<p>The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste.</p><p>In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster.</p><p>In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.</p>

The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste. In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster. In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.

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Master of Science / Department of Biochemistry / John M. Tomich / Biologically based adhesives offer many industrial advantages over their chemically synthesized counterparts, not the least of which are reduced environmental impact and limited toxicity. They also represent a renewable resource. In addition, nanoscale biomaterials also show an incredibly large potential for biomedical uses, including possible drug delivery and novel wound bandaging, as well as tissue engineering. Understanding the adhesion mechanisms at work in peptide-based nanomaterials is key for producing viable industrial and clinical biomimetic compounds. Our previous work has shown that small hydrophobic oligopeptide segments flanked by short tri-lysine sequences display adhesion strength that is dependent on the formation of β-structure and large-scale association of monomers. In this study, three oligopeptides were synthesized based on putative amyloid fibril nucleation sites. Two of the sequences originate from the Alzheimer’s beta amyloid peptide Aβ1-40, while the third sequence comes from a nucleation site for islet amyloid polypeptide (IAPP). These peptides show unusual structural properties associated with adhesive ability. Furthermore, they represent a third category of requirements for β-structure formation. In addition, I report the first morphological evidence for the previously predicted structural mechanism underlying our previous peptide based adhesives.

Alzheimer’s disease (AD), the most common form of dementia is associated with fibril formation of amyloid-ß peptides (Aß). Aß, proteolytically derived from Aß precursor protein (AßPP), is the major component of amyloid plaques in AD brains. Familial British and Danish dementias (FBD and FDD) share pathological and clinical characteristics with AD, and the underlying mechanisms are associated with amyloid formation of mutant peptides released from the Bri2 protein. Bri2 interacts with AßPP and its BRICHOS domain has been shown to delay Aß40 and Aß42 fibril formation and toxicity in vitro and in vivo. This makes Bri2 BRICHOS a promising anti-amyloid chaperone and a potential treatment strategy for AD. Furthermore, Bri2 BRICHOS possesses a general chaperone activity as it suppresses non-fibrillar aggregation of destabilized citrate synthase (CS). Recent findings show that Bri2 BRICHOS produced in E.coli can form different molecular weight assemblies, ranging from monomers to dimers and poly-disperse oligomers. The oligomers inhibit CS aggregation, whereas the monomers and dimers are more efficient against Aß42 fibrillation and neurotoxicity, respectively. The work in this thesis shows that similar Bri2 BRICHOS quaternary structures are formed in eukaryotic cells as in E.coli. Larger BRICHOS oligomers were found in cell media, derived from proteolytically processed endogenous Bri2 in SH-SY5Y cells, as well as in human embryonic kidney (HEK293) cells transfected with a Bri2 BRICHOS construct. Recombinant human Bri2 BRICHOS mutants with one or none of the two strictly conserved cysteine residues were studied. All mutant monomers become proteolytically degraded during purification, but form stable oligomers. Single Cys to Ser mutants form stable disulfide-dependent dimers that differ in ability to prevent Aß42 fibrillation, the most stable mutant (C164S) being even more efficient than the wildtype Bri2 BRICHOS dimer. This result suggests that intra or intermolecular disulfide(s) and oligomerization affect Bri2 BRICHOS stability and activity towards Aß42 fibril formation.

No / ß2-microglobulin (ß2m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How ß2m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on ß2m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (¿N6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that ¿N6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of ß2m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type ß2m with that of ¿N6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.