ABD-Derived Affinity Proteins (ADAPTs) is a novel class of small engineered scaffold proteins based on albumin-binding domain (ABD) of streptococcal protein G. High affinity ADAPT  binders against various therapeutic targets can be selected.  In this thesis, we report a development of ADAPT-based radionuclide imaging agents providing high sensitivity and specificity of molecular imaging of HER2 expression in disseminated cancers. We investigated the feasibility of the use of ADAPTs as imaging agents and influence of molecular design and radiolabeling chemistry on in vivo targeting and biodistribution properties of the tracers. In Paper I we demonstrated the feasibility of the use of anti-HER2 ADAPT6 molecule as a high contrast imaging agent; In Paper II we evaluated the influence of composition of histidine-containing tag on in vivo biodistribution of ADAPT-based tracers labeled with 99mTc using 99mTc(CO)3 binding to histidine-containing tags and 111In using DOTA chelator at N-terminus; In Paper III we evaluated the influence of different aspects of N-terminus leading sequence on targeting including effect of sequence size on clearance rate and effect of the composition of the sequence on biodistribution profile; In Paper IV, we evaluated the influence of residualizing properties and positioning of the label on biodistribution and targeting; and In Paper V, we compared tumor-targeting properties of the ADAPT6 labeled at C-terminus with 99mTc using N3S chelator and 111In using DOTA chelator. In conclusion, ADAPTs constitute a very promising class of targeting probes for molecular imaging providing high contrast. Molecular design of the ADAPT proteins and chelators/linkers for labeling has an appreciable effect on their imaging properties.

Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.

Spolu s narastajúcimi nárokmi na kvalitu liečby v oblasti popálenin a plastickej chirurgie existuje možnosť ako uplatniť nové technologické riešenie na liečbu porúch s celkovou stratou kožnej vrstvy. Diplomová práca sa zaoberá prípravou nanoštrukturovaného, dvojvrstvového skafoldu pre využitie v tkánivovom inžinierstve, ktorý nahrádza kožnú časť dermis (dolná porézna vrstva) a bazálnu membránu (horná a tenká nanovlákenná vrstva). Zákaldom dolnej pórovitej vrstvy je kolagén, charakterizovaný v prítomnosti ďalších polysacharidových aditív: chitosan, vápenatá soľ oxidovanej celulózy (CaOC), sodná soľ karboxymetylcelulózy (NaCMC). Zároveň prídavok dopamínu a fibroblastového rastového faktoru (FGF), s cieľom zlepšiť biomechanické vlastnosti, regulovať a podporovať hojenie kože. Tenká nanovlákenná vrstva je zložená zo želatíny, polycaprolaktónu (PCL) a CaOC. Sú navrhnuté dva rôzne mechnizmy prípravy skafoldu, ktoré sa odlišujú sa v prítomnosti sieťovaných a nesieťovaných nanovláken. Skafoldy boli charakterizované z hľadiska biomechanických, štruktúrnych vlastností a in vitro. Vrchná nanovlákenná vrstva poskytuje mechanickú podporu, ktorá je výrazne zvýšená prítomnosťou polydopamínu (PDA). Test botnania poréznej vrstvy skafoldu ukázal na dostatočne veľké póry, umožňujúce filtráciu buniek. Táto botnatosť bola znížená v prítomnosti PDA, ktorý má zároveň významný vplyv na časové predĺženie degrádácie v prítomnosti kolagenázy a lyzozýmu. Spolu s FGF výrazne podporil proliferizáciu a životaschopnosť myších fibroblastov. Nanoštrukturovaný, dvojvrstvový skafold má potenciál pre budúce aplikácie pri hojení rán, kedže sa vyznačuje dobrými mechanickými vlastnosťami a umožňuje bunkám adherovať, proliferovať a formovať extra celulárny matrix.

Les acides nucléiques riches en guanines ou en cytosines peuvent se replier sur eux-mêmes et former des systèmes tétramériques tels que les G-quadruplexes (G4) ou les i-motifs. Ces motifs, abondamment représentés dans certaines régions du génome humain semblent contribuer à la régulation cellulaire et suscitent depuis plusieurs années un intérêt grandissant. Ils sont notamment présents dans la région télomérique, mais aussi dans les promoteurs d’oncogènes ou au sein des génomes viraux et sont impliqués dans certaines pathologies humaines. Ils représentent ainsi des cibles thérapeutiques et diagnostiques potentielles. Cependant, les G4 adoptent in-vitro des topologies variées qui compliquent le développement de ligands spécifiques et affins. Dans ce contexte, le laboratoire a développé le concept du TASQ pour ‘‘Template Assembled Synthetic G-Quadruplex’’ dans le but d'accéder à des G4 se structurant en une topologie définie.Le premier chapitre décrit l’assemblage de mimes de motifs G4 contraints en une topologie unique. En utilisant un gabarit cyclodécapeptide rigide et différentes méthodes de conjugaison, nous avons assemblé des motifs G4 ARN parallèle et hybride ADN/ARN dérivant de la séquence télomérique ainsi qu’un motif G4 d’ADN présent dans la séquence promotrice du VIH-1. L’utilisation du concept TASQ nous a également permis de préparer un motif G-triplexe (G3), intermédiaire à la formation des motifs G4. Nous avons montré une forte stabilisation de tous les édifices G4 contraints ainsi préparés.Le second chapitre concerne les études de caractérisation et de sélection de ligands vis-à-vis des motifs G4 et G3 contraints. La caractérisation repose sur l’évaluation de l’affinité et de la sélectivité de différentes familles de ligands pour ces édifices, par résonance plasmonique de surface ou par interférométrie bio-couche. La sélection de ligands a été réalisée par la méthode SELEX dans le but d’obtenir des aptamères affins et spécifiques d’un motif G4 contraint. / Guanines or cytosines rich nucleic acids can fold into tetrameric G-quadruplexes (G4) or i-motifs structures. G4 motifs are found within the human genome and should contribute to cellular regulation. In particular G4 are found at telomeric region and also in promoters of oncogenes or within viral genomes. They are suspected of participating in the regulation of human pathologies and have therefore been envisioned as potential therapeutic and diagnostic targets. However, the intrinsic conformational polymorphism of G4 motifs complicates the development of specific and affine ligands. In this context, the laboratory has developed the TASQ concept for "Template Assembled Synthetic G-Quadruplex" with the aim to obtain a defined G4 topology.The first chapter reports on the assembly on the peptide template of RNA and DNA:RNA hybrid G4 structures that derive from the human telomeric sequence as well as of DNA G4 structure found within the HIV virus promoter. G-triplex (G3) motif which is supposed to be an intermediate during the formation of the G4 motifs has also been prepared. By using appropriate ligations of the oligonucleotide strands on the peptide template we were able to control the folding of G-quadruplex motifs and stabilize them.The second chapter reports the studies for the characterization and the selection of ligands against G4 and G3 motifs. The evaluation of the affinity and selectivity of different families of ligands for these constrain motifs was performed by using surface plasmon resonance or by bio-layer interferometry. The selection of ligands was carried out by the SELEX method in order to obtain affine and specific aptamers of a constrained G4 motif.

No description available.

Scaffolding proteins belonging to the Membrane Associated GUanylate Kinase (MAGUK) superfamily function as adaptors linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal transduction. We have identified a novel Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2. Similar to its vertebrate counterpart, Varicose localizes to pleated Septate Junctions (pSJs) of all embryonic, ectodermally derived epithelia and peripheral glia. In vari mutants, essential SJ proteins NeurexinIV and FasciclinIII are mislocalized basally and the cells develop a leaky paracellular seal. Localization of SJ protein Discs Large is not affected, indicating Vari is not involved in cell polarization. In addition, vari mutants display irregular tracheal tube diameters and have reduced lumenal protein accumulation suggesting involvement in tracheal morphogenesis. We found that Vari is distributed in the cytoplasm of optic lobe neuroepithelium and is required for proper ommatidial patterning. As well, Vari is expressed in a subset of neuroblasts and differentiated neurons of the nervous system. We also present a novel MAGUK function in wing hair alignment during adult morphogenesis. We conclude that Varicose is involved in scaffold assembly at the SJ and has a role in patterning adult epithelia and in nervous system development. / Thesis / Doctor of Philosophy (PhD)

DNA damage is a constant threat to our genomes which drives genome instability and contributes to cancer progression. DNA damage interferes with important DNA transactions such as transcription and replication. DNA lesions are removed by repair pathways that ensure genome stability during transcription and replication. Here, we identify and characterize distinct roles for the ultra violet stimulated scaffold protein A (UVSSA) in the maintenance of genome stability during transcription in human cells. First, we unravel a novel function for UVSSA in transcription-coupled repair of DNA interstrand crosslinks (ICLs), genotoxic adducts that covalently bind opposing strands of the DNA and block transcription and replication. UVSSA knockout cells are sensitive to ICL inducing drugs, and UVSSA is specifically required for transcription-coupled repair of ICLs in a fluorescence-based reporter assay. Based on analysis of the UVSSA protein interactome in crosslinker treated cells we propose a model for transcription-coupled ICL repair (TC-ICR) that is initiated by stalling of transcribing RNA polymerase II (Pol II) at an ICL. Stalled Pol II is first bound by CSA and CSB, followed by UVSSA which recruits TFIIH to initiate downstream lesion removal steps. Second, we establish that UVSSA counteracts MYC dependent transcription stress to promote genome stability in cells aberrantly expressing the cMYC oncogene. UVSSA knockdown sensitizes cells to MYC expression, resulting in synthetic sickness and increased doubling time. UVSSA knockdown impacts Pol II dynamics in MYC activated cells. We conclude that UVSSA is required for regulation of Pol II during MYC induced transcription to prevent transcription stress. Together, these studies expand our understanding of UVSSA’s role in genome stability during transcription and elucidates the poorly understood transcription-coupled ICL repair pathway.

Strukturbestämning av proteiner är viktigt för att kunna förstå deras funktion och en snabbt utvecklande metod inom fältet är kryoelektronmikroskopi. Storleksbegränsningar förhindrar en bredare applikation av metoden eftersom små proteiner har för låg signal i förhållande till bakgrund för att kunna visualiseras som enstaka partiklar i elektronmiksoskopibilder. Hypotesen för projektet är att det är möjligt att avbilda väldigt små proteiner och kringgå den konventionella storleksbegränsningen genom att använda ett bärarprotein ((Putrescine Aminotransferase; YgjG) som kopplas till en affibody (Zwt) genom “helical fusion” och sedan binda ett litet målprotein till denna större struktur. Komplexet ska ge en tillräcklig storlek, symmetri och rigiditet för en lyckad elektronmikroskopi av bärare tillsammans med det lilla icke kovalent bundna målproteinet. För att karaktärisera den föreslagna bäraren, genomförs stabilitetstester genom CD, verifiering av inbindning av målproteinet i SPR, renhetsundersökning med SEC och slutligen kryoelektronmikroskopi för att testa konceptet. Det lilla målproteinet som kommer att avbildas i konceptstudien är en annan affibody (Z963), som i så fall skulle vara det minsta proteinet som någonsin har lösts med kryogenelektronmikroskopi. Resultaten visar att den undersökta tetramera-bäraren är väldigt stabil (Tm~ 85 oC) och kan tolerera en affibody-fusion med bibehållen bindning av multipla säten. Proteinet kan uttryckas rekombinant och renas till högt utbyte och bildar tetramerer även med fuserad affibody. De slutgiltiga resultaten från den kryoelektronmikroskopiska analysen inväntas fortfarande, men lovande griddar har skapats och en partikelselektion har gett klara 2-D klasser som också framhäver att det lilla målproteinet har bundit. Sammanfattningsvis har biofysikalisk karaktärisering indikerat att YgjG är en lovande bas för ett “imaging scaffold” och att preliminära enstaka-partikel kryoelektronmikroskopi analyser visar att den föreslagna strategin att undersöka små målproteiner är möjlig. / Determining structures of proteins is important to understand protein functions, and a rapidly evolving technique in this field is cryogen electron microscopy. However, size limitations are preventing wider applications of the technique because small proteins have poor signal to noise ratios and are not possible to distinguish in single-particle images. The hypothesis of this project is that it is possible to image very small proteins, bypassing the conventional size limitations of single-particle cryo-EM, by utilizing a carrier protein-scaffold (Putrescine Aminotransferase; YgjG) connected through helical fusion to an affibody (Zwt) that can bind to a small protein of interest. The complex provides a sufficient size, symmetry, and rigidity for successful electron microscopy also of the non-covalently bound small protein of interest. To characterise the proposed scaffold, thermal stability through CD, binding of target protein in SPR, purity through SEC and experiments towards proof-of-concept in cryo-EM will be performed. The small protein of interest to be imaged in the proof-of-concept setup is another affibody, called Z963, that would be the smallest protein ever solved with cryo-EM. The results show that the investigated tetrameric protein scaffold is a highly stable protein (Tm~85oC) that can tolerate affibody fusion with retained binding function of multiple sites. The protein can be recombinantly expressed and purified in high yield and forms tetramers also when fused to affibody. The cryo-EM results are still pending, but promising grids have been created and in an initial particle selection clear 2-D classes that also reveal the small bound protein of interest have been generated. To conclude, biophysical characterization indicates that YgjG is a promising base structure for an imaging scaffold and preliminary single-particle cryo-EM analyses show that the proposed strategy to investigate structures of small proteins of interest is feasible.

No description available.

No description available.