Design and production of protein nanostructures for biomolecular detection

Particulate nanostructures are increasingly used for analytical purposes. Such particles are often generated by chemical synthesis from non-renewable raw materials. Generation of uniform nanoscale particles is challenging and particle surfaces must be modified to make the particles biocompatible and water-soluble. Usually nanoparticles are functionalized with binding molecules (e.g., antibodies or their fragments) and a label substance (if needed). Overall, producing nanoparticles for use in bioaffinity assays is a multistep process requiring several manufacturing and purification steps. This study describes a biological method of generating functionalized protein-based nanoparticles with specific binding activity on the particle surface and label activity inside the particles. Traditional chemical bioconjugation of the particle and specific binding molecules is replaced with genetic fusion of the binding molecule gene and particle backbone gene. The entity of the particle shell and binding moieties are synthesized from generic raw materials by bacteria, and fermentation is combined with a simple purification method based on inclusion bodies. The label activity is introduced during the purification. The process results in particles that are ready-to-use as reagents in bioaffinity. Apoferritin was used as particle body and the system was demonstrated using three different binding moieties: a small protein, a peptide and a single chain Fv antibody fragment that represents a complex protein including disulfide bridge.If needed, Eu3+ was used as label substance. The results showed that production system resulted in pure protein preparations, and the particles were of homogeneous size when visualized with transmission electron microscopy. Passively introduced label was stably associated with the particles, and binding molecules genetically fused to the particle specifically bound target molecules. Functionality of the particles in bioaffinity assays were successfully demonstrated with two types of assays; as labels and in particle-enhanced agglutination assay. This biological production procedure features many advantages that make the process especially suited for applications that have frequent and recurring requirements for homogeneous functional particles. The production process of ready, functional and watersoluble particles follows principles of “green chemistry”, is upscalable, fast and cost-effective.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Preconditions for Successful Global Sourcing and Production Ramp up

Työn tavoitteena oli tutkia The Switch Drive Systems Oy:n edellytyksiä globaalille hankintatoiminnalle ja tuotannon aloittamiselle Kiinassa. Lisäksi pyrittiin keräämään empiiristä tietoa ja palautetta yrityksen prosesseista niiden kehittämistä varten. Yrityksen tuotantostrategia perustuu mallitehdaskonseptiin ja yrityksellä on kaksi mallitehdasta Suomessa. Työ keskittyy tutkimaan näiden mallitehtaiden valmiuksia aloittaa globaali ja ulkoistettu sarjatuotanto. Tutkimus suoritettiin empiirisenä tutkimuksena haastattelemalla yhteensä yhdeksää edustajaa yrityksen kolmesta yksiköstä Suomessa. Empiirinen tutkimus perustui riskienhallinnan keinoihin. Kvantitatiivisen tutkimuksen tarkoituksena oli kartoittaa merkittävimmät riskit tarkempaa tarkastelua varten. Kvalitatiivisen tutkimuksen tavoitteena oli löytää syitä merkittävimmille riskeille. Tutkimuksen keskeisimmäksi tulokseksi on saatu lista merkittävimmistä syistä, jotka vaikeuttavat yrityksen tuotantostrategian toteuttamista. Lisäksi tuloksia ovat kvantitatiiviset tulokset merkittävimmistä riskeistä ja niihin liittyvät syyseuraussuhteet.

Effect of light intensity and growth substratum on plant development and production of secondary metabolites in Cordia curassavica (Jacq.) Roem. & Schult

Cordia curassavica (Jacq.) Roem. & Schult. (Boraginaceae), also referred to as Cordia verbenacea DC, has been traditionally used for medicinal purposes. This study was driven to verify the behavior of the species in similar conditions to its natural environment, such as high light intensity and sandbank soil, and in conditions of low light intensity and fertilized substratum (dystroferric red nitosoil plus earthworm humus). The growth of the plant, the income of leaf crude extracts and, in the alcoholic extract, the number of substances found in thin layer cromatography and the toxicity of the substratum was observed. The results indicated that the growth of the root biomass, stem and leaves in discharge or lower light intensity was similar, but smaller in sandbank soil than in fertilized soil. The relative income of extracts in ether of petroleum and alcohol was larger in high light intensity and fertilized substratum. The light intensity and the substratum type didn't affect the number of substances detected in the alcoholic extract or the toxicity of this extract. Stains corresponding to the rosmarinic acid were only evidenced in some samples of the alcoholic extract, not allowing the verification of the effect of the treatments about its production.

Work and non-work : on work and meaning

I den politiska retoriken florerar debattinlägg om arbetslivet, och målsättningen är för det mesta att så många som möjligt ska arbeta så länge som möjligt. Att skapa jobb och minimera arbetslösheten ses som de främsta utmaningarna. På ett individuellt plan beskrivs arbete ofta som personligt självförverkligande eller som en nödvändig form av inkomst som kräver att livet struktureras enligt behovet av sysselsättning. I avhandlingen diskuteras hur de här till synes självklara rollerna som arbete spelar kan sägas neutralisera arbete. Med detta menas att kontroverser kring arbetets mening åsidosätts så att politiskt och etiskt grundläggande frågor om ett gott liv och mänskliga gemenskaper inte ges plats. Genom att gå i dialog med kritiska tänkare som Hannah Arendt, André Gorz, Kathi Weeks, Karl Marx och Richard Sennett diskuteras riskerna med att behandla arbete som en resurs, ett gemensamt samhällsprojekt eller individuellt självförverkligande. Vad är nödvändigt arbete? Vad innebär det att arbete är en vara? Vad är problemet med fragmentering av arbete? Det sägs ofta att arbete har blivit alltmer gränslöst – är detta något positivt eller negativt? Utifrån sådana frågor tar avhandlingen fasta på spänningar i arbetets roll i våra liv och i samhället. Genom att artikulera spänningar utmanas en neutralisering av arbetets etiskt och politiskt brännbara dimensioner. Avhandlingen syftar därmed till att lyfta fram ständigt pågående sätt att ifrågasätta och reflektera kring arbetets mening.

Adhesion and production of degrading enzymes by bacteria isolated from biofilms in raw milk cooling tanks

Biofilms in milk cooling tanks compromise product quality even on farms. Due to the lack of studies of this topic, this study evaluated the microbiological conditions of raw milk cooling tanks on farms and characterized the microorganisms isolated from these tanks. Samples were wiped off with sterile swabs from seven milk cooling tanks in three different points in each tank. Mesophiles and psychrotrophic counts were performed in all samples. The isolation of Pseudomonas spp., Bacillus cereus and atypical colonies formed on selective media were also performed, totalizing 297 isolates. All isolates were tested for protease and lipase production and biofilm formation. Of the total isolates, 62.9% produced protease, 55.9% produced lipase, and 50.2% produced biofilm. The most widespread genus inside the milk cooling tank was Pseudomonas since it was not possible to associate this contamination with a single sampling point in the equipment. High counts of microorganisms were found in some cooling tanks, indicating poor cleaning of the equipment and providing strong evidences of microbial biofilm presence. Moreover, it is worth mentioning the milk potential contamination with both microbial cells and their degrading enzymes, which compromises milk quality.

Order and membrane organization in chlorhexidine-lipid mixtures /

Formulations of a general bactericidal agent, chlorhexidine, mixed with a phospholipid at different concentrations are investigated using ^H NMR spectroscopy on a chain-deuterated lipid analog. Lipid-chlorhexidine formulation is known to release the drug into an aqueous medium slowly, maintaining a comparable concentration of the drug for up to four times longer than a direct aqueous solution. The NMR data does not support the proposed liposomal entrapment of chlorhexidine in lipid compartments. Complex thermal history of the lipid-chlorhexidine preparations is investigated in detail. In preparation for a counterpart measurement, using ^H NMR of deuterated chlorhexidine mixed with protonated lipid, the synthesis of a deuterated analog of chlorhexidine is performed.

Child care by choice or by default? : examining the experiences of unregulated home-based child care for women in paid work and training

This thesis aims to uncover the dynamics, causes and outcomes of women's reliance on unregulated home-based child care in Ontario, Canada, and the implications ofthis form of care for women's equality. Drawing on a longitudinal qualitative study, I examine the diverse experience of 14 women using home-based child care and engaged in both paid work/training and care work for children under the age of six, and draw comparisons with users of other forms of child care. I argue that home-based child care involves high levels of instability for continuity of care and is chosen largely as a default position based on economic considerations. It represents a compromise between the demands of social reproduction and paid work/training that entangles mothers in relations of exploitation with care providers. Doing so leaves both mothers and care providers socially and economically vulnerable and relying on social networks to fill in the gaps.

Social Economy and Developmental Disabilities: An Employment Option that Promotes a Form of Authentic Work and Fosters Social Inclusion

Typical employment options for people with developmental disabilities are insufficient. Most employment opportunities that are community-based provide typical workplace and geographical inclusion but tend not to support social inclusion and "belonging". This study explored the innovative employment alternative of social businesses and considered this form of employment for persons with a developmental disability as a viable avenue for meaningful work and social inclusion. A total of six business partners with a developmental disability were interviewed; two partners from three separate worker owned businesses. The partners' descriptions of their job and their workplace composed the interpretative findings. The social businesses provided an avenue for this group of people who tend to be segregated in isolated workshops or marginalized in mainstream work environments and who feel a sense of being "outsiders" to participate in meaningful work in community settings. This group of partners described their job as authentic "work" and discussed the many skills and the work ethic learned from their employment opportunity. In addition to the instrumental aspects of the job, the partners also discussed the group autonomy and self-determination of being their own "bosses". The partners confidently expressed feeling valued, understood in the context of others with similar life experiences, attached to the workplace and connected to a larger community as important outcomes of their businesses. These criteria of social inclusion (Hall, 2010) were complemented by teamwork, friendship and ultimately, with a feeling of being genuine "insiders". Replication of this innovative employment model would be recommended for groups of marginalized people with DD in other geographic areas.

Receipt from Chatfield and Neelon, Specialties, Steamboat Work and Engineers’ Supplies, St. Catharines

Receipt from Chatfield and Neelon, Specialties, Steamboat Work and Engineers’ Supplies, St. Catharines for various fittings, March 31, 1887.

Convergence of corporate governance : comparative study in Canada and in China

"Mémoire présenté à la Faculté des études supérieures En vue de l'obtention du grade de Maître en droit (LL.M.)"