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<p>A novel approach is proposed for modeling of structurally variable regions in proteins. In this approach, a prerequisite sequence-structure alignment is examined for regions where the target sequence is not covered by the structural template. These regions, extended with a number of residues from adjacent stem regions, are submitted to fold recognition. The alignments produced by fold recognition are integrated into the initial alignment to create a multiple alignment where gaps in the main structural template are covered by local structural templates. This multiple alignment is used to create a protein model by existing protein modeling techniques.</p><p>Several alternative parameters are evaluated using a set of ten proteins. One set of parameters is selected and evaluated using another set of 31 proteins. The most promising result is for loop regions not located at the C- or N-terminal of a protein, where the method produces an average RMSD 12% lower than the loop modeling provided with the program MODELLER. This improvement is shown to be statistically significant.</p>

Most amino acid sequences are predicted to specify a single three-dimensional protein structure. However, the identification of "metamorphic" proteins, which can adopt two folds from a single amino acid sequence, has challenged the one sequence/one structure paradigm. Polar-to-hydrophobic substitutions have been suggested computationally as one mechanism to decrease structural specificity, allowing the population of novel folds. Here, we experimentally investigate the role of polar-to-hydrophobic substitutions on structural specificity in the homodimeric ribbon-helix-helix protein Arc repressor. Previous work showed that a single polar-to-hydrophobic surface substitution in the strand region of Arc repressor (Arc-N11L) populates the wild-type fold and a novel dimeric "switch" fold. In this work, we investigate an Arc repressor variant with the N11L substitution plus two additional polar-to-hydrophobic surface substitutions (Arc-S-VLV). We determine that this sequence folds into at least three structures: both dimer forms present in Arc-N11L, and a novel octamer structure containing higher stability and less helicity than the dimer folds. We are able to isolate and stabilize a core of the S-VLV octamer by limited trypsinolysis and deletion mutagenesis (Arc-VLV 4-44). The shortened construct contains only the octameric structure by removing disordered C-terminal segments nonessential for this fold. A two-dimensional NMR spectrum of VLV 4-44 and subsequent trypsinolysis of this construct suggests that at least two types of subunits comprise the S-VLV octamer: subunits structured from residues 4 to 44 and subunits structured from residues 4 to 31. Crystal trials of trypsinolyzed Arc-VLV 4-44 yielded several leads, suggesting that obtaining a high resolution structure of the S-VLV octamer is possible. Relatedly, we determine that the proline residues flanking the Arc repressor strand act in concert as "gatekeepers" to prevent aggregation in the S-VLV sequence. We also find that three highly hydrophobic surface substitutions in the Arc repressor strand region are necessary and sufficient to promote higher-order oligomer formation. In summation, this work reveals in an experimental context that progressive increases in polar-to-hydrophobic surface substitutions populate increasingly diverse, structurally degenerate folds. These results suggest that "metamorphic" as well as "polymetamorphic" proteins, which adopt numerous folds, are possible outcomes for a single protein sequence.

Exploration for large zinc silicate deposits is more challenging than zinc sulfide deposits, as they do not exhibit similar geophysical anomalies. The Vazante deposit, which is the world’s largest zinc silicate deposit, occurs in brecciated dolomite and comprises mainly willemite with various proportions of hematite, and minor franklinite and sphalerite. In the Vazante region, the exploration challenge is enhanced as outcrops are rare, bedrock generally sits below 10s of metres of laterite cover and barren hematite-rich breccias have a similar geophysical signature to willemite ore bodies. In order to evaluate the applications of geophysical surveys in the exploration of this type of deposit, data from 475 samples were investigated from drill holes representative of the various types of ore, host rocks and zones of known geophysical anomalies in the Vazante District. Geochemical (ICP-MS and XRF) and mineralogical (optical, EMPA, SEM and MLA) data were integrated with physical rock properties (density, magnetic susceptibility and K-U-Th gamma ray spectrometry) to assist in finding new ore zones. The most distinct physical property of the ore is density (3.0-4.3 g/cm3), compared with the host rocks (2.7-3.0 g/cm3). This is due to high proportion of denser minerals (hematite and willemite) in the ore. However, barren hematite breccias also have high densities (3.0-4.5 g/cm3). The zinc ore and hematite breccias yielded higher magnetic susceptibilities (0.1-38 x10-3 SI) than the surrounding host rocks, with the highest values associated with greater proportions of franklinite and magnetite (7-38 x10-3 SI). The zinc ore has an elevated U concentration (up to 33ppm) relative to the various host rocks (up to 7 ppm), yielding higher gamma spectrometric values. The results of this investigation indicate that an integration of magnetic, gravimetric and radiometric surveys would be required to identify zinc silicate ore zones and potentially differentiate them from barren hematite breccias and host rocks. / Thesis (Master, Geological Sciences & Geological Engineering) -- Queen's University, 2014-03-27 13:32:54.132

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Understanding the involvement of the central nervous system (CNS) in voice production is essential to incorporating principles of neuroplasticity into therapeutic practice for voice disorders. Early steps to attaining this goal require the identification of specific neural biomarkers of the changes occurring in the CNS from a voice disorder and its subsequent treatment. In the absence of an adequate animal vocalization model, the larynx has not been acutely and reversibly perturbed to concurrently examine the effect on both peripheral and central processing of the altered input/output. Using a unique, reversible perturbation approach, it was the purpose of this study to perturb the larynx to mimic a voice disorder and study short-term neuroplastic response. Functional magnetic resonance imaging (fMRI) was the neuroimaging tool of choice for this study due to its superior spatial and temporal resolution. The voice was perturbed by anesthetizing the right recurrent laryngeal nerve, with a solution of lidocaine hydrochloride and epinephrine to induce a temporary right vocal fold paralysis. The paralysis lasted for approximately 90 minutes and had an overt presentation similar to that of a true vocal fold paralysis. Behavioral and fMRI data were obtained at three time points- baseline, during the vocal fold paralysis and one hour after recovery. Patterns of activity on fMRI during the three time points were found to be distinct on both subjective examination and statistical analysis. The regions of interest examined had distinct trends in activity as a function of the paralysis. Interestingly, males and females responded differently to the paralysis and its subsequent recovery. Strong correlation was not observed between the behavioral measures and fMRI activity reflecting a disparity between the overt presentation and recovery of vocal fold paralysis and cortical activity as seen on fMRI. The fictive paralysis model employed in this study provided a perturbation model for phonation that allowed us to examine behavioral and central neural correlates for disordered phonation in a controlled environment. Although this data is representative of acute changes from a transient paralysis, it provides an insight into the response of the cortex to sudden perturbation at the peripheral phonatory mechanism.

In the recent years, a rapidly increasing amount of experimental data has been generated by high-throughput technologies. Despite of these large quantities of protein-related data and the development of computational prediction methods, the function of many proteins is still unknown. In the human proteome, at least 20% of the annotated proteins are not characterized. Thus, the question, how to predict protein function from its amino acid sequence, remains to be answered for many proteins. Classical bioinformatics approaches for function prediction are based on inferring function from well-characterized homologs, which are identified based on sequence similarity. However, these methods fail to identify distant homologs with low sequence similarity. As protein structure is more conserved than sequence in protein families, structure-based methods (e.g. fold recognition) may recognize possible structural similarities even at low sequence similarity and therefore provide information for function inference. These fold recognition methods have already been proven to be successful for individual proteins, but their automation for high-throughput application is difficult due to intrinsic challenges of these techniques, mainly caused by a high false positive rate. Automated identification of remote homologs based on fold recognition methods would allow a signi cant improvement in functional annotation of proteins. My approach was to combine structure-based computational prediction methods with experimental data from genome-wide RNAi screens to support the establishment of functional hypotheses by improving the analysis of protein structure prediction results. In the first part of my thesis, I characterized proteins from the Ska complex by computational methods. I showed the benefit of including experimental information to identify remote homologs: Integration of functional data helped to reduce the number of false positives in fold recognition results and made it possible to establish interesting functional hypotheses based on high con dence structural predictions. Based on the structural hypothesis of a GLEBS motif in c13orf3 (Ska3), I could derive a potential molecular mechanism that could explain the observed phenotype. In the second part of my thesis, my goal was to develop computational tools and automated analysis techniques to be able to perform structure-based functional annotation in a high-throughput way. I designed and implemented key tools that were successfully integrated into a computational platform, called StrAnno, which I set up together with my colleagues. These novel computational modules include a domain prediction algorithm and a graphical overview that facilitates and accelerates the analysis of results. StrAnno can be seen as a first step towards automatic functional annotation of proteins by structure-based methods. First, the analysis of long hit lists to identify promising candidates for further analysis is substantially facilitated by integration and combination of various sequence-based computational tools and data from functional databases. Second, the developed post-processing tools accelerate the evaluation of structural and functional hypotheses. False positives from the threading result lists are removed by various filters, and analysis of the possible true positives is greatly enhanced by the graphical overview. With these two essential benefits, fold recognition techniques are applicable to large-scale approaches. By applying this developed methodology to hits from a genome-wide cell cycle RNAi screen and evaluating structural hypotheses by molecular modeling techniques, I aimed to associate biological functions to human proteins and link the RNAi phenotype to a molecular function. For two selected human proteins, c20orf43 and HJURP, I could establish interesting structural and functional hypotheses. These predictions were based on templates with low sequence identity (10-20%). The uncharacterized human protein c20orf43 might be a E3 SUMO-ligase that could be involved either in DNA repair or rRNA regulatory processes. Based on the structural hypotheses of two domains of HJURP, I predicted a potential link to ubiquitylation processes and direct DNA binding. In addition, I substantiated the cell cycle arrest phenotype of these two genes upon RNAi knockdown. Fold recognition methods are a promising alternative for functional annotation of proteins that escape sequence-based annotation due to their low sequence identity to well-characterized protein families. The structural and functional hypotheses I established in my thesis open the door to investigate the molecular mechanisms of previously uncharacterized proteins, which may provide new insights into cellular mechanisms.

Superimposition of contractional structures is widely observed in different scales in the world. Superimposed structures form due to different processes: change in strain accommodation from one type of structure to another during a single progressive shortening; successive coaxial shortening phases separated by an unconformity; superimposition of different non-coaxial shortening phases. Using results of a series of systematic analogue models and detailed field structural mapping, this thesis focuses on the geometry and kinematics of such superimposed structures that are formed by these three processes. During a single progressive folding, thrusts develop within a fold to accommodate stain variations in different regime of the fold. Limited displacement along these thrusts does not significantly modify the geometry of the fold. However, during multiple shortening phases (coaxial or non-coaxial), early formed structures are modified by the later phase ones. The later thrusts can cut and displace the pre-existing structures. The early folds are tightened or interfered by the later folding phase. Pre-existing thrusts may be reactivated either in dip direction and/or along strike during the later shortening. The pre-existing structures in turn influence development of the later structures, which results in change in structure spacing. An angular unconformity between two shortening phases clearly truncates the early phase structures and separates structures of different levels. Unlike in the post-erosional layers, in the layers below the unconformity, complicated superimposed structures are visible. This thesis shows that geometry and sequence of structures formed during one progressive shortening or multiple shortening phases strongly depend on the mode of the superimposition (coaxial, orthogonal or oblique) and the orientation of pre-existing structures.

The Early Permian strata of the Hongliuhe Group, NW China, experienced a thin-skinned fold-and-thrust belt style of deformation that recorded the final stages of amalgamation of the Beishan orogenic collage, a part of the Central Asian Orogenic Belt. The Hongliuhe Group was syn-orogenically deposited on an undetermined foreland, with the Mazongshan arc terrane acting as the hinterland. In this study results from detailed mapping combined with a regional analysis elucidate involvement of a northward-dipping subduction system with the collision. Well-preserved fold-and-thrust belt style deformation mapped in the upper stratigraphy of the Hongliuhe Group exhibits dominantly south-southeast verging structure, including shear folding, low-angle thrust ramping, imbrication and duplexing. Restoration of a portion of a mapped outcrop-scale cross-section estimates the accommodation of a minimum of 24% shortening. Lower stratigraphy shows discrete, steeper, north-over-south dip-slip ductile shear zones that bound packages of less deformed Hongliuhe Group strata. Fault displacement is considered to have been prolonged enough to juxtapose basal formations in northerly hangingwalls against upper formations in southerly footwalls. Faulting is closely associated with the creation of large-scale brittle-ductile eye-fold structures that are postulated to be sheath folds. The most examined and mapped structure, 16km wide, is a synclinal structure with axes plunging steeply towards its center. The ellipticity of the exposed bedding traces increases towards the center of the eye-fold, implying a structural relationship with metamorphic shear zones. Except for large-scale folding, the bulk of its strata remain relatively undeformed and have preserved primary soft-sediment deformation structures indicating younging towards the center on both limbs of the synclinal structure. Stratigraphic reconstruction of the Hongliuhe Group that considers the significant faulting shows that the Group's basal conglomerates unconformably overlie a Late-Carboniferous volcanic assemblage. The clast lithotypes of the conglomeratic successions change from polymictic metamorphic rocks at the base to monomictic granitoid clasts mid-section, showing the gradual unroofing sequence of the provenance. The stratigraphic reconstruction shows a general fining upward sequence, transitioning from terrestrial to nearshore marine depositional environments that, and in conjunction with the conglomeratic successions, suggests that the tectonic setting for deposition of the Hongliuhe Group is a foreland basin. Considering the deformation styles reported in this study, the Hongliuhe Group is interpreted to be a foreland fold-and-thrust belt. Stratigraphic reconstruction of the Hongliuhe Group that considers the significant faulting shows that the Group’s basal conglomerates unconformably overlie a Late Carboniferous volcanic assemblage. The clast lithotypes of the conglomeratic successions change from polymictic metamorphic rocks at the base to monomictic granitoid mid-section, showing the gradual unroofing sequence of the provenance. The stratigraphic reconstruction shows a general fining upward sequence through nearshore depositional environments that, and in conjunction with the conglomeratic successions, give interpretation that the tectonic setting for deposition of the Hongliuhe Group is a foreland basin. Considering the deformation styles reported in this study the Hongliuhe Group is interpreted to be a foreland fold-and-thrust belt.

Tissue engineering (TE) approaches have emerged as an alternative to traditional tissue and organ replacements. The aim of this work was to contribute to the understanding of the effects of cell-material and endothelial cell (EC) paracrine signaling on cell responses using poly(ethylene glycol) diacrylate (PEGDA) hydrogels as a material platform. Three TE applications were explored. First, the effect of glycosaminoglycan (GAG) identity was evaluated for vocal fold restoration. Second, the influence of GAG identity was explored and a novel approach for stable endothelialization was developed for vascular graft applications. Finally, EC paracrine signaling in the presence of cyclic stretch, and hydrophobicity and inorganic content were studied for osteogenic applications. In terms of vocal fold restoration, it was found that vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by GAG identity. VFF phenotype was preserved in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). Furthermore, collagen I deposition, fibronectin production and smooth muscle alpha-actin (SM-alpha-actin) expression in PEG-HA, PEG-chondroitin sulfate C and PEG- heparan sulfate (HS) gels suggest that CSC and HS may be undesirable for vocal fold implants. Regarding vascular graft applications, the impact of GAG identity on smooth muscle cell (SMC) foam cell formation was explored. Results support the increasing body of literature that suggests a critical role for dermatan sulfate (DS)-bearing proteoglycans in early atherosclerosis. In addition, an approach for fabricating bi-layered tissue engineering vascular grafts (TEVGs) with stable endothelialization was validated using PEGDA as an intercellular "cementing" agent between adjacent endothelial cells (ECs). Finally, mesenchymal stem cell (MSC) differentiation toward osteogenic like cells was evaluated. ECM and cell phenotypic data showed that elevated scaffold inorganic content and hydrophobicity were indeed correlated with increased osteogenic differentiation. Moreover, the present results suggest that EC paracrine signaling enhances MSC osteogenesis in the presence of cyclic stretch.