A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.

Importance of considering food waste in the development of sustainable food packaging systems

Meeting the needs of both present and future generations forms the foundation of sustainable development. Concern about food demand is increasing alongside the continuously growing population. In the pursuit of food security preventing food waste is one solution avoiding the negative environmental impacts that result from producing food unnecessarily. Packages offer one answer to preventing food waste, as they 1) preserve and protect food, 2) introduce the user to the correct way to handle and use the food and package and 3) allow the user to consume the food in its entirety. This thesis aims to enhance the sustainability of food packages by giving special emphasis to preventing food waste. The focus of this thesis is to assist the packaging designer in being able to take into account the requirements for the sustainability of food packages and to be able to integrate these requirements into the product development process. In addition, life cycle methods that can be used as a tool in the packaging design process or in assessing the sustainability of finished food-packaging combinations are evaluated. The methods of life cycle costing (LCC) and life cycle working environment (LCWE) are briefly discussed. The method of life cycle assessment (LCA) is examined more thoroughly through the lens of the literature review of food-package LCA case studies published in the 21st century in three relevant journals. Based on this review and on experiences learned from conducting LCAs, recommendations are given as to how the LCA practitioner should conduct a food packaging study to make most of the results. Two case studies are presented in this thesis. The first case study relates the results of a life cycle assessment conducted for three food items (cold cut (ham), sliced dark bread (rye) and Soygurt drink) and the alternative packaging options of each. Results of this study show that the packaging constitutes only 1–12 % of the total environmental impacts of the food-packaging combination. The greatest effect is derived from the food itself and the wasted food. Even just a small percentage of wasted food causes more environmental impacts than does the packaging. The second case study presents the results of LCC and LCWE analysis done for fruit and vegetable transport packages. In this thesis, the specific results of the study itself are not the focus, but rather the study methods and scope are analysed based on how these complement the sustainability assessment of food packages. This thesis presents reasons why prevention of food waste should be more thoroughly taken into account in food packaging design. In addition, the task of the packaging designer is facilitated by the requirements of sustainable food packaging, by the methods and step-by-step guidance on how to integrate sustainability issues into the design process, and by the recommendations on how to assess the sustainability of food packages. The intention of this thesis is to express the issues that are important in the field of the food packaging industry. Having recognised and implemented these issues, businesses can better manage the risks that could follow from neglecting these sustainability aspects.

The role of cathepsin K in lung development and newborn chronic lung injury.

Chronic lung diseases, specifically bronchopulmonary dysplasia (BPD), are still causing mortality and morbidity amongst newborn infants. High protease activity has been suggested to have a deleterious role in oxygen-induced lung injuries. Cathepsin K (CatK) is a potent protease found in fetal lungs, degrading collagen and elastin. We hypothesized that CatK may be an important modulator of chronic lung injury in newborn infants and neonatal mice. First we measured CatK protein levels in repeated tracheal aspirate fluid samples from 13 intubated preterm infants during the first two weeks of life. The amount of CatK at 9-13 days was low in infants developing chronic lung disease. Consequently, we studied CatK mRNA expression in oxygen-exposed wild-type (WT) rats at postnatal day (PN) 14 and found decreased pulmonary mRNA expression of CatK in whole lung samples. Thereafter we demonstrated that CatK deficiency modifies lung development by accelerating the thinning of alveolar walls in newborn mice. In hyperoxia-exposed newborn mice CatK deficiency resulted in increased number of pulmonary foam cells, macrophages and amount of reduced glutathione in lung homogenates indicating intensified pulmonary oxidative stress and worse pulmonary outcome due to CatK deficiency. Conversely, transgenic overexpression of CatK caused slight enlargement of distal airspaces with increased alveolar chord length in room air in neonatal mice. While hyperoxic exposure inhibited alveolarization and resulted in enlarged airspaces in wild-type mice, these changes were significantly milder in CatK overexpressing mice at PN7. Finally, we showed that the expression of macrophage scavenger receptor 2 (MSR2) mRNA was down-regulated in oxygen-exposed CatK-deficient mice analyzed by microarray analysis. Our results demonstrate that CatK seems to participate in normal lung development and its expression is altered during pulmonary injury. In the presence of pulmonary risk factors, like high oxygen exposure, low amount of CatK may contribute to aggravated lung injury while sustained or slightly elevated amount of CatK may even protect the newborn lungs from excessive injury. Besides collagen degrading and antifibrotic function of CatK in the lungs, it is obvious that CatK may affect macrophage activity and modify oxidative stress response. In conclusion, pulmonary proteases, specifically CatK, have distinct roles in lung homeostasis and injury development, and although suggested, broad range inhibition of proteases may not be beneficial in newborn lung injury.

Data analysis tools and methods for DNA microarray and high-throughput sequencing data

The recent rapid development of biotechnological approaches has enabled the production of large whole genome level biological data sets. In order to handle thesedata sets, reliable and efficient automated tools and methods for data processingand result interpretation are required. Bioinformatics, as the field of studying andprocessing biological data, tries to answer this need by combining methods and approaches across computer science, statistics, mathematics and engineering to studyand process biological data. The need is also increasing for tools that can be used by the biological researchers themselves who may not have a strong statistical or computational background, which requires creating tools and pipelines with intuitive user interfaces, robust analysis workflows and strong emphasis on result reportingand visualization. Within this thesis, several data analysis tools and methods have been developed for analyzing high-throughput biological data sets. These approaches, coveringseveral aspects of high-throughput data analysis, are specifically aimed for gene expression and genotyping data although in principle they are suitable for analyzing other data types as well. Coherent handling of the data across the various data analysis steps is highly important in order to ensure robust and reliable results. Thus,robust data analysis workflows are also described, putting the developed tools andmethods into a wider context. The choice of the correct analysis method may also depend on the properties of the specific data setandthereforeguidelinesforchoosing an optimal method are given. The data analysis tools, methods and workflows developed within this thesis have been applied to several research studies, of which two representative examplesare included in the thesis. The first study focuses on spermatogenesis in murinetestis and the second one examines cell lineage specification in mouse embryonicstem cells.