Terminally functionalised oligo(vinyl ether)s were produced in ab initio cationic polymerisations. Various polymerisations and chain end functionalisation systems were investigated. MALDI-TOF mass spectrometry was applied to analyse the obtained oligomers and thus the polymerisation and chain end functionalisation process. Oligo(isobutyl vinyl ether), also oligo(ethyl vinyl ether) and oligo(methyl vinyl ether) were synthesised in cationic polymerisation. Silyl enol ethers were added to the polymerisation as end-capping agents before initiations and they compete with monomer to cap the carbocationic chain end. The methodology relies on a comparable end capping rate to chain propagation rate so that oligomers can still be produced in the presence of reactive end-capping agents whilst other side reactions are suppressed. Polymerisation temperatures investigated range from -78°C to the room temperature (21 °C), 4 out of 6 silyl enol ethers were applied and are proved to be reactive in the end-capping. Initiation systems investigated include iBVEHCl/Yb(OTf)3 and iBVE-HCl/SnC14 and both produced oligomers with high chain end functionalities. Generally silyl enol ether functionalised oligo(vinyl ether)s have lower molecular weights and broader molecular weight distributions than their identical control polymerisations without end-capping. The iBVE-HCl/SnCI4 initiation system produced functionalised oligomers with narrower molecular weight distribution than iBVE-HCl/Yb(OTf)3 initiation system. Different silyl enol ether reactivities in end-capping were observed. It was also observed that ab initio chain end functionalisation by reactive silyl enol ethers largely suppressed the majority of side reactions during polymerisation. This suppression was attributed to the higher rates of end-capping than side reaction rates. When (1-tert-butyl-vinyloxy)trimethyl-silane was applied as end-capping agent, the polymerisation system produced oligomers with narrower molecular weight distributions than the control polymerisations without end-capping while the chain end functionalities were also obtained. This indicates the possibility of setting up a controlled ab initio chain end functionalisation cationic polymerisation system in which the oligomer's molecular weight, polydispersity and chain end functionality can be regulated at the same time. MALDI-TOF MS, NMR and SEC are mainly applied in the oligomer characterisation. Side reactions in this polymerisation system were examined from these analyses. Under less critical polymerisation conditions 7 different chain ends from side reactions are observed in MALDI-TOF mass spectra. Based on the MS and NMR observation the various chain end structures are identified. Side reactions are also postulated which mainly include ß-proton elimination, water capping of the carbocationic chain end and combinations of these. Sample preparation for MALDI-TOF MS analysis of oligo(vinyl ether)s is investigated. Direct laser desorption of oligo(isobutyl vinyl ether) was observed for the samples of molecular weight of up to 2k Daltons. Complementary information of oligo(isobutyl vinyl ether)s obtained from ESI MS indicates a serious mass discrimination in MALDI-TOF MS technique and thus it is regarded that MALDITOF MS can not provide reliable molecular weight distributions for polymers with broad molecular weight distributions. Combination of SEC and MALDI-TOF MS to calibrate SEC columns and quantitative application of MALDI-TOF MS to analyse the oligomers' chain end functionalities were also explored.

Phospholipase A2 (PLA2) extensively exists in various snake venom. Till now, a controversy remained to elucidate whether the PLA2 activity exclusively associates with the manifestation of the pharmacological activities. In the present study, we used liposome to imitate cell membrane for excluding the effects of receptor and membrane proteins, and estimating the molecular mechanism of snake venom phospholipase A2 on damaging liposome. Although a greater membrane damaging activity of Naja naja atra phospholipase A2 (NNA-PLA2) and notexin was noted in the presence of Ca2+, inhibitions of PLA2 activity by Sr2+ and Ba+2 were unable to abolish the membrane damaging effect. In addition, modification of Lys-82 and Lys-115 of notexin retained the full PLA2 activity, but the membrane damaging activity notably decreased. Fluorescence quenching studies, CD measurement, and tryptophan fluorescence lifetime assay indicated that liposome induced the £\-helix conformation change and the tryptophan residues microenviroment change with the addition of Ca2+, Sr2+ or EDTA. Rhodamine quenching assay revealed that NNA-PLA2 and notexin formed oligomers when they bound with liposome. Besides, the modified PLA2 (BPB-PLA2) only formed monomer when it bound with liposome and lost the membrane damaging activity. Taken together, these results indicate that the membrane damaging effects of NNA-PLA2 and notexin are not critically caused by their enzymatic activitys and are probably associated with oligomerization.

The morphological investigation of AFR-PEPA-N and the development of a new polyimide have been established herein. AFR-PEPA-N is an imide oligomer that was created out of the need to attain a high temperature polyimide that is also resistant to hygrothermal and thermooxidative degradation. Previously, AFR700B was implemented in aerospace applications, but it was found to be hygrothermally unstable. It experienced a severe drop in its glass transition temperature and composite blistering. AFR700B was improved upon, by altering the chemical structure of the polyimide. The nadic end-cap was removed and replaced by a more hydrolytically stable end-cap. However this phenylethynyl-terminated end-group could possibly create semi-crystallinity or liquid-crystalline characteristics within the polymer. Previous research suggests further study of the relationships between AFR-PEPA-N's oligomer crystallinity and the properties of phenylethynyl-terminated polyimides. This understanding is valuable in processing AFR-PEPA-N by resin transfer molding (RTM) to obtain its optimum properties. The investigation included the identification of a processing window, temperature overlap between the melting of residual crystals and crosslinking reactions, and liquid crystallinity behavior. These reactions were investigated primarily through birefringence. The residual crystals were found to be innate in the oligomer powder and not created by preliminary thermal processing. Therefore a reasonable processing window was found based upon the reduction of crystal size by appropriate dissolution techniques. Possible nematic liquid-crystalline characteristics were found to be present at 360oC. A new imide oligomer, which was based upon AFR-PEPA-N's original structure, was synthesized. The non-linear, flourinated backbone of AFR-PEPA-N was replaced with a co-linear backbone, pyromellitic dianhydride (PMDA). These modifications were made in hopes to improve upon the network structure by it becoming more regular and resistance to nano-sized defects in the final crosslinked structure. The initial characterization found that the new polyimide, AFR-P3, displayed a cure temperature at 350oC. The degree of cure reaches about 80 to 90 percent complete based upon the consumption of the carbon-triple bond. AFR-P3 did not show signs of liquid-crystalline behavior. However, there will be future work in creating a more rigid-rod, self-assembling oligomer that can attain optimum thermal and mechanical properties.

No description available.

In many DNA processes, action at a distance is required for signaling across long distances on DNA. These pathways, generally have an initiation site (site 1) that signals an event at a second location (site 2). Such a paradigm is found in processes such as transcription, replication, and DNA repair. To overcome long distances on DNA, proteins can utilize translocation, oligomerization, and DNA looping to bridge the distance between the initiating signal at site 1 and the site of action at site 2. The utilization of these mechanisms for action at a distance is crucial in eukaryotic mismatch repair. In this pathway, MutS homologs scan DNA and recognize mis-paired bases. The MutS protein then recruits the endonuclease MutLα, which nicks the nascent strand of DNA containing a mis-incorporated DNA base. The MutLα-generated nick leads to downstream mis-pair removal through excision by an exonuclease or strand displacement activities of a DNA polymerase working together with a flap endonuclease. Although, previous models have suggested that MutL homolog endonucleases can form oligomeric complexes on DNA, the role of a MutLα oligomeric complex and how it might facilitate action at a distance has been unclear. Here, I present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations, and it can use this activity to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, I determined that a MutLα oligomer associates two DNA duplexes and that this activity can stimulate MutLα’s endonuclease function. I also show evidence that MutLα enhances a topoisomerase without nicking the DNA itself. These behaviors of MutLα could localize nicking on DNA near a mismatch and help overcome barriers that could inhibit additional repair proteins from activating MutLα and facilitating efficient DNA repair. The endonuclease activity of MutLα is critical for efficient mismatch repair, but in addition to this activity, MutLα is also an ATPase, although the crosstalk between the two enzymatic functions has been largely unexplored. It has been shown previously that the ATPase activity of MutLα allows the protein to undergo conformational changes and in vivo is necessary for efficient mismatch repair. Mechanistically, how this activity supports MutLα’s functions in the mismatch repair pathway remains unclear. Using DNA binding and photo-crosslinking experiments, I provide evidence that MutLα recognizes and localizes itself to a nick. Additionally, through DNA protection assays and photo-crosslinking I provide evidence of a signaling mechanism initiated at the nick for a MutLα oligomer to undergo its ATP cycle. These data provide insight into how MutLα uses ATP to signal events for mismatch removal. These data also provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair and send signals for additional repair processes after the protein nicks DNA that help explain new models for action at a distance. / Chemistry

No description available.

Transthyretin (TTR)-associated amyloidoses are diseases wherein wild-type or mutant TTR forms amyloid fibrils that infiltrate multiple organs. Wild-type TTR amyloidosis, ATTRwt, is a sporadic disease characterized by deposits that occur mainly in the heart. Alternatively, >100 TTR mutants cause inherited forms, ATTRm, frequently featuring cardiac amyloid deposits. The goals of this research were to create a cell-based model of ATTR amyloidosis, to define the mechanism of cardiac TTR-associated amyloid at the cellular level, and to study several agents that could interrupt the amyloid process. We hypothesized that TTR oligomers were cardiotoxic and played a role in the mechanism of ATTR amyloidosis, and that cytotoxicity could be inhibited by diflunisal, doxycycline, and Kiacta®. Focusing on TTR proteins associated with cardiac amyloidosis (wild-type, L55P, V30A, and V122), we developed a thermal denaturation method for creating TTR oligomers that allowed us to study the direct effect of oligomers on cells. Congo red and thioflavin T analyses confirmed that the oligomers were on pathway to amyloid fibril formation. We tested the effect of TTR oligomers on rat and human cardiac cells by measuring cell viability and stress response (through live protease activity and qPCR). TTR-L55P oligomers elicited a cytotoxic effect; fluorescent microscopy indicated cellular uptake of the oligomers and continued intra-cellular aggregation. Cytotoxicity was blocked when TTR was heated in the presence of doxycycline; the drug appeared to dissociate TTR aggregates or stabilize the monomeric forms. We also investigated retinol-binding protein (RBP), a natural binding partner of TTR. By immuno-histochemistry, RBP was demonstrated in ATTRwt and ATTRm `non-amyloid' transplant heart tissues, localized to areas containing amyloid or in the case of the transplant tissue, regions that appeared to display ischemic damage. Serum RBP levels were significantly different in ATTR vs. age-matched controls (p = 0.03), and in ATTRwt vs. ATTRm (p <0.0001) by ELISA. These data provide evidence that TTR oligomers are cardiotoxic, possibly due to cellular internalization and progressive intracellular aggregation. Furthermore, our results support the use of doxycycline as a therapeutic in ATTR to target these amyloidogenic oligomers, and suggest that RBP may have potential as a disease biomarker.

This thesis was made to get a deeper understanding of how chaperones interact with unstable, aggregation prone, misfolded proteins involved in human disease. Over the last two decades, there has been much focus on misfolding diseases within the fields of biochemistry and molecular biotechnology research. It has become obvious that proteins that misfold (as a consequence of a mutation or outer factors), are the cause of many diseases. Molecular chaperones are proteins that have been defined as agents that help other proteins to fold correctly and to prevent aggregation. Their role in the misfolding disease process has been the subject for this thesis. Transthyretin (TTR) is a protein found in human plasma and in cerebrospinal fluid. It works as a transport protein, transporting thyroxin and holo-retinol binding protein. The structure of TTR consists of four identical subunits connected through hydrogen bonds and hydrophobic interactions. Over 100 point mutations in the TTR gene are associated with amyloidosis often involving peripheral neurodegeneration (familial amyloidotic polyneuropathy (FAP)). Amyloidosis represents a group of diseases leading to extra cellular deposition of fibrillar protein known as amyloid. We used human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Various conformers of TTR were incubated with the cells for different amounts of time. The experiments showed that early prefibrillar oligomers of TTR induced apoptosis when neuroblastoma cells were exposed to these species whereas mature fibrils were not cytotoxic. We also found increased expression of the molecular chaperone BiP in cells challenged with TTR oligomers. Point mutations destabilize TTR and result in monomers that are unstable and prone to aggregate. TTR D18G is naturally occurring and the most destabilized TTR mutant found to date. It leads to central nervous system (CNS) amyloidosis. The CNS phenotype is rare for TTR amyloid disease. Most proteins associated with amyloid disease are secreted proteins and secreted proteins must pass the quality control check within the endoplasmic reticulum (ER). BiP is a Hsp70 molecular chaperone situated in the ER. BiP is one of the most important components of the quality control system in the cell. We have used TTR D18G as a model for understanding how an extremely aggregation prone protein is handled by BiP. We have shown that BiP can selectively capture TTR D18G during co-expression in both E. coli and during over expression in human 293T cells and collects the mutant in oligomeric states. We have also shown that degradation of TTR D18G in human 293T cells occurs slower in presence of BiP, that BiP is present in amyloid deposition in human brain and mitigates cytotoxicity of TTR D18G oligomers. / Denna avhandling handlar om proteiner. Särskilt de som inte fungerar som de ska utan har blivit vad man kallar ”felveckade”. Anledningen till att proteiner veckas fel beror ofta (men inte alltid) på mutationer i arvsmassan. Felveckade proteiner kan leda till sjukdomar hos människor och djur (man brukar tala om amyloidsjukdomar), ofta av neurologisk karaktär. Exempel på amyloidsjukdomar är polyneuropati, där perifera nervsystemet är drabbat, vilket leder till begränsad rörelseförmåga och senare till förlamning; och Alzheimer´s sjukdom, där centrala nervsystemet är drabbat och leder till begränsad tankeförmåga och minnesförluster. Studierna som presenteras i denna avhandling har gått ut på att få en bättre förståelse för hur felveckade proteiner interagerar med det som vi har naturligt i cellerna och som fungerar som skyddande, hjälpande proteiner, så kallade chaperoner. Transtyretin (TTR) är ett protein som cirkulerar i blodet och transporterar tyroxin (som är ett hormon som bland annat har betydelse för ämnesomsättningen) samt retinol-bindande protein (vitamin A). I TTR genen har man funnit över 100 punktmutationer, vilka har kopplats samman med amyloidsjukdomar, bland annat ”Skellefteåsjukan”. Mutationer i TTR genen leder ofta till att proteinet blir instabilt vilket leder till upplösning av TTR tetrameren till monomerer. Dessa monomerer kan därefter sammanfogas på nytt men denna gång på ett sätt som är farligt för organismen. I denna avhandling har fokus legat på en mutation som kallas TTR D18G, vilken har identifierats i olika delar av världen och leder till en dödlig form av amyloidos i centrala nervsystemet. Det chaperon som vi har studerat benämns BiP och är beläget i en cellkomponent som kallas för det endoplasmatiska retiklet (ER). I ER finns cellens kontrollsystem i vilket det ses till att felveckade proteiner inte släpps ut utan istället bryts ned. Denna avhandling har visat att BiP kan fånga upp TTR D18G inuti celler och där samla mutanten i lösliga partiklar som i detta fall är ofarliga för cellen. Avhandligen har också visat att nedbrytningen av TTR D18G sker mycket långsammare när BiP finns i riklig mängd.

This dissertation describes the work done on two distinct projects. In the first part I sought to unravel the mechanisms that underlie the activity-dependent modulation of response in the excitation-secretion coupling of the neurohypophysis. In the second part, I optically monitored and analyzed the secondary structure changes accompanying amyloid fibril formation along multiple pathways, under both denaturing and near-physiological conditions. Neuronal plasticity plays an important role in regulating various biological systems by modulating release of hormones or neurotransmitters. The changing response to the same stimulus, depending on the context and previous stimulation events, is also the basis of learning and all higher order brain functions. The mechanisms behind this modulation are widely varied, and are often poorly understood in specific tissues. In this work, we examined excitation-secretion coupling in the neurohypophysis, a tissue composed of densely packed axons that secretes the hormones arginine vasopressin and oxytocin. The release of hormones depends not only on the overall level of activity in the gland, but also upon the specifics of the temporal pattern of stimulation. By optically monitoring the electrical activity using voltage sensitive dyes, we were able to investigate this plasticity in the intact gland. Varying extracellular potassium concentration in the bath, increasing interstitial space via hypertonic saline, and retarding potassium reuptake with ouabain all showed that extracellular potassium accumulation drives the depression of excitability. This effect is hidden from glass micro-electrode recordings because of the inevitable damage sustained by the surrounding tissue. Furthermore, no calcium mediated release mechanism played any significant role in the depression. Numerical simulations confirmed the findings and give more insight to the details of the mechanism. Deposits of amyloid fibrils, long, unbranched polymeric protein aggregates, are the molecular hallmark for a variety of human diseases, including Alzheimer's disease, Parkinson's disease, and type II diabetes. While the amyloid fibrils all share a characteristic cross-beta sheet structure, the proteins that make up the aggregates have no unifying theme in either native structure or function. In this research, I characterized the structural reordering that accompanies this aggregation using Fourier transform infrared spectroscopy (FTIR). Hen egg white lysozyme forms fibrillar aggregates with two distinct morphologies, depending on the growth conditions. At acidic pH with low ionic concentrations, lysozyme forms the fibrils with standard amyloid morphology. These aggregates are long and stiff but with the cross sectional area of a single monomer. At higher salt concentrations, the aggregation follows another pathway, under which oligomers initially form and later assemble into protofibrils. The oligomeric protofibrils are thicker than the monomeric filaments, but are much more curvilinear. These fibrils are not universally recognized as amyloidogenic aggregates. Using FTIR, I showed that both this aggregates are indeed amyloid structures, but that they are structurally distinct. While it is generally accepted that partial unfolding of the protein is a prerequisite for amyloid fibril formation, we found that native protein can be the substrate for amyloid growth when seeded with preformed oligomeric or protofibrillar aggregates. These seeded fibrils grown under near-physiological conditions are structurally indistinguishable from those grown from partially unfolded protein under denaturing conditions. This incorporation and restructuring of native monomers is characteristic of prion-like assembly.

The covalent modification of proteins with a suite of molecular tags, a process termed post-translational modification, is a powerful means to enhance the proteomic complexity of an organism far beyond that which is directly encoded by its genome. A particularly widespread form of modification involves the conjugation of the protein ubiquitin to specified substrates, which serves to regulate numerous cellular processes. The mechanism of ubiquitin conjugation, known as ubiquitylation, requires E3 ubiquitin ligases that specify and recruit substrate proteins for ubiquitin conjugation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. This thesis describes the dimeric structure of the E3 ligase adaptor protein SPOP in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. Here, it is demonstrated that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. These results reveal a mechanism through which adaptor protein self-assembly may provide a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.