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.

Polyphenol oxidases (PPOs) are ubiquitous enzymes that oxidize phenols to quinones in the presence of molecular oxygen, often leading to tissue discolouration. They are sometimes considered as defense proteins but other functions, for example in phenolic compound biosynthesis, have also been found. In this thesis, bioinformatic searches were conducted to identify putative PPO genes from available genomes representing five Viridiplantae lineages: chlorophytes, bryophytes, lycophytes, monocotyledonous anthophytes and eudicotyledonous anthophytes. Duplicated PPO genes were found in most land plant genomes. A detailed investigation of the poplar (Populus trichocarpa) PPO gene family found nine genes that exhibit differential expression profiles during development and following stress, of which PtrPPO1 was the only significant wound-inducible PPO gene. A phylogenetic reconstruction of the poplar PPOs identified PtrPPO13 to be an unusual PPO homolog and it was studied in detail. Experimental evidence indicated that PtrPPO13 is expressed in most organs, and unlike most PPOs, is localized to the vacuole. Together, the phylogeny, gene expression and subcellular localization studies suggest that PPOs are likely to have variable physiological functions in plants and that PtrPPO13 is distinct from most typical PPOs. / Graduate / 0309

Current filter validation methods require 48 hours culture for results to become available, which creates time delays within the manufacturing process and quality control back-logs The thesis compares alternative methods for the production of filter challenge test data Within 24 hours to the desired test sensitivity, using bioluminescent and fluorescent genetically engineered strains of the test organism Brevundzmonas dzmznuta The recombinant strains were produced using a Tn5 transposon system, using a filter mating method. The genes cloned into the bacterial chromosome were the biolummescence lux_ genes, taken from the marme bacteria, Photorhabdus lummescens or Vzbno harveyz, and the gene encoding green fluorescent protein taken from the marine jelly fish Aequoria victoria The cloned strains were found to show no difference to the w1ld type strain With respect to their surface hydrophobicity, according to a bacterial adherence to hydrocarbons assay, and surface charge, according to an electro-static interaction chromatography method. Furthermore, the cell size according to Transmission Electron Microscopy was not significantly different to the wild type strain, which had cell dimensions of 1 05 x 0 52 Jlm The retention of cells by 0 45 mtcron rated filters, was shown to be not significantly different to the wild type All strains were retained by 0 2 Jlm filters These data confirmed that the cloned strains were suitable for challenge testing Four methods were used to detect microcolonies of the recombinant strains on filters. The advantage of the microcolony detection system was that it showed that the cells detected downstream of the filter were viable and culturable. The best detection method was with an epifluorescent microscope and the fluorescent strain after 24 hours, for which the sensitivity was 98.1 %. Two CCD camera systems were used to detect the bioluminescent strains on filters. The sensitivity of these systems were 80.1% and 83 9%, for the Nucleovision and Nightowl CCD camera systems, respectively, after 24 hours In addition, the Bwprobe photomultipherbased system was shown to achieve the detection sensitivity of one microcolony after 24 hours. Also, steps were made to study transcription Initiation signals for gene expression in fluorescent recombinant Brevundzmonas dzmmuta. Various putative promoter sequences were Identified in one fluorescent strain, using a DNA sequencmg method. These sequences showed homology to previously identified E colr and Brevundzmonas promoter sequences. Finally, an attempt was made to produce recombinant fluorescent and bioluminescent Acholeplasma lazdlawu, however this was unsuccessful and further work will be required to achieve this objective.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 199-213). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 199-213). Also available on the Internet.

xiv, 82 p., ill. (some col.) / Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS). GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide. Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design. This dissertation includes my unpublished co-authored materials. / Adviser: S. James Remington

Increased understanding of the molecular mechanisms underlying cancer development has shifted drug discovery towards target driven drug development the last decades, but the development of effective cancer drugs has been hampered by the lack of predictive preclinical models. 3-D cultures, considered to more accurately reflect solid tumors in vivo, have been proposed as one way to increase the predictability of clinical efficacy in cancer drug discovery and development. The aims of this thesis were to improve preclinical models for cancer drug development, with focus on colorectal cancer (CRC) and use of multicellular tumor spheroids (MCTS), and also to mechanistically characterize some potentially new anticancer drugs (papers I – IV). The most important technical improvement was the development of direct measurement of green fluorescent protein (GFP) marked cells in spheroids, simplifying live collection of viability data and enabling high-throughput screening (HTS) in the MCTS model (paper I). In paper III and IV, the 3-D model was adapted to enable studies on the interaction between drugs and radiation. Two potentially new anticancer drugs, VLX50 and VLX60, were mechanistically characterized. VLX60, a novel copper containing thiosemicarbazone, induced reactive oxygen species (ROS) formation, was selectively active against BRAF mutated colon cancer cells and exhibited anticancer activity in vivo (paper II). Furthermore, two potentially new anticancer drugs were found suitable for further development for use in combination with radiation (papers III and IV). In paper III, synergy with radiation in spheroids compared to monolayer cultured colon cancer cells was shown with the novel iron-chelating inhibitor of oxidative phosphorylation, VLX600. In paper IV, the antiprotozoal drug nitazoxanide was shown to sensitize quiescent clonogenic colon cancer cells to radiation. In conclusion, introduction of measurement of fluorescence of GFP marked cells in spheroids makes clinically relevant 3-D models feasible for HTS experiments and characterization of candidate drugs and radiosensitizers in early cancer drug discovery and development. VLX60 has several characteristics suitable for further development into a cancer drug, notably against BRAF mutated colorectal cancer cells. VLX600 and nitazoxanide show radiosensitizing properties making them promising for further development for use as cancer drugs in combination with radiation.

Monomeric red fluorescent proteins (RFPs) are used extensively for applications in molecular biology research, and are especially suited for whole body imaging applications due to their longer excitation and emission wavelengths, which are less damaging and penetrate deeper into animal tissue. However, these proteins suffer from reduced brightness compared to other fluorescent proteins, and require further engineering, which is often achieved through random methods, incurring large time and resource costs. Here we propose a rational design approach to improve the quantum yield of RFPs by reducing conformational variability of the chromophore. We engineered mRojoA, a mutant containing a π-stack involving Tyr197 and the chromophore phenolate, to include the P63F/H/Y mutations on its other side, by simultaneously mutating neighbouring positions 16, 143, and 163. The brightest mutants that we found in each library, mRojo-VYGV, mRojo-VFAV, and mRojo-VHSV, exhibited 1.8- to 2.4-fold increases in brightness, and quantum yield increases of up to 2.1-fold. In all three mutants, the increases in brightness were predominantly due to improvements in the quantum yield and not the extinction coefficient. Solving the crystal structures of two of these mutants along with a dim variant allowed us to strongly infer a link between rigidity of the chromophore and increased quantum yield. In addition, back-mutating position 63 in the highest quantum yield mutant, mRojo-VYGV, reversed the improvement in quantum yield, indicating that Y63 was the primary residue responsible for the improved brightness of the protein. Unfortunately, the mCherry-VYGV mutant did not achieve a similar increase in quantum yield or brightness. This is likely due to the lack of a second bulky aromatic residue at position 197, which is present in mRojoA. Nevertheless, this rational approach could be applied to some other RFPs whose chromophores exhibit increased conformational variability in order to further improve their brightness.

BCG é uma das vacinas mais usadas no mundo. Avanços na manipulação genética têm permitido o seu uso como carreador de antígenos heterólogos, porém o aprimoramento dos sistemas de expressão se faz necessário, sendo o promotor um importante elemento, uma vez que regula o nível de produção do antígeno, induzindo uma resposta imunológica adequada. Avaliamos a atividade de diferentes promotores de micobactérias, como o PAg, PAN, PBlaF*, Phsp60 e um promotor ainda não caracterizado do micobacteriófago L5, usando o gene gfp como repórter da expressão, todos clonados no vetor extracromossomal, pLA71. Foi possível avaliar as cepas de M. smegmatis e BCG fluorescentes para quase todas as construções e alguns plasmídeos pLA71-p mostraram características diferentes dependentes da micobactéria transformada. Numa escala de força de expressão, os diferentes promotores se apresentaram como fraco (pLA71-PAN-gfp), médio (pLA71-PBlaf*-gfp) e forte (pLA71-Phsp60-gfp). Os rBCG foram usados para infecção de macrófagos e a atividade dos promotores não foi afetada após a internalização. Para ensaio de localização, camundongos foram inoculados com BCG e foi possível confirmar a presença de colônias (recombinantes ou não) nos pulmões após 1 e 3 dias de inoculação, por plaqueamento em meio sólido e por microscopia confocal. / BCG is one of the most widely used vaccines in the world. Advances in genetic manipulation have allowed their use as a carrier for heterologous antigens, however the improvement of systems of expression is necessary, the promoter being an important element, since it regulates the expression level of the antigen, inducing an adequate immune response. We evaluated the activity of different promoters of mycobacteria, such as PAg, PAN, PBlaF* and Phsp60, and the not yet characterized promoter of the micobacteriophage L5, using GFP as a reporter gene expression activity, all cloned in the extrachromosomal vector, pLA71. It was possible to evaluate promoters in the M. smegmatis and BCG strains, fluorescent for almost all constructions and some pLA71-p plasmids showed different characteristics dependent on the transformed mycobacterium. The different promoters showed expression levels as weak (pLA71-PAN-gfp), medium (pLA71-PBlaf*-gfp) and strong (pLA71-Phsp60-gfp). The rBCG were used for infection of macrophages and the activity of the promoters wasnt affected after internalization. For BCG location test, mice were inoculated and it was possible to confirm the presence of colonies (recombinant or not) in the lungs after 1 and 3 days after inoculation by plating on solid medium and by confocal microscopy.

The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.