Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

Determining how biological cells respond to external factors in the environment can aid in understanding disease progression, lead to rational design strategies for tissue engineering, and contribute to understanding fundamental mechanisms of cellular function. Dynamic mechanical forces exist in vivo and are known to alter cellular response to other stimuli. However, identifying the roles multiple external factors play in regulating cell fate and function is currently impractical, as experimental techniques to mechanically stimulate cells in culture are severely limited in throughput. Hence, determining cell response to combinations of mechanical and biological factors is technically limited. In this thesis, microfabricated systems were designed, implemented and characterized to screen for the effects of mechanical stimulation in a high-throughput manner. Realizing these systems required the development of a fabrication process for precisely-aligned multilayer microstructures, and the development of a method to integrate non-traditional and clinically-relevant biomaterials into the microfabrication process. Three microfabricated platforms were developed for this application. First, an array was designed for experiments with high mechanical throughput, in which cells cultured on a surface experience a range of cyclic, uniform, equibiaxial strains. Using this array, a novel time- and strain-dependent mechanism regulating nuclear β-catenin accumulation in valve interstitial cells was identified. Second, a simpler system was designed to screen for the effects of combinatorially manipulated mechanobiological parameters on the pathological differentiation of valve interstitial cells. The results demonstrate functional heterogeneity between cells isolated from different regions of the heart valve leaflet. Last, a microfabricated platform was developed for high-throughput mechanical stimulation of cells encapsulated in a three-dimensional biomaterial, enabling the study of mechanical forces on cells in a more physiologically relevant microenvironment. Overall, these studies identified novel biological phenomena as a result of designing higher-throughput systems for the mechanical stimulation of cells.

In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.

Over a decade after the completion of the human genome, researchers around the world are still wondering what information is hidden in the genome. Although the sequences of all human genes are known, it is still almost impossible to determine much more than the primary protein structure from the coding sequence of a gene. As a result of that, the need for recombinantly produced proteins to study protein structure and function is greater than ever. The main objective of this thesis has been to improve protein production, particularly using Escherichia coli. To improve protein production in Escherichia coli there are a number of different parameters to consider. Two very important parameters in the process of protein production are transcription and translation. To study the influence of differences in transcription rate, target proteins with different characteristics were produced under control of three promoters of different strength (lacUV5, trc and T7). Analyzing the total amount of target protein as well as the amount of soluble protein demonstrated the benefits of using a strong promoter such as T7. However, protein production is also highly dependent on translational efficiency, and a drawback associated with the use of Escherichia coli as host strain is that codons rarely used in this host can have a negative effect on the translation. The influence of using a strain supplied with genes for rare codon tRNAs, such as Rosetta(DE3), instead of the standard host strain BL21(DE3), was therefore evaluated. By using Rosetta(DE3) an improved protein yield for many of the poorly produced proteins was achieved, but more importantly the protein purity was significantly increased for a majority of the proteins. For further understanding of the underlying causes of the positive effects of Rosetta(DE3), the improved purity was thoroughly studied. The cause of this improvement was explained by the fact that Rosetta(DE3) has a significantly better read through of the full sequence during translation and thereby less truncated versions of the full-length protein is formed.  Moreover, the effect of supplementation of rare tRNAs was shown to be highly dependent on the target gene sequence. Surprisingly, it was not the total number of rare codons that determined the benefit of using Rosetta(DE3), instead it was shown that rare arginine codons and to some extent also rare codon clusters had a much bigger impact on the final outcome. As a result of the increased interest in large-scale studies in the field of proteomics, the need for high-throughput protein production pipelines is greater than ever. For that purpose, a protein production pipeline that allows handling of nearly 300 different proteins per week was set up within the Swedish Human Protein Atlas project. This was achieved by major and minor changes to the original protocol including protein production, purification and analysis. By using this standard setup almost 300 different proteins can be produced weekly, with an overall success rate of 81%. To further improve the success rate it has been shown that by adding an initial screening step, prior high-throughput protein production, unnecessary protein production can be avoided. A plate based micro-scale screening protocol for parallel production and verification of 96 proteins was developed. In that, protein production was performed using the EnBase® cultivation technology followed by purification based on immobilized metal ion affinity chromatography. The protein products were finally verified using matrix-assisted laser desorption ionization time-of-flight MS. By using this method, proteins that will be poorly produced can be sorted out prior high-throughput protein production. / <p>QC 20131120</p>

Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.

In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.

Microbial fuel cells (MFCs) are novel green technologies that convert chemical energy stored in biomass into electricity through microbial metabolisms. Both fossil fuel depletion and environmental concern have fostered significant interest in MFCs for both wastewater treatment and electricity generation. However, MFCs have not yet been used for practical applications due to their low power outputs and challenges associated with scale-up. High throughput screening devices for parallel studies are highly necessary to significantly improve and optimize MFC working conditions for future practical applications. Here in this research, microfabricated platforms of microbial fuel cell array as high throughput screening devices for MFC parallel studies have been developed. Their utilities were described with environmental sample screening to uncover electricigens with higher electrochemical activities. The first version of the MFC arrays is a batch-mode miniaturized 24-well MFC array using ferricyanide as catholyte. Several environmental species that showed higher electricity generation capabilities than Shewanella oneidensis MR-1 (SO) were uncovered using the developed MFC array, with one environmental electricigen, Shewanella sp. Hac353 (dq307734.1)(7Ca), showing 2.3-fold higher power output than SO. The second MFC array platform developed is an air-cathode MFC array using oxygen in air as electron acceptor, which is sustainable compared to ferricyanide that depletes over time. Environmental electricigen screenings were also conducted, showing parallel comparison capabilities of the developed array. The third MFC array platform is a microfluidic-cathode MFC array that enables long-term operations of miniature MFC arrays with improved power generation abilities. The capability of the microfluidic-cathode MFC array to support long-term parallel analysis was demonstrated by characterizing power generation of SO and 7Ca, proving extended operation time and improved power outputs compared to batch-mode MFC array. The fourth MFC array platform enables both catholyte and anolyte replenishments for long-term characterization of various carbon substrate performances. Finally, the 24-well microfluidic MFC array was further scaled up to 96 wells, which greatly increased the throughput of MFC parallel studies. The developed MFC arrays as high throughput screening platforms are expected to greatly impact how current MFC studies are conducted and ultimately lead to significant improvement in MFC power output.

Staphylococcus aureus is a common and versatile opportunistic pathogen in humans. Increases in the incidence of community acquired and nosocomial infections, coupled with the emergence of antibiotic resistant strains, are causing new treatment challenges for health care professionals. S. aureus readily binds to the endothelial cell surface and utilizes host cell endocytosis to evade host cell immune responses. Inhibition of endocytosis may cause S. aureus to remain unprotected at the host cell surface, allowing host immune systems and other therapeutics more time to clear an infection. Simvastatin inhibits host cell endocytosis. We hypothesize that using simvastatin to inhibit S. aureus invasion of host cells, a high throughput, small molecule screen can be developed. The high throughput screen will evaluate the National Institutes of Health small molecule library for compounds that better inhibit endocytosis. Additionally, 2-dimensional gel electrophoresis will be performed to elucidate the pathway simvastatin alters to inhibit endocytosis. / Department of Biology

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