small remainder of praire that does exist is further threatened by the encroachment of woody plant species. Woody plant encroachment may not only alter prairie ecosystem function, but also prairie microbial communities responsible for these functional processes. Further, prairies are high disturbance ecosystems, especially prairie streams which are hydrologically harsh. They support communities that frequently undergo succession due to recurring flood and drought conditions, yet little is known about the response of microbial communities to these disturbances. In my dissertation, I first address the degree of woody vegetation expansion in riparian corridors (parallel to streams) in watersheds with variable fire frequency and grazing. I found that the rate of riparian woody expansion declines with higher fire intervals and is not affected by grazing, but even annual burns may not prevent woody plant expansion in riparian zones from occurring. Second, I quantified the effect of using restorations of riparian corridors, through removal of woody plants, on physical, chemical, and microbial community (bacteria and fungi) dynamics across stream to upslope soils. Removal restoration causes a decrease in NH₄⁺ and soil water content, and causes streams and upslope soils to become similar in fungal community richness unlike forested landscapes. Bacterial communities were minimally impacted by removals, but were highly structured among stream to upslope soils due to multiple environmental gradients (i.e., pH, NO₃⁻, soil moisture). Lastly, I examined the successional development of biofilm-associated microbial communities in a prairie stream from both a functional and structural perspective. I found that biofilm microbes exhibited strong successional trajectories, with communities developing towards net autotrophy and therefore becoming reliant upon in-stream derived carbon. Further, bacterial communities displayed spatial differences, but much stronger temporal patterns in community composition were detected. These studies highlight how woody plant encroachment may influence stream ecosystems in addition to spatiotemporal trends in microbial community assembly.

Dynamics of proteins are increasingly recognised as key features as they can contribute to the function of the protein. Structural dynamics manifest as protein folding, protein domain movement and small allosteric responses. Hence, investigating and understanding atomistic motions in proteins to elucidate their implication in protein function mark a crucial paradigm shift from a structure-function relation to a structure-dynamics-function relation. The human histone deacetylase 8 is a key hydrolase in gene regulation and has been identified as a drug target for the treatment of several cancers. I used molecular dynamics simulations to propose a mechanism by which dynamic loop interactions can influence the activity of the human histone deacetylase 8. Subsequently I substantiated this hypothesis by using experimental techniques such as biochemical assays and single point mutations. Furthermore, I studied the structure and dynamics of the histone deacetylase 8 using nuclear magnetic resonance techniques. The proposed mechanism of loop interaction yields a mechanistic rationale for phenomena that could not be explained on a molecular level before.

The present work is concerned with projective prepositions, which express the relation between two objects by referring to a direction in three-dimensional space. The projective prepositions have been regarded as expressing simple schematic relations of a geometric nature. A theory of the apprehension of projective relations can account for their meanings when they express strictly geometric relations. However, many studies have shown that the appropriateness of the prepositions also depends on the functional relation between the objects and that a number of functional factors influence the comprehension of English prepositions. This experimental study investigates if the acceptability of the Swedish prepositions över, under, ovanför and nedanför are influenced by functional factors as well, and whether acceptability judgments about över and under are more sensitive to functional influences than judgments about ovanför and nedanför, as has been shown for the corresponding English prepositions over and under, and above and below, respectively. It also investigates how the shapes and the parts of the related objects influence their functional interaction, and how the acceptability of the prepositions is in consequence influenced by the shapes of the objects. It was found that the theory of apprehension can indeed account for the acceptability of the prepositions when the relation between the objects is strictly geometric. It was further found that acceptability judgments about them are influenced by functional factors in a similar manner to the corresponding English prepositions when the objects are functionally related, although judgments about under and nedanför are not differentially influenced by these factors. Furthermore, the shapes and the parts of both of the related objects influence acceptability judgments about the prepositions in predictable manners. An extension of the theory of apprehension is suggested which can account for the functional influences indicated in the present study.

In hippocampal CA1 and CA3 regions, various properties of neuronal activity follow skewed, lognormal-like distributions, including average firing rates, rate and magnitude of spike bursts, magnitude of population synchrony, and correlations between pre- and postsynaptic spikes. In recent studies, the lognormal features of hippocampal activities were well replicated by a multi-timescale adaptive threshold (MAT) neuron network of lognormally distributed excitatory-to-excitatory synaptic weights, though it remains unknown whether and how other neuronal and network properties can be replicated in this model. Here we implement two additional studies of the same network: first, we further analyze its burstiness properties by identifying and clustering neurons with exceptionally bursty features, once again demonstrating the importance of the lognormal synaptic weight distribution. Second, we characterize dynamical patterns of activity termed neuronal avalanches in in vivo CA3 recordings of behaving rats and in the model network, revealing the similarities and differences between experimental and model avalanche size distributions across the sleep-wake cycle. These results show the comparison between the MAT neuron network and hippocampal readings in a different approach than shown before, providing more insight into the mechanisms behind activity in hippocampal subregions.
Nas regiões CA1 e CA3 do hipocampo, várias propriedades da atividade neuronal seguem distribuições assimétricas com características lognormais, incluindo frequência de disparo média, frequência e magnitude de rajadas de disparo (bursts), magnitude da sincronia populacional e correlações entre disparos pré- e pós-sinápticos. Em estudos recentes, as características lognormais das atividades hipocampais foram bem reproduzidas por uma rede de neurônios de limiar adaptativo (multi-timescale adaptive threshold, MAT) com pesos sinápticos entre neurônios excitatórios seguindo uma distribuição lognormal, embora ainda não se saiba se e como outras propriedades neuronais e da rede podem ser replicadas nesse modelo. Nesse trabalho implementamos dois estudos adicionais da mesma rede: primeiramente, analisamos mais a fundo as propriedades dos bursts identificando e agrupando neurônios com capacidade de burst excepcional, mostrando mais uma vez a importância da distribuição lognormal de pesos sinápticos. Em seguida, caracterizamos padrões dinâmicos de atividade chamados avalanches neuronais no modelo e em aquisições in vivo do CA3 de roedores em atividades comportamentais, revelando as semelhanças e diferenças entre as distribuições de tamanho de avalanche através do ciclo sono-vigília. Esses resultados mostram a comparação entre a rede de neurônios MAT e medições hipocampais em uma abordagem diferente da apresentada anteriormente, fornecendo mais percepção acerca dos mecanismos por trás da atividade em subregiões hipocampais.

The three-dimensional structure of a protein, formed as a result of amino-acid sequences folding into compact domains, is regarded as a key factor in its biological function. How and why proteins fold into specific topologies, remain the key focus of scientific research in the field of biophysics. By stripping down complex reactions down to the most basic elements, biophysicists aim to develop simplified models for biological phenomena such as antibody discrimination, viral fusion or self-assembly. Focusing on small model peptide systems, rather than the full proteins from which they were derived, was hoped to result in accurate structural measurements and provide a more transparent comparison between simulation and experiment. The aim of this research was therefore to investigate how accurate these models were when compared against experiment. Furthermore, while breaking down the complex biological phenomena into simple models, there was also a conscious effort to ensure that the models were representative of real biological systems, and a major focus was therefore aimed at determining whether any meaningful biomedical insight may be extrapolated from such models. Peptides found in hormones (human chorionic gonadotropin, luteinizing hormone), viruses (HIV) and amyloid diseases (transthyretin) were selected in order to probe a variety of questions in relation to the aforementioned biological phenomena. Namely, how the primary sequence influenced the three-dimensional structure (and thus its biological function), how its environment could influence such a confirmation, and how these systems aggregated. This doctoral study has made use of a combination of computer simulations and experimental techniques to investigate a selection of biologically relevant peptides; utilising classical atomistic molecular dynamics (MD) simulations to characterise the free-energy landscapes of the chosen peptides, and compare these findings with the secondary structure content predicted by spectroscopic methods such as circular dichroism and infrared spectroscopy. The peptide systems studied within, were found to be characterised by rugged free-energy landscapes unlike their protein counterparts (defined by singular, deep minima). Furthermore, these landscapes were found to be highly plastic and sensitive to changes in the local environment.

Theoretical studies based on first-principles theory are presented for a number of different magnetic systems. The first part of the thesis concerns spin dynamics and the second part concerns properties of magnetic multilayers. The theoretical treatment is based on electronic structure calculations performed by means of density functional theory.

A method is developed for simulating atomistic spin dynamics at finite temperatures, which is based on solving the equations of motion for the atomic spins by means of Langevin dynamics. The method relies on a mapping of the interatomic exchange interactions from density functional theory to a Heisenberg Hamiltonian. Simulations are performed for various magnetic systems and processes beyond the reach of conventional micromagnetism. As an example, magnetization dynamics in the limit of large magnetic and anisotropy fields is explored. Moreover, the method is applied to studying the dynamics of systems with complex atomic order such as the diluted magnetic semiconductor MnGaAs and the spin glass alloy CuMn. The method is also applied to a Fe thin film and a Fe/Cr/Fe trilayer system, where the limits of ultrafast switching are explored. Current induced magnetization dynamics is investigated by calculating the current induced spin-transfer torque by means of density functional theory combined with the relaxation time approximation and semi-classical Boltzmann theory. The current induced torque is calculated for the helical spin-density waves in Er and fcc Fe, where the current is found to promote a rigid rotation of the magnetic order.

Properties of magnetic multilayers composed of magnetic and nonmagnetic layers are investigated by means of the Korringa-Kohn-Rostocker interface Green's function method. Multilayer properties such as magnetic moments, interlayer exchange coupling and ordering temperatures are calculated and compared with experiments, with focus on understanding the influence of interface quality. Moreover, the influence on the interlayer exchange coupling of alloying the nonmagnetic spacer layers with small amounts of a magnetic impurity is investigated.

Enzymes are proteins that accelerate intracellular chemical reactions often by factors of 10(5) - 10(12)s(-1). We propose the structure and function of enzymes represent the thermodynamic expression of heritable information encoded in DNA with post-translational modifications that reflect intra- and extra-cellular environmental inputs. The 3 dimensional shape of the protein, determined by the genetically-specified amino acid sequence and post translational modifications, permits geometric interactions with substrate molecules traditionally described by the key-lock best fit model. Here we apply Kullback-Leibler (K-L) divergence as metric of this geometric "fit" and the information content of the interactions. When the K-L 'distance' between interspersed substrate p(n) and enzyme r(n) positions is minimized, the information state, reaction probability, and reaction rate are maximized. The latter obeys the Arrhenius equation, which we show can be derived from the geometrical principle of minimum K-L distance. The derivation is first limited to optimum substrate positions for fixed sets of enzyme positions. However, maximally improving the key/lock fit, called 'induced fit,' requires both sets of positions to be varied optimally. We demonstrate this permits and is maximally efficient if the key and lock particles p(n), r(n) are quantum entangled because the level of entanglement obeys the same minimized value of the Kullback-Leibler distance that occurs when all p(n) approximate to r(n). This implies interchanges p(n) reversible arrow br(n) randomly taking place during a reaction successively improves key/lock fits, reducing the activation energy E-a and increasing the reaction rate k. Our results demonstrate the summation of heritable and environmental information that determines the enzyme spatial configuration, by decreasing the K-L divergence, is converted to thermodynamic work by reducing Ea and increasing k of intracellular reactions. Macroscopically, enzyme information increases the order in living systems, similar to the Maxwell demon gedanken, by selectively accelerating specific reaction thus generating both spatial and temporal concentration gradients.

Initial forms of analytical models created to simulate real engineering structures may generally yield dynamic response predictions different than those obtained from experimental tests. Since testing a real structure under every possible excitation is not practical, it is essential to transform the initial mathematical model to a model which reflects the characteristics of the actual structure in a better way. By using structural model updating techniques, the initial mathematical model is adjusted so that it simulates the experimental measurements more closely. In this study, a sensitivity-based finite element (FE) model updating method using experimental frequency response (FRF) data is presented. This study bases on a technique developed in an earlier study on the computation of the so-called Mis-correlation Index (MCI) used for identifying the system matrices which require updating. MCI values are calculated for each required coordinate, and non-zero numerical values indicate coordinates carrying error. In this work a new model updating procedure based on the minimization of this index is developed. The method uses sensitivity approach. FE models are iteratively updated by minimizing MCI values using sensitivities. The validation of the method is realized through some case studies. In order to demonstrate the application of the method for real systems, a real test data obtained from the modal test of a scaled aircraft model (GARTEUR SM-AG19) is used. In the application, the FE model of the scaled aircraft is updated. In the case studies the generic software developed in this study is used along with some commercial programs.

Pluripotent stem cells (PSCs) are characterised by their ability to self-renew and to differentiate into tissues comprising the three developmental germ layers of the body. This makes them ideal sources of cells for use in regenerative medicine. However, their use for such purposes is hampered for a number of reasons. Both the reprogramming of somatic cells into PSCs and their differentiation for use in research and the clinic are extremely low yielding. This is in part likely to be a result of adaptation to the artificial culture environment, known to force cell structural away from the native state, resulting in altered gene expression. The mechanisms by which PSCs, specifically, integrate cues from their physical microenvironment remain largely unexplored. This project aimed to improve the culture environment of PSCs for their enhanced differentiation capacity. It was hypothesised that by altering the physical geometry of the culture substrate, the native PSC structure could be maintained, to improve performance upon differentiation. This work utilised the varying topographies provided by three commercial polyHIPE scaffolds (Alvetex®), and the human EC cell line, TERA2.cl.SP12, as a model PSC lineage. The three scaffolds of differing topography were considered for their ability to maintain pluripotency; a micro-topography resulted in immediate evidence of differentiation. A mixed micro/nano and nano-topography substrate appeared to maintain pluripotency. Due to its ability to allow complete cell removal, the smaller void size scaffold was used to develop a method for long-term PSC culture. This, however, resulted in gradual cell differentiation, suggesting that a mixed-topography was more suited for PSC growth. Alvetex® Strata has been previously shown to be a suitable substrate for the long-term maintenance of PSCs for enhanced differentiation (up to approximately 50 days). Here, we show that the same effect is possible from a single 10-day conditioning step, we term "priming", with evidence of enhanced differentiation in vitro and in vivo. The mechanisms of mechanotransduction in PSCs are poorly understood; the culture system developed here allowed for a direct comparison between two-dimensional (2D, conventional) and three-dimensional (3D) culture with substrate geometry as the only variable. The structure of the actin cytoskeleton, as well as that of intermediate filaments and microtubules, was less-developed after 3D culture. This was attributed to a marked decrease in the nucleation of actin, providing a correlative link to the enhanced differentiation possible after 3D priming. Additionally, there were significant differences in the presence of cell-matrix adhesions as well as evidence of reorganisation of the nuclear lamina. This process was successfully applied to the culture of mouse embryonic stem cells, with comparable effect, demonstrating amenability to functionalisation and scale-up development. Overall this system has allowed for the culture of PSCs for enhanced differentiation and allowed for novel insights into how they interact with their microenvironment through mechanotransduction.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

This study advances understanding of how the changes in ecosystem structure and function associated with woody shrub encroachment in semi-arid grasslands alter ecosystem carbon (C) dynamics. In terms of both magnitude and dynamism, dryland ecosystems represent a major component of the global C cycle. Woody shrub encroachment is a widespread phenomenon globally, which is known to substantially alter ecosystem structure and function, with resultant impacts on C dynamics. A series of focal sites were studied at the Sevilleta National Wildlife Refuge in central New Mexico, USA. A space-for-time analogue was used to identify how landscape structure and function change at four stages over a grassland to shrubland transition. The research had three key threads: 1. Soil-associated carbon: Stocks of organic and inorganic C in the near-surface soil, and the redistribution of these C stocks by erosion during high-intensity rainfall events were quantified using hillslope-scale monitoring plots. Coarse (>2 mm) clasts were found to account for a substantial proportion of the organic and inorganic C in these calcareous soils, and the erosional effluxes of both inorganic and organic C increased substantially across the vegetation ecotone. Eroded sediment was found to be significantly enriched in organic C relative to the contributing soil with systematic changes in OC enrichment across the vegetation transition. The OC enrichment dynamics observed were inconsistent with existing understanding (derived largely from reductionist, laboratory-based experiments) that OC enrichment is largely insignificant in the erosional redistribution of C. 2. Plant biomass: Cutting-edge proximal remote sensing approaches, using a remotely piloted lightweight multirotor drone combined with structure-from-motion (SfM) photogrammetry were developed and used to quantify biomass carbon stocks at the focal field sites. In such spatially heterogeneous and temporally dynamic ecosystems existing measurement techniques (e.g. on-the-ground observations or satellite- or aircraft-based remote sensing) struggle to capture the complexity of fine-grained vegetation structure, which is crucial for accurately estimating biomass. The data products available from the novel SfM approach developed for this research quantified plants just 15 mm high, achieving a fidelity nearly two orders of magnitude finer than previous implementations of the method. The approach developed here will revolutionise the study of biomass dynamics in short-sward ecogeomorphic systems. 3. Ecohydrological modelling: Understanding the effects of water-mediated degradation processes on ecosystem carbon dynamics over greater than observable spatio-temporal scales is complicated by significant scale-dependencies and thus requires detailed mechanistic understanding. A process-based, spatially-explicit ecohydrological modelling approach (MAHLERAN - Model for Assessing Hillslope to Landscape Erosion, Runoff and Nutrients) was therefore comprehensively evaluated against a large assemblage of rainfall runoff events. This evaluation highlighted both areas of strength in the current model structure, and also areas of weakness for further development. The research has improved understanding of ecosystem degradation processes in semi-arid rangelands, and demonstrates that woody shrub encroachment may lead to a long-term reduction in ecosystem C storage, which is contrary to the widely promulgated view that woody shrub encroachment increases C storage in terrestrial ecosystems.

The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties.  In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases form a complex at the stalled replication fork called the mutasome. This complex is structurally organized by the replication accessory factor PCNA and the non-classical DNA polymerase Rev1. One of the non-classical DNA polymerases within the mutasome then catalyzes replication through the damage. Each non-classical DNA polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical DNA polymerase within the mutasome is chosen to bypass the damage. In this thesis, I discuss how the most appropriate polymerase is chosen. In so doing, I examine the components of the mutasome; the structural motifs that mediate the protein interactions in the mutasome; the methods used to study translesion synthesis; the definition of a cognate lesion; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome. Taken together, this thesis provides and inclusive review of the current state of what is known about translesion synthesis with conclusions at its end suggesting what major questions remain and ideas of how to answer them.