Biomaterials used in biomedical implants, diagnostic devices, and in-situ sensors, all face the issue of biofouling. Surface modification of biomaterial surfaces with antifouling polymers can prevent non-specific adsorption of proteins and other bio-foulants onto these surfaces. Although there are many antifouling polymers to chose from, getting the polymers onto different materials is challenging as the surface modification process is dependent on the substrate’s surface chemistry. This limits the kinds of materials that are able to be modified, especially in devices made with several materials that must be modified as a single unit. Therefore, the goal of this research is to develop an effective antifouling surface modification that is compatible with different types and classes of biomaterials. A three-step modification approach was taken to form dense antifouling polymer brushes. The surfaces were first activated using oxygen plasma to increase the density of surface hydroxyl groups. Next, a silane coupling agent with an Atom Transfer Radical Polymerization (ATRP) initiator was attached to the activated surfaces. Finally, an antifouling zwitterionic monomer was polymerized on the surface using an aqueous controlled living radical polymerization technique, Surface Initiated - Activators Regenerated by Electron Transfer – Atom Transfer Radical Polymerization (SI-ARGET-ATRP). Two zwitterionic antifouling polymers, poly(carboxybetaine methacrylate) (pCBMA), and poly(sulfobetaine methacrylate) (pSBMA) were investigated. Clinically- and environmentally-relevant materials were studied and include poly(dimethylsiloxane) (PDMS), poly(ether ether ketone) (PEEK), titanium, silicon, and 3D printed stainless steel. Water contact angle (WCA) analysis showed that surfaces modified with zwitterionic polymers became more hydrophilic. WCA analysis may not be suitable for evaluating non-modified 3D printed surfaces due to their poor surface finish, and this material requires further surface topography characterization. Atomic force microscopy (AFM) and ellipsometry showed that the zwitterionic polymer layers did not necessarily have to be thick to produce their hydrophilic effect. AFM also revealed that each step of the surface modification process produced different roughness effects on all of the different surfaces. The zwitterionic layer with the smoother surface tended to better resist bovine serum albumin (BSA) adsorption. Radiolabelled BSA experiments showed reduced fouling on all 2D samples but to different degrees. The pCBMA modification was not successful in preventing BSA fouling on 3D printed 316L stainless steel. Full or partial BSA fouling may be due to the hydrolytic instability of the silane coupling agent, used to form covalent bonds between the antifouling polymers and the different surfaces, although further investigation is required to validate this hypothesis. Improving the long-term stability of silanes or research with other multi-surface compatible coupling agents that have better long-term stability in aqueous solutions should be pursued. / Thesis / Master of Applied Science (MASc)

L'atrophie musculaire bulbo-spinale (SBMA) est une dégénérescence lente et progressive des motoneurones causée par l'élongation du triplet nucléotidique (CAG) dans le gène codant pour le récepteur aux androgènes (RA) localisé sur le chromosome X. Dans la SBMA, ce récepteur à extension polyglutaminique (polyQ) pathogène s'accumule de manière ligand dépendante dans le cytoplasme sous forme d'agrégats mais également dans le noyau y créant des corps d'inclusions nucléaires considérés comme la marque identitaire histologique, dont le caractère cytotoxique est aujourd'hui remis en question. Nous avons développé un modèle SBMA in vitro basé sur l'expression inductible d'un RA51Q dans la lignée hybride NSC34, qui est comparé au modèle normal NSC34 exprimant un RA contenant 20Q. Nous avons démontré que l'expression du RA51Q entraîne une diminution de la viabilité ainsi qu'une altération de la croissance neuritique sans formation d'agrégats insolubles dans le noyau ou le cytoplasme des cellules. Le RA en tant que membre de la superfamille des récepteurs nucléaires est un facteur de transcription mais peut également induire des voies de signalisation non génomiques via sa localisation membranaire. Après avoir montré une localisation du RA20Q et du RA51Q dans les « lipid rafts », nous avons corrélé la diminution de la viabilité et de la pousse neuritique induite par le RA51Q à une altération de la signalisation cellulaire non génomique. Les résultats obtenus mettent en évidence une dérégulation des voies de signalisation PI3K/Akt et JNK/c-jun induite par l'expression du RA muté dans notre modèle SBMA. / Spinal Bulbar Muscular Atrophy (SBMA) is a progressive inherited motoneuron disease caused by the expansion of a trinucleotide (CAG) repeat in the gene coding for the androgen receptor (AR) located on the X chromosome. This rare disease causes muscle weaknesses, hypotonia, hyporeflexia, fasciculations of facial muscles in male patients. The androgen-dependent formation of cytoplasmic aggregates and nuclear inclusions are pathological hallmarks of this polyglutamine disease but their potential neurotoxicity is still under debate. We developed a SBMA model based on a doxycycline-inducible AR51Q expression system in the NSC34 hybrid cell line. We have shown that the expression of the mutated AR leads to a reduced viability and to an alteration of neurite outgrowth compared to cells expressing the normal AR20Q. The AR belongs to the nuclear receptor superfamily of transcription factors. However, recent data have put in evidence a membrane localization of AR initiating non-genomic signaling pathways. Because we have not observed insoluble aggregates, reduced viability and neurite outgrowth could not be correlated to AR aggregation. We hypothesized that motoneuron death is not only due to aggregate formation but also to the alteration of AR signaling pathways. We focused on a correlation between the AR localization in lipid rafts and the observed phenotypes. Our results highlight the deregulation of PI3K/Akt and JNK/c-jun signaling pathways induced by the expression of AR51Q in our SBMA model.

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Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Although androgens are known to exert anabolic effects in skeletal muscle, overexpression of androgen receptor (AR) selectively in this tissue causes androgen dependent motor deficits and muscular atrophy. The cellular and subcellular changes underlying this phenotype are unknown. Therefore, this study aimed to elucidate the ultrastructural and histologic changes accompanying myopathy and to determine the importance of androgens and overexpression level for myopathic features. Transmission electron microscopy revealed augmented mitochondrial content and reduced myofibril width in androgen exposed transgenics. Additionally, male transgenics demonstrated increased glycogen content. Histochemical analyses confirmed sex-specific changes in glycogen content and revealed a surprising loss in the proportion of oxidative fibers in symptomatic animals. However, increased mitochondrial content was confirmed by the presence of ragged red fibers. Overexpression of AR in muscle fiber results in mitochondrial pathology and dysregulation of glycogen metabolism, possibly reflecting normal but exaggerated function of androgens in skeletal muscle fibers.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluoresceinlabeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and DsbmA and DmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.