published_or_final_version / Biochemistry / Master / Master of Philosophy

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has demonstrated that the regulatory domain of CaN (RD) is disordered. The binding of CaM to CaN results in RD folding. Folding of the RD in turn causes the autoinhibitory domain (AID) located C-terminal to the RD to be ejected from CaN’s active site. This binding-induced disorder-to-order transition is responsible for the activation of CaN by CaM. In this work, we explore the nature of the disorder in the RD and its transition to an ordered state, demonstrating that the RD exists in a compact disordered state that undergoes further compaction upon CaM binding. We also demonstrate that a single CaM molecule is responsible for binding to and activating CaN. Finally, we determine that the CaM binding to CaN induces an amphipathic helix (the distal helix) C-terminal to the CaM binding region. The distal helix undergoes a hairpin-like chain reversal in order to interact with the surface of CaM, resulting in the removal of the AID from CaN’s active site. We employ site-directed mutagenesis, size-exclusion chromatography, protein crystallography, circular dichroism spectroscopy, fluorescence anisotropy and correlation spectroscopy, and phosphatase activity assays to investigate the ordering of CaN’s regulatory domain, the stoichiometry of CaN:CaM binding, and the impact of the distal helix on CaM activation of CaN.

<p>Examensarbetet handlar om en jämförelsestudie av beräkningseffektivitet med avseende på energi- och tidsförbrukning mellan grafikkort och processorer i persondatorer och PlayStation 3.</p><p>Problemet studeras för att göra allmänheten uppmärksam på att det går att lösa en del av energiproblematiken med beräkningar genom att öka energieffektiviteten av beräkningsenheterna.</p><p>Undersökningen har genomförts på ett explorativt sätt och studerar förhållandet mellan processorer, grafikkort och vilken som presterar bäst i vilket sammanhang. Prestandatest genomförs med molekylberäkningsprogrammet F@H och med filkomprimeringsprogrammet WinRAR. Testerna utförs på MultiCore- och SingleCorePCs och PS3s av olika karaktär. I vissa test mäts effektförbrukning för att kunna räkna ut hur energieffektiva vissa system är.</p><p>Resultatet visar tydligt hur den genomsnittliga effektförbrukningen och energieffektiviteten för olika testsystem skiljer sig vid belastning, viloläge och olika typer beräkningar.</p> / <p>The thesis is a comparative study of computational efficiency in terms of energy and time consumption of graphics cards and processors in personal computers and Playstation3’s.</p><p>The problem is studied in order to make the public aware that it is possible to solve some of the energy problems with computations by increasing energy efficiency of the computational units.</p><p>The audit was conducted in an exploratory way, studying the relationship between the processors, graphics cards and which one performs best in which context. Performance tests are carried out by the molecule calculating F@H-program and the file compression program WinRAR. Tests performed on MultiCore and SingleCore PC’s and PS3’s with different characteristics. In some tests power consumption is measured in order to figure out how energy-efficient certain systems are.</p><p>The results clearly show how the average power consumption and energy efficiency for various test systems at differ at load, sleep and various calculations.</p><p> </p>

The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.

Baigiamajame magistro darbe tirta popieriaus paviršiaus ir mechaninių savybių kaita spausdinant fleksografiniais dažais ir keičiant dažų sluoksnio storį. Tyrimams buvo pasirinktos šešios kreidinio ir nekreidinio popieriaus rūšys. Buvo tirtos šių mechaninių ir paviršiaus savybių kaita: spalvinės charakteristikos, atsparumas tempimui ir pailgėjimas, atsparumas lankstymui, PPS šiurkštumas, trinties savybės, popieriaus sugertis. Buvo nustatyta, kad fleksografiniai dažai didina popieriaus šiurkštumą, trūkio jėga ir atsparumas lankstymui visada yra didesni išilgine popieriaus kryptimi, atsparumas tempimui nepriklauso nuo dažų sluoksnio storio, o atsparumas lankstymui mažėja. Darbą sudaro 6 dalys: įvadas, literatūros apžvalga, tyrimo metodika, tyrimų rezultatai ir jų aptarimas, išvados ir siūlymai, literatūros sąrašas. Darbo apimtis – 83 psl. teksto be priedų, 82 iliustracijos, 1 lentelė, 28 bibliografinių šaltinių. / In the Master thesis were investigated the change of the surface and mechanical properties during flexo printing with different ink thickness. 6 papers coated and uncoated grades were tested. Following properties were investigated: colour properties, tension strength and elongation, folding strength, PPS roughness and friction properties. It was found that flexographic ink increases paper PPS roughness, tensile strength and folding resistance is higher in the machine direction in all cases, tensile strength does not depend on the ink thickness and folding resistance becomes smaller. Master thesis includes 6 chapters: introduction, publications review, experimental methods, results and discussion, conclusions and recommendations, references. Master thesis consists of 83 p. (without appendixes), 82 fig., 1 table, and 28 references.

Two global optimization methods are proposed in this thesis. They are the multicanonical basin hopping (MUBH) method and the basin paving (BP) method. <br /><br /> The MUBH method combines the basin hopping (BH) method, which can be used to efficiently map out an energy landscape associated with local minima, with the multicanonical Monte Carlo (MUCA) method, which encourages the system to move out of energy traps during the computation. It is found to be more efficient than the original BH method when applied to the Lennard-Jones systems containing 150-185 particles. <br /><br /> The asynchronous multicanonical basin hopping (AMUBH) method, a parallelization of the MUBH method, is also implemented using the message passing interface (MPI) to take advantage of the full usage of multiprocessors in either a homogeneous or a heterogeneous computational environment. AMUBH, MUBH and BH are used together to find the global minimum structures for Co nanoclusters with system size <em>N</em>&le;200. <br /><br /> The BP method is based on the BH method and the idea of the energy landscape paving (ELP) strategy. In comparison with the acceptance scheme of the ELP method, moving towards the low energy region is enhanced and no low energy configuration may be missed during the simulation. The applications to both the pentapeptide Met-enkephalin and the villin subdomain HP-36 locate new configurations having energies lower than those determined previously. <br /><br /> The MUBH, BP and BH methods are further employed to search for the global minimum structures of several proteins/peptides using the ECEPP/2 and ECEPP/3 force fields. These two force fields may produce global minima with different structures. The present study indicates that the global minimum determination from ECEPP/3 prefers helical structures. Also discussed in this thesis is the effect of the environment on the formation of beta hairpins.

Le dépliement thermique et chimique est raisonnablement compris, mais pas les effets déstabilisants de la pression. Dans une tentative de caractérisation des facteurs à la base des effets de la pression sur les protéines, nous avons étudié le dépliement sous pression d'une protéine modulaire, le domaine ankyrine du récepteur Notch (Nank1-7*), ainsi que plusieurs mutants en nombre de motifs. Nos expériences montrent un dépliement à deux états sous pression. La dépendance à la température des sauts de pression a montré qu'à faibles températures l'ensemble d'état de transition(TSE) est proche en volume de l'état replié alors que son énergie est proche de celle de l'état déplié,ce qui est significatif d'une déshydratation importante de la barrière énergétique, et a montré que l'augmentation de la température conduit à un TSE plus grand en volume que l'état replié. Ce comportement révèle une grande plasticité du TSE de Nank1-7*. L'étude des mutants (délétion demotifs) nous a montré que le ΔV n'est déterminé ni par l'hydratation des liaisons peptidiques ni par l'hydratation différentielle des acides aminés, mais par l'existence de vides internes exclus du solvant. Seule une petite fraction des cavités est déterminante pour le ΔV, celles qui ne sont pas significativement solvatées dans les coeurs hydrophobes. Finalement, nous avons déterminé que le TSE possède même des cavités plus grandes, et nous émettons l'hypothèse que l'énergétique et la dynamique contribuent aux effets de la pression sur les protéines. / Thermal and chemical unfolding of proteins are reasonably well understood, but the destabilizingeffects of pressure are not. In an attempt to characterize the factors at the basis of pressure effects,we investigated the pressure unfolding of a modular protein, the ankyrin domain of the Notch receptor(Nank17*) and several of its deletion constructs. All our experiments were consistent with a simpletwo-state folding/unfolding transition under pressure. The temperature dependence of pressure-jumpsdemonstrated that at low temperature, the transition state ensemble (TSE) lies close in volume to thefolded state despite its unfolded-like energetics, consistent with significant dehydration at the barrier,and that increasing temperature leads to a volume of the TSE larger than that of the folded state. Thisbehavior reveals a high degree of plasticity of the TSE of Nank17*. Studies of the deletion mutantsshowed us that ΔV is determined not by hydration of peptide bonds or by differential hydration ofresidues, but by the existence of internal solvent-excluded void volumes. Only a small fraction of theinternal cavities are relevant of the ΔV, those that are not significantly solvated in the hydrophobiccore. Finally, we determined that the transition state ensemble show even larger cavities, and wehypothesize both energetics and dynamics contribute to pressure effects on proteins.

In the early 1990s, South Africa’s re-entry into the competitive global marketplace and the first non-racial elections brought significant changes to an industry previously plagued by the racialisation of the labour process. South Africa’s post-apartheid work order led to the restructuring of the gold mining workplace, with a greatly increased emphasis on efficiency, productivity and equity. This period saw a number of gold mines reorganising work through new forms of working practices aimed at creating new kinds of workers who could identify with the goals of the company by expending rather than withdrawing effort at the point of production. There was a shift in the attitude of worker responses to managerial practices, from coercion to consent in the day-to-day running of the production process. This thesis examines worker responses to the reorganisation of work and their impact on worker and workplace productivity in a deep-level gold mine. At the core of this thesis are the perceptions, views, experiences and reactions displayed by underground work teams to management initiatives. The thesis highlights the significance of worker agency in managerially defined work structures – the capacity of underground gold miners to reshape and adapt management strategies in ways that make sense and enable them to maintain control over production and the effort-bargain. The findings presented in this thesis, particularly the gold miners’ informal or coping strategy of making a plan (planisa), reveal that underground work teams are not merely passive or docile reactors to management initiatives. They find opportunity to manipulate (and where necessary, avoid) new forms of management control in a variety of innovative ways that enable them to reassert their power and autonomy over their working day. Underground gold miners are not simply appendages to nor alienated beings in the production process but are able to take control of the production process, independent of management prescriptions, in ways which may embody resistance, consent or a subtle combination of the two. The thesis calls attention to workers’ subjective orientation, agency and resilience to new work structures – not just as recipients but also as shapers of such new work structures within the politics, limits and contradictions of capitalist production systems.

This thesis considers all of Ernest McClain's published writings, from March, 1970, to September, 1976, from the standpoint of their present-day acoustical significance. Although much of the material comes from McClain's writings, some is drawn from other related musical, mathematical, and philosophical works. The four chapters begin with a biographical sketch of McClain, presenting his background which aided him in becoming a theoretical musicologist. The second chapter contains a chronological itemization of his writings and provides a synopsis of them in layman's terms. The following chapter offers an examination of some salient points of McClain's work. The final chapter briefly summarizes the findings and contains conclusions as to their germaneness to current music theory, thereby giving needed exposure to McClain's ideas.

<p>Protein biomarkers can facilitate the diagnosis of many diseases such as cancer and they can be important for the development of effective therapeutic interventions. Current large-scale biomarker discovery and disease state characterization studies have largely focused on the global analysis of gene and protein expression levels, which are not directly tied to function. Moreover, functionally significant proteins with similar expression levels go undetected in the current paradigm of using gene and protein expression level analyses for protein biomarker discovery. Protein-ligand interactions play an important role in biological processes. A number of diseases such as cancer are reported to have altered protein interaction networks. Current understanding of biophysical properties and consequences of altered protein interaction network in disease state is limited due to the lack of reproducible and high-throughput methods to make such measurements. Thermodynamic stability measurements can report on a wide range of biologically significant phenomena (e.g., point mutations, post-translational modifications, and new or altered binding interactions with cellular ligands) associated with proteins in different disease states. Investigated here is the use of thermodynamic stability measurements to probe the altered interaction networks and functions of proteins in disease states. This thesis outlines the development and application of mass spectrometry based methods for making proteome-wide thermodynamic measurements of protein stability in multifactorial complex diseases such as cancer. Initial work involved the development of SILAC-SPROX and SILAC-PP approaches for thermodynamic stability measurements in proof-of-concept studies with two test ligands, CsA and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). In these proof-of-principle studies, known direct binding target of CsA, cyclophilin A, was successfully identified and quantified. Similarly a number of known and previously unknown ATP binding proteins were also detected and quantified using these SILAC-based energetics approaches. </p><p>Subsequent studies in this thesis involved thermodynamic stability measurements of proteins in the breast cancer cell line models to differentiate disease states. Using the SILAC-SPROX, ~800 proteins were assayed for changes in their protein folding behavior in three different cell line models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. Approximately, 10-12% of the assayed proteins in the comparative analyses performed here exhibited differential stability in cell lysates prepared from the different cell lines. Thermodynamic profiling differences of 28 proteins identified with SILAC-SPROX strategy in MCF-10A versus MCF-7 cell line comparison were also confirmed with SILAC-PP technique. The thermodynamic analyses performed here enabled the non-tumorigenic MCF-10A breast cell line to be differentiated from the MCF-7 and MDA-MB-231 breast cancer cell lines. Differentiation of the less invasive MCF-7 breast cancer cell line from the more highly invasive MDA-MB-231 breast cancer cell line was also possible using thermodynamic stability measurements. The differentially stabilized protein hits in these studies encompassed those with a wide range of functions and protein expression levels, and they included a significant fraction (~45%) with similar expression levels in the cell line comparisons. These proteins created novel molecular signatures to differentiate the cancer cell lines studied here. Our results suggest that protein folding and stability measurements complement the current paradigm of expression level analyses for biomarker discovery and help elucidate the molecular basis of disease.</p> / Dissertation