Self-assembly has emerged as a promising technique for fabrication of novel hybrid materials and nanostructures. The work presented in this thesis has been focused on developing nanoscale materials based on synthetic de novo designed polypeptides. The polypeptides have been utilized for the assembly of gold nanoparticles, fibrous nanostructures, and for sensing applications. The 42-residue polypeptides are designed to fold into helix-loop-helix motifs and dimerize to form four-helix bundles. Folding is primarily driven by the formation of a hydrophobic core made up by the hydrophobic faces of the amphiphilic helices. The peptides have either a negative or positive net charge at neutral pH, depending on the relative abundance of Glu and Lys. Charge repulsion thus prevents homodimerization at pH 7 while promoting hetero-dimerization through the formation of stabilising salt bridges. A Cys incorporated in position 22, located in the loop region, allowed for directed, thiol-dependent, immobilization on planar gold surfaces and gold nanoparticles. The negatively charged (Glu-rich) peptide formed homodimers and folded in solution at pH < 6 or in the presence of certain metal ions, such as Zn2+. The folding properties of this peptide were retained when immobilized directly on gold, which enabled reversible assembly of gold nanoparticles resulting in aggregates with well-defined interparticle separations. Particle aggregation was found to induce folding of the immobilized peptides but folding could also be utilized to induce aggregation of the particles by exploiting the highly specific interactions involved in both homodimerization and hetero-association. The possibility to control the assembly of polypeptide-functionalized gold nanoparticles was utilized in a colorimetric protein assay. Analyte binding to immobilized ligands prevented the formation of dense particle aggregates when subjecting the particles to conditions normally causing extensive aggregation. Analyte binding could hence easily be distinguished by the naked eye. Moreover, the peptides were utilized to assemble gold nanoparticles on planar gold and silica substrates. Fibrous nanostructures were realized by linking monomers through a disulphide-bridge. The disulphide-linked peptides were found to spontaneously assemble into long and extremely thin peptide fibres as a result of a propagating association mediated by folding into four-helix bundles. / Ingenjörer och vetenskapsmän har ofta inspirerats av naturen i sökandet efter lösningar på tekniska problem. Allt ifrån byggnadskonstruktioner, flygplansvingar, kompositmaterial till kardborrebandet har skapats med utgångspunkt från förebilder i naturen. Många av de material och konstruktioner som återfinns i naturen har åtråvärda egenskaper som är svåra att erhålla i syntetiska matrial med traditionell teknik. Även om vi i flera fall kan härma sammansättningen och formen blir resultatet inte nödvändigtvis det samma. Den största skillnaden mellan syntetiska material och material producerade av levande organismer är hur deras komponenter sinsemellan är organiserade och sammansatta. I syntetiska material är komponenterna ofta inbördes mer eller mindre slumpvis ordnade medan de i biologiska material är organiserade med en oerhörd precision som sträcker sig ända ned på molekyl- och atomnivå. Naturens byggstenar har genom evolutionens gång förfinats för att spontant kunna organisera sig och bilda komplexa material  och strukturer. Denna process, som styrs genom att många svaga krafter inom och mellan byggstenarna samverkar, kallas ofta för självorganisering och är en förutsättning för allt liv. Självorganisering har också blivit en allt viktigare metod inom nanotekniken för att konstruera material och strukturer med nanometerprecision. I den här avhandlingen beskrivs en typ av självorganiserande material där byggstenarna utgörs av nanometerstora guldpartiklar och syntetiska proteiner. De syntetiska proteinerna är designade för att efterlikna naturliga biomolekyler och antar en välbestämd tredimensionell struktur när två av dem interagerar med varandra. Denna interaktion är mycket specifik men kan styras genom att variera kemiska parametrar som surhet och jonstyrka vilket ger en möjlighet att påverka och kontrollera proteinernas struktur. Proteinerna har vidare modifierats för att spontant organisera sig till fibrer som är flera mikrometer långa men endast några nanometer tjocka. Proteinfibrer utgör en mycket viktig typ av strukturer i biologiska system och finns i alltifrån spindelväv till muskler. Syntetiska proteinfibrer är därför både ett intressant modellsystem och ett material med många potentiellt intressanta användningsområden. Genom att fästa de syntetiska proteinerna på ytan av guldnanopartiklar går interaktionerna mellan partiklarna att kontrollera på samma sätt som interaktionerna mellan proteinerna. Krafterna mellan proteinerna och interaktionerna involverade i proteinernas veckning har använts för att reversibelt aggregera och organisera nanopartiklarna. Ett antal olika byggstenar har studerats och utvecklats till något som liknar ett mycket enkelt nano-Lego, som på en given signal spontant bygger ihop sig eller trillar isär. Guldnanopartiklar är intressanta eftersom de är stabila och lätta att modifiera kemiskt men också på grund av deras optiska egenskaper som ger dem en ovanligt vacker vinröd färg. Färgen uppstår på grund av partiklarnas ringa storlek och varierar naturligt med egenskaperna hos den omgivande miljön. Detta gör det enkelt att studera hur partiklarna interagerar eftersom de byter färg när de närmar sig varandra, men gör dem också intressanta för sensortillämpningar. En enkel och robust sensor beskrivs i avhandlingen där syntetiska proteiner, speciellt utformade för att upptäcka och binda andra molekyler, har fästs på nanopartiklarna. Med partiklarnas hjälp går det att med blotta ögat detektera ett mänskligt protein i koncentrationer under ett tusendels gram per liter. En tidig diagnos av sjukdomstillstånd kan i de flesta fall avsevärt underlätta behandlingen och behovet av enkla sensorer för att bestämma närvaro och koncentration av medicinskt intressanta molekyler är därför mycket stort.

Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events in neurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) disruption is associated with cerebral ischemia and leads to vasogenic edema, a primary cause of stroke-associated mortality. To date, only a single drug has received US Food and Drug Administration (FDA) approval for treatment of acute ischemia/reperfusion injury, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is re-established. Therefore, there is a critical need for novel therapeutic approaches that can "rescue" salvageable brain tissue and/or protect BBB integrity during cerebral hypoxia and subsequent reoxygenation stress (H/R). One approach that may enable neural tissue rescue following H/R is CNS delivery of drugs with brain protective effects such as HMG-CoA reductase inhibitors (i.e., statins). Our present in vivo data demonstrates that atorvastatin, a commonly prescribed statin, attenuates poly (ADP-ribose) polymerase (PARP) cleavage in the brain following H/R, suggesting neuroprotective efficacy. However, atorvastatin use as a CNS therapeutic is limited by poor blood-brain barrier (BBB) penetration. Therefore, we examined regulation and functional expression of the known statin transporter Oatp1a4 at the BBB under H/R conditions. In rat brain microvessels H/R (6% O₂, 60 min followed by 21% O₂, 10 min) increased Oatp1a4 expression. Brain uptake of taurocholate (i.e., Oap1a4 probe substrate) and atorvastatin were reduced by Oatp inhibitors (i.e., estrone-3-sulfate, fexofenadine), suggesting involvement of Oatp1a4 in brain drug delivery. Pharmacological inhibition of TGF-β/ALK5 signaling with the selective inhibitor SB431542 increased Oatp1a4 functional expression, suggesting a role for TGF-β/ALK5 signaling in Oatp1a4 regulation. Taken together, our novel data show that targeting an endogenous BBB drug uptake transporter (i.e., Oatp1a4) may be a viable approach for optimizing CNS drug delivery for treatment of diseases with an H/R component.

<p>Traditional small molecule chemotherapeutics show limited effectiveness in the clinic as their poor pharmacokinetics lead to rapid clearance from circulation and their exposure to off-target tissues results in dose-limiting toxicity. The objective of this dissertation is to exploit a class of recombinant chimeric polypeptides (CPs) to actively target drugs to tumors as conjugation to macromolecular carriers has demonstrated improved efficacy by increasing plasma retention time, reducing uptake by healthy tissues, and enhancing tumor accumulation by exploiting the leaky vasculature and impaired lymphatic drainage characteristic of solid tumors. CPs consist of two principal components: (1) a thermally responsive elastin-like polypeptide (ELP) that displays a soluble-to-aggregate phase transition above a characteristic transition temperature (Tt); and (2) a cysteine-rich peptide fused to one end of the ELP to which small molecule therapeutics can be covalently attached (the conjugation domain). This work describes the development of CP drug-loaded nanoparticles that can be targeted to solid tumors by the external application of mild regional hyperthermia (39-43°C). </p><p>Highly repetitive ELP polymers were assembled by Plasmid Reconstruction Recursive Directional Ligation (PRe-RDL), in which two halves of a parent plasmid, each containing a copy of an oligomer, were ligated together to dimerize the oligomer and reconstitute the functional plasmid. Chimeric polypeptides were constructed by fusing the ELP sequence to a (CGG)8 conjugation domain, expressed in Escherichia coli, and loaded with small molecule hydrophobes through site specific attachment to the conjugation domain. Drug attachment induced the assembly of nanoparticles that retained the thermal responsiveness of the parent ELP in that they experienced a phase transition from soluble nanoparticles to an aggregated phase above their Tt. Importantly, the Tt of these nanoparticles was near-independent of the CP concentration and the structure of the conjugated molecule as long as it displayed an octanol-water distribution coefficient (LogD) > 1.5. </p><p>A series of CP nanoparticles with varying ratios of alanine and valine in the guest residue position was used to develop a quantitative model that described the CP transition temperature in terms of three variables - sequence, chain length, and concentration - and the model was used to identify CPs of varying molecular weights that displayed transition temperatures between 39°C and 43°C. A murine dorsal skin fold window chamber model using a human tumor xenograft was used to validate that only the thermoresponsive CP nanoparticles (and not the controls) exhibited a micelle-to-aggregate phase transition between 39-43°C in vivo. Furthermore, quantitative analysis of the biodistribution profile demonstrated that accumulation of these thermoresponsive CP nanoparticles was significantly enhanced by applying heat in a cyclical manner. It is hoped that this work will provide a helpful resource for the use of thermoresponsive CP nanoparticles in a variety of biomedical applications.</p> / Dissertation

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

Thesis (Ph. D.)--University of Göteborg, 1994. / Published dissertation.

Type II diabetes mellitus (T2D) and Alzheimer's disease (AD) share aetiology and have a high incidence of co-morbidity. Evidence suggests that both diseases are caused by the pathogenic aggregation of an intrinsically disordered native amyloid peptide. Furthermore, T2D and AD share risk factors such as age, obesity and vascular health. Recent studies demonstrate that amylin, an amyloidogenic pancreatic hormone deposited in the pancreas in T2D, is also deposited in the brain in AD. We hypothesised that amylin directly contributes to AD through deposition in the brain and activation of pathogenic signalling cascades. We provide evidence to validate that amylin is deposited in the brain parenchyma and vasculature. Furthermore, we present data demonstrating amylin (IAPP) expression in the brain is significantly elevated in AD; and that amylin treatment increases amyloid-beta (AB) secretion in neuronal culture. Soluble oligomeric species of AB cause AD by initiation of downstream signalling cascades that dysregulate kinase activity, promote tau phosphorylation and result in neuronal death. One such pathway involves AB oligomer activation of the Src-family kinase Fyn, through binding to the cellular prion protein (PrPC) receptor complex. We provide evidence that amylin activates Fyn in neuroblastoma and stem cell derived neurons, this activation is possibly mediated through PrPC. Together the data presented in this thesis demonstrate multiple modes of action whereby amylin may directly propagate or indirectly exacerbate AD-associated processes. Amylin aggregation, deposition, up-regulation and signalling should be considered one of several links between T2D and AD. The pathogenic actions of AB and amylin are mediated by oligomer species. Therefore therapeutics which prevent oligomerisation or oligomer action may be valuable in AD and T2D. One such class of therapeutic are flavonoids. Our collaborators have recently demonstrated the flavonoids rutin and quercetin reduce amylin aggregation and extend lifespan in diabetic animal models. As a result of this we investigated the anti-amyloidogenic and anti-oligomeric properties of the flavonoid quercetin against AB. Quercetin treatment prevented AB oligomerisation, cell binding of pre-formed AB oligomers and also reduced APP processing in cell models. These data suggest quercetin is a multimodal therapeutic with potential utility in AD and T2D and should be explored for further drug development.

No description available.

Övervikt och fetma sprider sig likt en epidemi över världen. Omkring 1,9 miljarder vuxna varöverviktiga år 2014 och av dessa klassificerades 600 miljoner som feta. Forskning kring fetmas uppkomst och nya former av behandlingsalternativ pågår. En viktig faktor för uppkomst av övervikt är aptitreglering, där t.ex. Peptide tyrosine tyrosine (PYY), Oxyntomodulin (OXM) och Glucosedependent insulinotropic polypeptide (GIP) har betydelse. En litteraturstudie genomfördes där totalt nio originalartiklar från PubMed utvärderades. Syftet var att undersöka om det finns något värde i att mäta dessa hormon postprandialt. Finns det någon skillnad mellan normalviktiga, överviktiga och obesa och finns det någon skillnad mellan individer med typ 2-diabetes mellitus (T2DM) och friska individer? Finns det någon pålitlig analysmetod? Samtliga studier var måltidsstudier där olika näringsämnens påverkan på den postprandiala responsen undersöktes. Peptide tyrosine tyrosine ochGlucose-dependent insulinotropic polypeptide mättes i sex resp. fem av artiklarna och OXM mättes ien artikel. Protein, fett och kolhydrater ger en postprandial respons på PYY och GIP. Responsen av PYY var starkast efter stimuli från fett och protein. Fett tycks ge starkast respons på GIP. Fastevärden av PYY och GIP var inte olika hos normalviktiga och överviktiga i de studier som undersöktes. Det fanns en signifikant skillnad (p=0,01) mellan normalviktiga och överviktiga tonårsflickor av den postprandiala utsöndringen av PYY efter fettrik måltid, där de obesa flickorna hade lägre procentuell ändring jämfört med de normalviktiga. Pålitliga analysmetoder vid koncentrationsbestämning av dessa tre hormon i plasma är Radioimmunoassay (RIA) och Enzyme-linked immunosorbent assay (ELISA).

In this thesis, a polypeptide binder concept is illustrated. By conjugation to a set of sixteen polypeptides, a small binding molecule can evolve into a polypeptide binder with increased affinity and selectivity. The concept was applied to 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) and acetazolamide (AZM) for development of high affinity binders targeting human C-reactive protein (CRP) and human carbonic anhydrase (HCA) II and IX respectively. In addition, peptididic macrocycles were developed as inhibitors of lysine specific demethylase 1 (LSD1). CRP is a well-known biomarker of inflammation in humans and binders recognizing it are therefore of large interest as medical diagnostics. Until now, phosphocholine (PCh) and derivatives are the only known small molecule binders for CRP, but they have low μM affinity and bind CRP in a Ca2+ dependent manner. The small molecule DQ was designed as a CRP binder that is structurally unrelated to PCh. Its polypeptide conjugate, 4-C25l22-DQ, was demonstrated as a strong, Ca2+ independent binder for CRP, and had an affinity approximately three orders of magnitude higher than DQ itself. HCA IX is a protein that is interesting for diagnosis of cancer. AZM is a small molecule inhibitor of HCAs with a dissociation constant of 38 nM for HCA II and 3 nM for HCA IX. Interestingly, polypeptide conjugate 4-C10L17-AZM displayed stronger binding to both HCA II (KD 4 nM) and HCA IX (KD 90 pM). This result provided evidence that the binder concept can be applied also for small molecules which already have high affinity for their protein receptors. LSD1 is an enzyme that regulates the methylation of Lys 4 of histone 3 via a PPI-like interaction and which is of therapeutic interest in certain cancers. Based on the structures of two peptidic ligands bound to LSD1, we sequentially prepared truncated, mono-substituted and macroyclic peptides in order to develop reversible inhibitors of LSD1. Some stapled cyclic peptides bound to LSD1 with 10-fold higher affinity than the corresponding linear parent peptide. Changing the staple into a lactam further improved the binding potency and the best lactams inhibited the enzymatic activity of LSD1 at low μM Ki values.

L'internalisation et le trafic intracellulaire sont des mécanismes cruciaux dans la régulation de la signalisation des récepteurs couplés aux protéines G (RCPG) dans lesquels les -arrestines jouent un rôle central. Des agonistes biaisés qui sont capables d'activer sélectivement les voies de signalisation dépendantes des protéines G ou dépendantes des -arrestines ont été récemment identifiés. D'autre part, le concept selon lequel la signalisation des RCPG serait limitée à la membrane cellulaire a été contesté sur la base des données qui démontrent que de nombreux RCPG induisent du signal aussi bien à partir d'endosomes qu'au niveau de la surface cellulaire. Le glucose-dependent insulinotropic polypeptide (GIP) est une hormone incrétine essentielle dans l'homéostasie glucidique postprandiale. Elle exerce ses fonctions en se liant à un récepteur couplé aux protéines G, le RGIP qui est impliquée dans divers processus physiologiques et physiopathologiques. À ce jour, l'internalisation et le trafic intracellulaire du RGIP ainsi que leurs mécanismes moléculaires sous-jacents n'ont pas été étudié en détail. Dans ce contexte, le but de notre travail était d'abord d'étudier ces mécanismes et ensuite de caractériser le profil d'internalisation du N-acétyl-GIP, un analogue du GIP connu pour être résistant à la dégradation par le DPP-IV. Enfin, nous avons étudié si, en plus de sa signalisation à la membrane cellulaire, le RGIP est capable d'induire une signalisation à partir d'endosomes. Dans cette étude, nous montrons d'abord que l'internalisation du RGIP est un processus impliquant la clathrine, le complexe AP-2 et la dynamine, mais pas la région C-terminale du récepteur, ni les -arrestines1/2. Nous avons également montré que le N-acétyl-GIP, qui présente une activité agoniste pleine sur la production d'AMPc et sur la sécrétion d'insuline dans les cellules MIN-6-B1, n'est pas capable de stimuler l'internalisation du RGIP. Cela suggère que le N-acétyl-GIP pourrait être un agoniste biaisé du RGIP induisant préférentiellement la voie d'activation de Gs comparativement à un adressage du récepteur vers des puits recouverts de clathrine. Nous avons également réussi à observer une persistance au cours du temps de la production d'AMPc induite par le GIP. Le signal persistant dépend de l'internalisation du RGIP et est irréversible après lavage du GIP de la membrane cellulaire. De plus, nous avons détecté d'une manière directe la forme active de Gs au niveau d'endosomes contenant le RGIP en utilisant des plasmides codant pour des Nanobodies fusionnés à la GFP. Enfin, en utilisant un biosenseur FRET d'AMPc dirigé à la surface des endosomes précoces, nous avons également pu détecter d'une manière directe la production d'AMPc spécifiquement à la surface des endosomes contenant le RGIP internalisé. À notre connaissance, cette dernière observation est la première de ce genre, prouvant le concept de signalisation à partir d'endosomes par une approche de détection directe. Les résultats de cette étude apportent des informations quant à la régulation pharmacologique de l'internalisation et de la signalisation du RGIP, ouvrant des perspectives prometteuses dans le domaine du GIP. / Internalization and trafficking are crucial mechanisms regulating G-protein coupled receptors (GPCRs) signaling in which -arrestins play a central role. Biased agonists which selectively activate either G protein or -arrestin signaling pathway were identified. On the other hand, the concept of GPCR signaling being restricted to cell membrane has been contested on the basis of data demonstrating GPCR signaling from endosomes as well as from the cell surface. Glucose insulinotropic polypeptide (GIP) is an incretin hormone essential in post-prandial glucose homeostasis. It exerts its functions through binding to a G protein-coupled receptor, GIPR which is involved in various physiological and pathological processes. To date, GIPR internalization and trafficking and the underlying molecular mechanisms have not been investigated in detail. In this context, the aim of our work was to study these mechanisms and to characterize the internalization profile of N-Acetyl-GIP, a GIP analogue resistant to DPP-IV degradation. Finally, we investigated if GIPR signaling can occur from endosomes alongside its signaling at the cell membrane. In this study, we first report that GIPR internalization involves clatherin, AP-2 and dynamin but not C-terminal region of the GIPR nor -arrestin1/2. Moreover, N-Acetyl-GIP, which fully stimulated cAMP production and insulin secretion from MIN-6-B1 cells, did not stimulate internalization of the GIPR. This suggests that N-Acetyl-GIP could be a biased GIPR agonist preferentially inducing Gs activation pathway over directing the receptor to clathrin-coated pits. We have also succeeded to witness a sustainability in GIP-induced cAMP production. The sustained signal was dependent on GIPR internalization and unreversed by GIP removal from the cell membrane. Moreover, we directly detected the active form of Gas in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody. Finally, using a FRET sensor of cAMP targeted to the surface of early endosomes, we also directly detected cAMP production specifically at the surface of endosomes containing internalized GIPR. The latter observation is the first of this kind, proving the endosomal signaling concept by a direct detection approach. This study brings new insights into the pharmacological regulation of GIPR internalization and signaling, opening promising perspectives in GIP field.