Doctor of Philosophy / Division of Biology / Jeroen Roelofs / The ubiquitin-proteasome pathway is the major pathway of selective protein degradation in the cell. Disruption of this pathway affects cellular protein homeostasis and contributes to diseases like cancer, and neurodegeneration. The end point of this pathway is the proteasome, a complex protease formed by 66 polypeptides. Structurally, it can be subdivided into the Core Particle (CP) and the Regulatory Particle (RP). The CP harbors the proteolytic sites, whereas, the RP contains six orthologous AAA-ATPases, the Rpt proteins. These Rpt’s are essential for proteasome function and are at the interface between RP and CP. The work in this thesis focuses on the Rpt subunit Rpt5 from yeast. The C-terminal tail of Rpt5 has been shown to contribute to the binding with the CP. However, our study showed it is also essential for the interaction with Nas2, one of nine proteasome-specific chaperones. Thus, Nas2 might function as a regulator of the Rpt5-CP interaction. Further analyses suggested that Nas2 has an additional function in assembly, and that mutating the tail of Rpt5 results in increased binding of the proteasome-associated protein Ecm29 to the proteasome. We showed that Ecm29 binds Rpt5 directly, thereby inducing a closed conformation of the CP substrate entry channel, and inhibiting proteasomal ATPase activity. Consistent with these activities, several proteasome mutant strains showed Ecm29-dependent accumulation of unstable substrates. Thus, Ecm29 is an inhibitor of the proteasome in vivo and in vitro. Interestingly, besides the Rpt5 mutants, several other proteasome mutants show increased levels of Ecm29, suggesting Ecm29 has a role in quality control. Consistent with this, we observed that Ecm29 associates preferably with specific mutants and nucleotide-depleted proteasomes. Based on our data we propose a model, where early in assembly Nas2 binds to the Rpt5 tail inhibiting the Rpt5-CP interaction directly. Later in assembly Ecm29 performs a quality control function, where it recognizes and remains bound to defective proteasomes. By inhibiting these proteasomes Ecm29 prevents the aberrant degradation of proteins.

Call number: LD2668 .T4 1964 D63 / Master of Science

LD2668 .T4 1966 D35 / Master of Science

At cable winding today one uses one portal, one caterpillar, one spooling boom and one tower and the same kind of set of equipment for unwinding. This kind of solution is both expensive and unpractical when one of the components of unwinding/winding is stationary for a long time without being used. To save unnecessary expenditure and maintenance it would have been practical to only use one set of caterpillar, tower, spooling boom and portal that later on can be rotated around the turntable when exporting the cable. The project that this work includes is to bring a concept that excludes two sets and thus provides the system with a rotatable spooling system. To get as much information about the product and of the manufacturing as possible several interviews has been done of the forging shop Ronneby Svets & Smide and also their fitters. At the concept generation several proposals was developed and to go further with only one of them a QFD and a SWOT analysis was made to get the best concept. Verification of solid mechanics on the construction with FEM-analysis has been implemented on beams, tubes and tube constructions. The project comprises constructing of details and drawings for structure of the concept and also commitment of assembly, maintenance, ergonomics and shipping. At the assembly has bolted joints been used on most attachments for fast assembling on site instead of welds. For commitment of maintenance it is thought that one should easily access for control and tighten afterwards of example the slewing ring. At the ergonomics the idea is put for example to accessing the workspace or maintenance areas in form of a ladder or a staircase. The shipping has played a major role for the construction parts and has then been divided for easiest and most practical way to be transported smoothly.

Inspired by the performance and evolutionarily-optimised natural molecular machines that carry out all the essential tasks contributing to the molecular basis of life, chemists aim towards fabricating synthetic molecular machines that mimic biological nanodevices. The use of rotaxanes as a prototype for molecular machines has emerged as a result of their ability to undergo translational motion between two or more co-conformations. Although biological machines are capable of complex and intricate functions, their inherent stability and operational conditions are restricted to in vivo. Synthetic systems offer a limitless number of building blocks and a range of interactions to be manipulated. Transition metal-ligand interactions are utilised as one strategy to control the directional movement of submolecular components in artificial machines due to their well-defined geometric requirements and significant strength. This thesis presents new externally addressable and switchable molecular elements for transition metal complexed-molecular machines involving an acid-base switch. The proton input that induces changes to cyclometallated platinum complexes can be exploited to control exchange between different coordination modes. The development of the pH-switchable metal-ligand motif for the stimuli-responsive platinum-complexed molecular shuttle has also been explored. The metal-directed self-assembly of tubular complexes were studied in order to develop self-assembled rotaxanes. A series of metal building blocks was explored to extend the scope for a tube self-assembly.

Inspired by nature’s use of self-assembled systems to carry out virtually all biological processes, chemists have taken to building simplified synthetic systems that mimic the biotic world. Although transition metal-ligand interactions are rarely used for the purpose of biological self-assembly, they have several advantages over other weak noncovalent interactions, such as pronounced directionality and significant strength. These particular attributes have allowed chemists to construct a comprehensive library of self-assembled polygons and polyhedra, using different transition metal-ligand motifs. Many of these supramolecular assemblies possess cavities of defined shape and size, which are able to accommodate guest molecules. It has further been realised that isolation of guest species from the bulk phase can lead to many interesting functions, such as containment, sensing and catalysis. Herein, a new self-assembly strategy has been used to construct novel cyclometalated Pt cages and assembly of the first known [Ir(ppy)2]-based capsule has also been achieved. Chapter 1 includes an introduction to metallosupramolecular assemblies, followed by a comprehensive review of three-dimensional architectures with accessible cavities, their synthetic strategies and applications. Chapter 2 reports on the construction of novel Pt(II)-based trigonal prisms using an unusual, kinetically controlled protocol. By exploiting asymmetric cyclometalated 2-phenylatopyridine based platinum corner units that possess both labile and non-labile cis-coordination sites, trigonal prismatic stereoisomeric architectures have been selectively prepared by altering the sequence of addition of ditopic 4,4′-bipy (4,4′-bipyridine) and tritopic tpt (2,4,6-tris(4-pyridyl)-1,3,5-triazine) molecular structural components using a template free method. Collision-induced-dissociation mass spectrometry experiments were used to differentiate between the structural isomers due to their significantly different fragmentation profiles. Chapter 3 describes the synthesis and characterisation of the first molecular capsule based on an [Ir(ppy)2]+ 90° metallosupramolecular acceptor unit. Initial work focused on pyridine-based donor ligands from which an Ir2L2 metallamacrocycle was assembled. However, when the highly conjugated tpt “panels” were used, due to postulated constraints in the dihedral angle, self-assembly of the Ir6tpt4 octahedral was unsuccessful. The constraints in the dihedral angle were eliminated by swapping pyridine for nitrile-based ligands and following the development of a method to resolve rac-[(Ir(ppy)2Cl)2] into its enantiopure forms, homochiral Ir6tcb4 (tcb = 1,3,5-tricyanobenzene) octahedral capsules where realised. Photophysical studies on the Ircapsules have shown that the ensemble of cooperative, weakly coordinating ligands can lead to luminescence not present in the comparative mononuclear analogues. X-ray crystallographic analysis revealed that the Ir capsules possess cavities large enough to accommodate 4 triflate counterions. Through a series of titration experiments the ability of the capsules to act as anion sensors was also exposed. Further exploration into the host-guest chemistry of the Ir6tcb4 capsule is reported in Chapter 4. Subsequent experiments have shown that self-assembly is highly dependent on the counterions associated with the system. While a number of different anions (OTf-, BF4 -, ClO4 -, PF6 -) facilitate the formation of the same octahedral scaffold, when triflimide was employed as a bulkier counterion, no capsule was observed. On subsequent addition of smaller counterions, such as triflate, the same Ir6tcb4 cage assembles, demonstrating that the anions also act as templates. Kinetic stability experiments, undertook by monitoring the rate of scrambling of the Δ and Λ-[Ir(ppy)2]+ components within the preformed ensembles, show that the Ir capsules are up to 1.4×104 times more stable than their mononuclear analogues. The counter anions were also observed to play a crucial role in the capsule’s stability with measured scrambling half-lives ranging from 4.7 mins with tetrafluoroborate to as long as 4.5 days with triflate. In contrast, the rate of ligand exchange in simple mononuclear complexes, as ascertained using EXSY NMR experiments, was found to be approximately independent of the associated anion.

Hydrogels are water-swellable cross-linked polymeric networks that are capable of incorporating a variety of functionalities and responsivities. The stable colloidal form of a hydrogel is known as a microgel and ranges in size from the nano- to the micrometer scale. Microgels can exhibit similar properties to hydrogels, but the colloidal size of the microgel creates differences in their responsive behavior, such as faster reaction kinetics, as compared to their macrogel counterpart. Microgels have been explored for a broad range of applications, either as individual entities or within large scale assemblies. Although these materials have shown a great deal of utility and versatility, microgels have also demonstrated a great deal of complexity due to the fact that they exhibit both polymeric and colloidal properties. This so-called polymer/colloid duality creates intricacies in characterizing the behavior of these materials, especially when coupled with an oppositely charged component within multilayered assemblies. In this dissertation, work is focused primarily on building a greater fundamental understanding of microgels and their behavior within large scale assemblies. This is done through the development of new characterization techniques or through a direct visualization of the interactions of microgels with their surrounding environment with emphasis on their interaction with an oppositely charged linear polyelectrolyte. From these studies, a more developed fundamental understanding of microgels and their assembly into complex structures is obtained, and these findings will aide in the development of future applications of microgel assemblies.

The aim of this thesis is to investigate and better understand the mechanisms of protein self-assembly. Specifically, I study three protein systems which form morphologically and structurally distinct brillar protein aggregates. The first of these studies is concerned with the self-assembly of amyloid brils formed from bovine insulin. Amyloid brils are associated with human diseases such as Alzheimers Disease and type-2 diabetes, and are also garnering interest in biomaterial applications. Fragmentation-dominated models for the self-assembly of amyloid brils have had important successes in explaining the kinetics of amyloid bril formation but predict bril length distributions that do not match experimental observations. Here I resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. I provide evidence for a structural transition demarcated by a critical bril mass concentration, or CFC, above which fragmentation of the brils is suppressed. Our simulations predict the formation of distinct bril length distributions above and below the CFC, which I confirm by electron microscopy. These results point to a new picture of amyloid bril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC. I further show that the CFC can be modulated by environmental conditions, pointing to possible in vivo strategies for controlling cytotoxicity. I probe the structural nature of the transition by performing small angle neutron scattering. Secondly, I study the formation of amyloid-like brils from the protein ovalbumin. I undertake kinetic experiments of self-assembly and find two key features emerge: the lack of a lag time and the existence of a slow growth regime in the long-time limit. I observe, using TEM, that these brils are worm-like in nature and form closed-loops. I find the growth kinetics are intimately connected to this particular morphology. I present a simple kinetic model which captures the features of the kinetics found in experiments by incorporating end-to-end association of brils. I comment on the ramifications this type of amyloid bril assembly may have on oligomeric toxicity. Thirdly, the DNA-mimic protein ocr is highly charged (-56e at pH 8) and forms non-amyloid brillar assemblies at very high ammonium sulphate concentrations (3.2M). The fact that ocr forms translucent brillar gels at such high salt concentrations is extremely unique. Typically under such high salt conditions, non-specific amorphous aggregates are formed. In order to better understand the mechanism of why ocr forms specific bril aggregates, I used variants of the wile-type protein in which extensive regions of surface have been removed or modified. The structural characteristics of gels formed from the variants were probed using microrheological techniques. I find that non-specific electrostatic charge screening plays an important role in ocr aggregation. However, I also locate a potentially important α-helical region which may play a part in establishing specific interactions so that ocr may form ordered brillar assemblies.

Gold nanoparticles have received significant interest recently due to their utility in biomedical imaging and therapy. Nanoparticles which exhibit intense extinction in the near infrared (NIR) region, where blood and tissue absorb light weakly, are of great interest as contrast agents for biomedical imaging applications. While strong NIR extinction often requires sizes greater than ~20-30 nm, effective clearance from the body to avoid toxic accumulation necessitates sizes below ~6 nm. Moreover, effective clearance depends upon lack of adsorption of serum proteins in the bloodstream onto the particles. Herein, this conflict is addressed by assembling sub-5 nm gold nanoparticles into clusters with controlled size and morphology, in order to provide intense NIR extinction. Furthermore, the surfaces of the primary gold nanoparticles are designed such that the particles avoid the adsorption of any serum proteins. Binary ligand monolayers of anionic citrate and appropriate amounts of either cationic lysine or zwitterionic cysteine are synthesized to completely prevent serum protein adsorption from undiluted fetal bovine serum. A mechanism is proposed whereby the zwitterionic tips which are present on both the lysine and cysteine ligands limit the interactions between serum proteins and the "buried" charges on the nanoparticle surfaces. These primary nanoparticles are subsequently assembled into biodegradable nanoclusters via "quenched assembly", wherein nanoclusters are assembled and subsequently quenched by the adsorption of a biodegradable polymer on the cluster surface. The sizes of completely reversible "quenched equilibrium" nanoclusters formed from gold nanoparticles coated with a mixture of lysine and citrate are tuned from 20 nm to 40 nm, and nanocluster size is semi-quantitatively predicted by a free-energy model. Additional control over nanocluster size and extinction is demonstrated by adding NaCl, which is shown to decrease the polymer adsorption on the clusters and thus decrease polymer bridging interactions. This nanocluster formation platform is extended to nanospheres capped with citrate and the thiolated, zwitterionic cysteine ligand. A general paradigm is presented whereby the sizes and optical properties of biodegradable gold nanoclusters formed from nanospheres which do not adsorb any serum proteins are tuned via control over van der Waals, electrostatic, depletion, and polymer bridging colloidal interactions. / text

The term “soft matter” applies to a variety of physical systems, such as liquids, colloids, polymers, foams, gels, and granular materials. The most fascinating aspect of soft matter lies in the fact that they are not atomic or molecular in nature. They are instead macromolecular aggregates, whose spatial extent lies in the domain 1 nm to 1 ¹m. Some of the most important examples of soft matter are polymers, which exhibit intriguing and useful physical properties. In this work, the adsorption and self assembly of linear and star polymers on smooth surfaces are studied using coarse-grained, bead-springmolecular models and Langevin dynamics computer simulations. The aim is to gain insight on atomic-forcemicroscopy images of polymer films on mica surfaces, adsorbed from dilute solution following a good solvent-to-bad solvent quenching procedure. In the case of linear polymers, under certain experimental conditions, a bimodal cluster distribution is observed. It is demonstrated that this type of distribution can be reproduced in the simulations, and rationalized on the basis of the polymer structures prior to the quench. In addition to providing insight on experimental observations, the simulation results support a number of predicted scaling laws such as the decay of the monomer density as a function of distance from the surface, and the scaling of the filmheight with the strength of the polymer-surface interactions. Star polymers represent a special class of polymers, in which one end of each linear chain is tethered to a small central core to forma single particle. The discovery of these molecules led to the synthesis of a wide range of new materials. Their structures are effectively considered as intermediate between those of colloids and linear polymers. We explore the behaviour of the star polymers (which are like “soft colloids”) in the proximity of a surface, using Langevin dynamics simulations. A number of different measurements such as the height, radius of gyration, and asphericity of adsorbed stars with different number of arms, are shown to provide valuable insights on experimental findings. The simplest soft matter systems consist of spherical, rigid colloidal particles. Examples of such particles are chemically synthesized polystyrene or silica particles. We investigated the neighbour distribution in a two-dimensional polydisperse harddisk fluid, corresponding physically to a colloidal monolayer. The disk diameter distribution was defined by a power-law with the aim of realizing a scale-free nearneighbour network. Scale-free (power-law) behaviour is found in many important networks, for example, in transportation systems, biochemical reactions, scientific and movie-actor collaborations, and sexual contacts. We have provided the first example of a scale-free network in amodel condensed-matter system. Finally, we use genetic algorithms, a method for efficiently searching for minima on energy landscapes, to investigate the ordered equilibrium structures formed by binary mixtures of anisotropic dipolar particles confined on a plane, under the presence of an external magnetic field. The anisotropy of the interparticle forces is controlled by tilting the external magnetic field with respect to the plane. Initially, as the field is tilted the structures are only slightly perturbed, but once the anisotropy exceeds a critical value, completely new structures emerge.