Bacteriophages (phages) are bacterial viruses. Phages offer remarkable diversity and can be found in many shapes and sizes; however, what they all have in common is that they are made of protein nano-shells that encase their genome (DNA or RNA). In other words, phages are proteinous bionanoparticles. In this work, we use the filamentous phage M13. M13 is a simple virus with a high aspect ratio. It has 11 genes and only 5 structural proteins. The phage filament is almost entirely made of 2700 copies of pVIII, the major coat protein, and is capped off on one end by five copies each of the proteins pIII and pVI, while the opposite end displays five copies each of the proteins pVII and pIX. M13 phage can be genetically engineered to display certain peptides with affinity toward cancer cells, specific tissue, or even minerals and polymers. These filaments can further self-organize to form liquid crystals at high concentrations. All these properties make M13 a unique building block for the bottom up synthesis of advanced bioactive material. The objective of my proposed research is to develop hydrogels using M13 phage. Hydrogels can absorb large quantities of water without dissolving. They mark a breakthrough in the field of biomaterials, owing to their high water content, porosity and soft consistency. I crosslinked M13 at liquid crystalline concentrations using glutaraldehyde. The resulting hydrogels were characterized for swelling and mechanical properties. These hydrogels exhibited self-healing and autofluorescence properties. In addition, I demonstrated that M13 can from self-healing hydrogels at lower concentrations by adding the small globular protein, BSA. The developed M13 hydrogels mark the first step in the development of bioactive hydrogels that could be utilized to direct cell destiny and genuinely mimic the natural tissue. / Thesis / Master of Applied Science (MASc) / Filamentous phage are viruses that infect bacteria. These bio-filaments are ~1 𝜇𝑚 long, 6-8 nm in diameter and can propagate themselves by infecting bacteria. This means one bio-filament can make 300-1000 particles only by infecting a bacterial host, a characteristic that drastically increases their utility over synthetic filamentous nanomaterial. Filamentous phage can be readily genetically engineered to express foreign receptors on their surface. In this thesis, I demonstrate how these bio-filaments can self-organize at high concentrations and can be crosslinked to make hydrogels that can adsorb up to 12 times their weight in water. These hydrogels can also heal themselves if broken or cut and exhibit autofluorescence, which are very useful properties for hydrogels used for biomedical applications. We further demonstrate that adding small proteins to the bio-filaments can expand the range of hydrogel formation, to the extent that even low concentrations of bio-filament can form hydrogels.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-30T21:26:19Z No. of bitstreams: 1 Mayara Ingrid Sousa Lima Seleção e caracterização de peptideos....pdf: 1915651 bytes, checksum: 4954d0969cc99ed5643d45ee27772173 (MD5) / Made available in DSpace on 2012-07-30T21:26:19Z (GMT). No. of bitstreams: 1 Mayara Ingrid Sousa Lima Seleção e caracterização de peptideos....pdf: 1915651 bytes, checksum: 4954d0969cc99ed5643d45ee27772173 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia,Brasil / A hanseníase é uma doença infecciosa crônica, causada pelo Mycobacterium leprae, que apresenta manifestações clínicas variadas. Essas variações refletem em diferenças que vão de uma forte resposta imune celular com controle do crescimento do bacilo, no pólo tuberculóide, a uma anergia em resposta celular, no pólo virchoviano. A caracterização do perfil antigênico do M. leprae frente a esse quadro de múltiplos aspectos clínicos representa uma ferramenta fundamental para o desenvolvimento de novas plataformas para um diagnóstico diferencial mais sensível e/ou desenvolvimento de unidades vacinais. Dessa forma, o objetivo desse trabalho foi selecionar e caracterizar peptídeos miméticos de antígenos do M. leprae reativos contra IgGs totais purificadas de pacientes com hanseníase. Para a seleção foi utilizada a tecnologia de phage display, usando bibliotecas randômicas de peptídeos expressos em fagos filamentosos. Foi realizada uma seleção com IgGs de pacientes Tuberculóides e outra com IgGs de pacientes Virchovianos. A validação dos peptídeos foi realizada utilizando o imunoensaio ELISA, o teste de redução de colônias e análise de bioinformática. Após a pré-validação e sequenciamento foram encontradas 17 mimotopos para o pólo Vichorviano e 12 no pólo Tuberculóide. Foram validados 4 peptídeos, sendo 2 do pólo Tuberculóide (T03, T04) e 2 do pólo Virchoviano (V06 e V13). Os peptídeos TALFPWL (T03) e YSTTLSY (T04) foram imunorreativos em soros de pacientes paucibacilares, bem como em pacientes Virchovianos, além de terem alinhado com proteínas de membrana do M. leprae com potencial antigênico. O peptídeo V06 apresentou especificidade de 100% e sensibilidade de 94,74%, o que se complementa com os dados do teste de redução da pIII, o qual obteve uma taxa de redução de 82% em soros Virchovianos. O peptídeo V13 também foi reativo e apresentou similaridades com chaperonas e proteínas de membrana. Este estudo aponta perspectivas para a identificação de novos antígenos, propiciando a descoberta de novos alvos biológicos com potencial diagnóstico e/ou terapêutico. / Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which has varied clinical manifestations. These variations reflect differences that spans from a strong cellular mediated immunity and bacili growth control the tuberculoid pole to a poor T cell immunity at the lepromatous pole. The antigenic profile characterization in both clinical forms represents a fundamental tool for the development of new platforms for a differential diagnosis more sensitive and/or development of vaccine units. Thus, the objective was to select and characterize mimetics peptides antigens of M. leprae reactive against total IgG purified from leprosy patients. The phage display technology was used for selection using random peptides libraries expressed on filamentous phages. A selection was performed with IgGs from tuberculoid patients and other IgGs of lepromatous patients. Peptides validation was performed using the ELISA immunoassay, the plaque reduction test and bioinformatics analysis. After the pre-validation and sequencing were found 17 valid sequences for the lepromatous pole and 12 tuberculoid pole. Four peptides were validated, two of tuberculoid pole (T03, T04) and two lepromatous pole (V06 and V13). The peptides TALFPWL (T03) and YSTTLSY (T04) were imunoreatives in sera from paucibacillary patients and in lepromatous patients. They had alignment with membrane proteins of M. leprae antigenic potential. The V06 peptide showed 100% specificity and 94.74% sensitivity, which is supplemented with the plaque reduction test, who obtained a reduction rate of 82% in lepromatous sera. The V13 peptide was also reactive and showed similarities with chaperones and membrane proteins. This study presents insights for new antigens identification, leading to discovery of new biological targets with potential diagnostic or therapeutic

M13 filamentous bacteriophage has been used as a biotemplate for the nucle- ation of materials. Phage is an ideal and diverse scaffold with its large aspect ratio and ability to display biomolecules to bind a range of targets. To form more complex patterned materials, interactions between the phage must be specific and reliable. We develop a phage labeling method using sortase enzymes to create multi-phage nanostructures. We exploit two sortases and functionalize the N-termini of the pIII, pIX, and pVIII proteins with small and large moieties. For the pVIII, we show a 100 fold improvement in display of GFP molecules on the phage surface. Taking advantage of orthogonal sortases, we simultaneously label two capsid proteins on a single phage particle. Using these N-terminal labeling techniques, we demonstrate fluorescent staining of cells and construct a lampbrush phage structure linking the pIII of one phage to the pVIII of another using a biotin-streptavidin linkage. To further expand our labeling repertoire, C-terminal sortase labeling of phage was pursued. To achieve this goal, we transfer a loop structure from cholera toxin to pIII and label it with a fluorophore and a multi-domain protein. With this archi- tecture, we form end-to-end dimers using sortase to conjugate the loop structure to phage containing the nucleophile motif. Lastly, we investigate DNA hybridization as a method for crosslinking phage. Using sortase, we label the pVIII on two sets of phage: one with ssDNA and the other with a complementary DNA oligonucleotide. We anneal these phages together and observe phage networks that are dispersed by heat and reform upon cooling.

Filamentous bacteriophages were engineered to express foreign genes with the ultimate purpose of displaying transmission control anti-malarial peptides as in phage display. It was hypothesized that expression of foreign genes would be possible using the phage’s promoters. This hypothesis was tested by assuming that promoters for the phage major coat protein (MCP) gene would also promote the expression of any foreign gene inserted downstream of the MCP gene. As proof of principle, the bacteriophages Pf3, Pf1, and M13 were engineered in this way to successfully synthesize Enhanced Green Fluorescent Protein (EGFP). Type 88 phage display on the EGFP recombinant Pf3 was attempted by fusing a second copy of its MCP gene to the existing EGFP gene. This resulted in a phage display Pf3 replacement vector which was then used to construct a phage for displaying an anti-malarial peptide.

This article investigates the determinants of entry and exit in the Austrian manufacturing sector based on 1981 to 1994 data. We study the response of entry, exit and other indicators of firm dynamics to changes in average plant size, size heterogeneity, concentration, incentives and vertical integration. By applying Bayesian simulation methods we estimate random coefficient models and study the symmetry of the determinants of entry and exit. Our empirical analysis shows that entry and exit rates are driven by the same determinants. The impacts of these determinants are nearly homogeneous for both, entry rates and exits rates, respectively. Moreover, we find (i) that changes in average plant size, size heterogeneity and concentration are not symmetric with respect to entry and exit, (ii) that changes in the growth of sales is weakly symmetric and (iii) that the growth rate of employment is strongly asymmetric across industries in Austrian manufacturing. Furthermore, we infer from the data that the turnover of firms influences the changes in the number of competitors. Low entry rates go hand in hand with low net entry rates and a low turnover. (author's abstract) / Series: Working Papers Series "Growth and Employment in Europe: Sustainability and Competitiveness"

The present paper provides first microlevel (indirect) empirical evidence on changes in the determinants of firm profitability, the role of fixed and sunk costs, as well as the nature of competition for a transition economy. We estimate size thresholds required to support different numbers of firms for four retail and professional service industries in a large number of geographic markets in Slovakia. The three time periods in the analysis (1995, 2001 and 2010) characterize different stages of the transition process. Specific emphasis is given to spatial spill-over effects between local markets. Estimation results obtained from a spatial ordered probit model suggest that entry barriers have declined considerably (except for restaurants) and the intensity of competition has increased. We further find that demand spill-overs and/or the effects associated with a positive correlation in unobservable explanatory variables seem to outweigh negative spill-over effects caused by competitive forces between neighboring cities and villages. The importance of these spatial spill-over effects differs across industries. / Series: Department of Economics Working Paper Series

Zahlreiche Arbeiten im Bereich der Entrepreneurship-Forschung versuchen aus unterschiedlichen disziplinären Perspektiven zu erklären, warum manche Menschen Opportunities wahrnehmen und unternehmerisch tätig werden und andere nicht. Dies wird in der Literatur als "Opportunity Recognition" (OR) bezeichnet und weitgehend als Kernelement im entrepreneurialen Prozess beschrieben. Aufgrund dieser Bedeutung von Opportunity Recognition entwickelten sich zahlreiche Erklärungsansätze, die als ökonomische, psychologische oder sozio-kognitive Perspektiven in die Entrepreneurship-Forschung eingeführt wurden. Die vorherrschende Trennung in disziplinenspezifische Erklärungsansätze hat zwar ihre Berechtigung, liefert jedoch bisher unbefriedigend integrative Erkenntnisse. In Abgrenzung zu anderen Arbeiten betrachtet dieser Beitrag Opportunity Recognition auf Basis subjektiver Theorien von Entrepreneuren. Der integrative Ansatz der subjektiven Theorien bietet das Potential disziplinenübergreifend Hinweise auf mögliche Einflussfaktoren der OR in subjektiven Theoriebeständen zu untersuchen. Ziel der Arbeit ist zu erforschen, welche inhaltlichen und strukturellen Charakteristika subjektive Theorien von Entrepreneuren aufweisen und inwiefern sich Logiken und/oder Strategien für die Identifikation einer Opportunity in ihnen widerspiegeln. Dazu wurde eine empirische Rekonstruktion der Strukturlogik subjektiver Theorien von Entrepreneuren auf Basis von narrativen Interviews und objektiv-hermeneutischen Interpretationsverfahren durchgeführt. Die Ergebnisse dieser Arbeit liefern vielfältige Erkenntnisse hinsichtlich der Strukturen und Inhalte von subjektiven Theorien von Entrepreneuren sowie ihrer Entwicklung. Sie verweisen auf die zentrale Bedeutung sozialer Interaktionskontexte des Entrepreneurs, sowohl für die Ausprägung subjektiver Theoriebestände im Allgemeinen als auch für die Schärfung von Opportunities und ihrer Verwertung im Speziellen. Zudem wird deutlich, dass sich subjektive Theoriebestände hinsichtlich ihres Abstraktions- und Komplexitätsgrades sowie ihrer zeitlichen Stabilität maßgeblich unterscheiden. Die Erkenntnisse der vorliegenden Arbeit lassen Zweifel an der Sinnhaftigkeit einer auf ein analytisches Konstrukt "Opportunity" fokussierten Forschung aufkommen. Als Alternative wird das umfassendere Konzept der subjektiven Unternehmertheorie vorgeschlagen, das die Relevanz mentaler Modelle von Unternehmern und das ihnen inhärente Integrationspotential für unterschiedliche Einflussfaktoren und Erklärungsansätze für OR verdeutlicht. (Autorenref.)

The successful fabrication and characterization of two chemiresistive platforms for biomolecule detection was demonstrated by this work. The Si/Silica based single walled nanotube thin film (SWNTTF) platform was developed to understand the effect of device geometry on pH and M13 bacteriophage sensing capabilities as well as the underlying mechanisms governing SWNTTF chemiresistive biosensors. The dominant mechanism of sensing switched from direct chemical doping to electrostatic gating when the target analyte changed from H+/OH- ions in pH testing to whole viruses. The experimental limit of detection for M13 for this platform was 0.5pM and an increased sensitivity as well as variability was observed in devices with smaller channel widths. Preliminary device calibration was completed in order to correlate a resistance response to a bulk M13 concentration. The polyethylene terephthalate (PET) based SWNTTF platform was developed to demonstrate the commercial potential of SWNTTF chemiresistive biosensors by detecting relevant concentrations of brain natriuretic peptide (BNP) on economically viable substrates. The pH response of these chemiresistors confirmed that chemical doping was the cause for resistance change in the SWNTTFs. The preliminary results demonstrated successful BNP detection at 50pg/mL using both aptamers and antibodies as recognition elements. Using SWNTTFs as the transducing element of chemiresistors allowed for further understanding of electrical mechanisms of sensing as well as achieving sensitive, real-time and reproducible electrical virus and biomolecule detection. Although these platforms do not achieve ultrasensitive limits of detection, they demonstrate the commercial potential of platforms using SWNTTFs as the transducing element of electrical biomolecule sensors.

Applications of biotechnology in drug delivery and medical instrumentation and energy storage have been gaining popularity. Especially, utilization of biotechnology for energy storage is attracting attention due to its environmentally friendly nature and cost efficiency. In this project, a filamentous bacteriophage, M13, to fabricate metal oxide battery electrodes. M13 phage is 6.5 nm wide and 800 nm long, and can act as a template to produce nano-sized metal oxide particles. A method to prepare manganese oxide electrodes was developed, where the phage is integrated with the oxide into a nanocomposite. The composite material was used to make a high capacity electrode for lithium ion batteries. The M13 templated manganese oxide, Mn3O4, could deliver a high initial capacity of 766 mAh/g, and recorded a stabilized discharge capacity of ~800 mAh/g even after 60 cycles.

The present article provides first microlevel (indirect) empirical evidence on changes in entry barriers, the determinants of firm profitability as well as the nature of competition for a transition economy. We estimate size thresholds required to support different numbers of firms for several retail and professional service industries in a large number of geographic markets in Slovakia. The 3 time periods in the analysis (1995, 2001 and 2010) characterize different stages of the transition process. Specific emphasis is given to spatial spill-over effects between local markets. Estimation results obtained from a spatial ordered probit model suggest that entry barriers have declined considerably (except for restaurants) and that the intensity of competition has increased on average. We further find that demand spill-overs and/or the effects associated with a positive correlation in unobservable explanatory variables seem to outweigh negative spill-over effects caused by competitive forces between neighbouring cities and villages. The importance of these spatial spill-over effects differs across industries.