Mycotoxins are toxic secondary metabolites produced by fungi that are a threat to the health of humans and domestic animals. The most important mycotoxin in the U.S. is deoxynivalenol (DON), which causes symptoms such as vomiting, feed refusal, and weight loss in farm animals. The fungus Fusarium graminearum produces DON in staple crops such as wheat, barley, and corn. It is estimated that the economic losses associated with DON contamination alone exceed $650 million per year in the U.S. New strategies are needed to mitigate DON and improve food safety in the U.S. The overall goal of my research is to discover and employ microorganisms and enzymes to detoxify DON. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) use a cell free protein synthesis (CFPS) system to study enzymes that modify DON, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food. In Objective 1, two mixed cultures were identified from environmental samples that converted DON into the less toxic 3-keto-deoxynivalenol (3-keto-DON). In Objective 2, a CFPS system was used to express three known acetyltransferase genes to convert DON to 3-acetyl-DON (3-A-DON). In Objective 3, we identified a potential DON transporter from a library of randomly amplified fragments from the genomes of mixed cultures of microbes isolated from the environment. In Objective 4, we developed and delivered a unique high school unit to educate high school students about potential mycotoxins in food and feed products. The work presented here represents new and improved methods for mitigating mycotoxin contamination in the United States. / Ph. D.

Biosensors are devices that use biological components to detect important analytes. Biosensing systems have various applications in areas such as medicine, environmental monitoring, and process control. Classical biosensors are often based on bacteria or purified enzymes that have limitations on efficiency or stability. I have developed several new biosensors to overcome these disadvantages. Two preliminary biosensors were first created based on the extremely strong and specific interaction between biotin and (strept)avidin. Both biosensors showed high sensitivity and reliability for measuring biotin with detection limits of 50-1000 pg/ml and 20-100 ng/ml, respectively. Following these, a new biosensor was developed by coupling a mobile, functionalized microsurface with cell-free expression approaches. This biosensor demonstrated a dynamic range of 1- 100 ng/ml. In addition, I also explored the possibility of combining these biosensing systems with engineered living cells. By leveraging the tools of synthetic biology, a genetic circuit was designed, constructed, and inserted into bacteria for enhanced biotin biosynthesis in vivo. Upon induction, a 17-fold increase in biotin production was measured in the engineered cells in comparison to wild type cells using the biosensors created herein. These new biosensors, particularly the mobile biosensing modality, form a building block for advanced biosensing and drug delivery systems due to enhancements in mobility and specificity. In the future, these biosensing and cellular production systems could impact a range of fields ranging from biomedicine to environmental monitoring. / Master of Science

Tous les organismes vivants convertissent leurs informations génétiques en protéines fonctionnelles par des mécanismes de transcription/traduction. Ces phénomènes fondamentaux de la biologie sont maintenant maitrisés au niveau biochimique, et il est possible par le biais de systèmes dits " cell free " de produire des protéines in vitro à partir de fragments d'ADN synthétiques. Ces systèmes minimaux d'expression génique représentent autant un outil fondamental pour la compréhension des mécanismes du vivant qu'une plateforme pour de nombreuses applications biotechnologiques. Néanmoins, le contrôle (déclenchement, régulation) de ces systèmes reste limité en comparaison de leurs analogues naturels. Dans cette thèse, j'explore tout d'abord des moyens de régulation de l'expression en système cell-free par voie métabolique, en étudiant l'influence de différents facteurs biochimiques ou énergétiques. Je m'intéresse ensuite à la mise en place d'un contrôle réversible en exploitant un couplage antibiotique/enzyme. Je montre également que l'inhibition produite par cet antibiotique peut être exploitée pour étudier les cinétiques de maturation des protéines fluorescentes. Enfin, je décris un protocole innovant de préparation de protéoliposomes géants fonctionnels permettant à la fois l'expression in situ de protéines membranaires eucaryotes et leur modification post-traductionnelle. Cette approche est appliquée en particulier pour la reconstitution de pores membranaires déclenchables par voie thermique ou enzymatique. / Throughout Transcription and Translation, every single living organism converts the genetic information it carries into functional proteins. These fundamental mechanisms are nowadays mastered up to their biochemical level, enabling us to produce in vitro proteins from synthetic DNA fragments, by the use of so called Cell-free systems. This minimalistic approach of gene expression represents both a tool to unravel biological mechanisms and a platform to develop new biotechnology applications. In spite of these credits, controlling these systems (triggering, regulation) is still scarcely achieved. The aim of the thesis is first of all to explore new means of metabolic regulation for gene expression in cell-free systems, by studying the influence on expression levels of biochemical and energetic factors. I then focus the research to establish a reversible control comprising the coupling of an enzyme and an antibiotic. Moreover, I demonstrate that the inhibition caused by this antibiotic can be used to study the maturation kinetics of fluorescent proteins. Finally, I describe an innovative, one-pot, protocol to prepare functional giant proteoliposomes, enabling simultaneously in situ expression of the eukaryotic membrane proteins and their post-translational modifications. This approach is then applied to reconstitute membranous pores triggered by thermal changes or specific enzymatic activity.

Therapeutic proteins have become a cornerstone of modern medicine since the FDA approval of recombinant human insulin in 1982. Likewise, biosensors transform chemical detection and disease diagnostics by identifying biomarkers, chemical contaminants, and infective agents. Long-standing methods for creating therapeutics and biosensors employ whole cells such as Escherichia coli (E. coli). Alternatively, cell-free protein synthesis (CFPS) employs the enzymatic reactions necessary for protein production and biosensing within a cell, but in an engineered reactor environment facilitating unprecedented access to and control over biochemical machinery, preservation by cryodesiccation for portable deployment, and functionality in cytotoxic applications. This dissertation reports advances in an E. coli CFPS production platform toward creating therapeutic proteins by this means. First, an endotoxin-free CFPS platform is created by optimizing fermentation and cell-extract harvest of an endotoxin-free E. coli strain. Next, liquid cell growth culture media is specially formulated to change chemical composition during cell culture and provide a streamlined method for producing high-yielding, endotoxin-free E. coli CFPS. Then, novel CFPS bioreactor formats are mathematically validated and developed which employ "hydrofoam" and oxygen to increase therapeutic protein production yield. Additionally, advances are reported in CFPS biosensing technology. First, a chimeric fusion protein incorporating the ligand binding domain of the human estrogen receptor is expressed in CFPS to detect estrogenic chemicals in the presence of human blood and urine. Next, the molecular mechanism of this protein construct is elucidated and the assay readout is optimized with mathematical simulations and CFPS. Then, CFPS is metabolically engineered to create a biosensor of L-glutamine, the most abundant amino acid in the body. Finally, this dissertation reports the development of a synergistic platform for potentially treating Acute Lymphoblastic Leukemia wherein CFPS is engineered to both produce the therapeutic protein crisantaspase and assess its activity in the presence of human serum for improved, potentially even personalized treatment of the disease. It is anticipated that the advances reported herein will contribute to the utility of in vitro or cell-free protein synthesis for therapeutic and diagnostic applications.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23772号 / 医博第4818号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 松田 文彦, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In recent years, a number of nuclear transcription factors have been shown to be present in the mitochondria where they have distinct roles in regulating mitochondrial function. Signal Transducer and Activator of Transcription 3 (STAT3), classically activated by the JAK family of receptor associated tyrosine kinases to drive nuclear gene expression, is one such transcription factor with a unique mitochondrial role. There, it has been shown to support oxidative phosphorylation, regulate mitochondrial-encoded transcripts, and be key for the transformation and growth of a number of different cancers. Despite its well-characterized functional importance at the level of the mitochondria, the mechanism through which mitochondrial STAT3 acts and how it is regulated has not been as well studied. Using various cell culture models, we now show that mitochondrial STAT3 is dynamically regulated by oxidative stress and cytokine treatment in the acute setting. Under these conditions we have observed a rapid loss of mitochondrial STAT3 that recovers to baseline conditions with time. During this recovery phase we have noted that mitochondrial STAT3 becomes competent to bind to Cyclophilin D (CypD), the key regulator and activator of the mitochondrial permeability transition pore (MPTP). This is particularly the case with oxidative insults, which we believe may represent an important homeostatic mechanism for the cell. Intriguingly, chronic stimulation with certain stressors seems to increase mitochondrial STAT3 levels suggesting differential regulation in the acute versus chronic setting. The regulation of mitochondrial STAT3 levels by various stimuli points to a novel signaling pathway potentially linking mitochondrial responses with those of the cell. Unification of responses throughout the cell would seem to serve a clear adaptive advantage, particularly in coupling nuclear regulation with metabolic demands as dictated by the mitochondria. Extramitochondrial signaling, also known as the mitochondrial retrograde response, has emerged as an important homeostatic mechanism in lower organisms, but its signaling components have not been well characterized at the mammalian level. Our results point to a role for mitochondrial STAT3 in sensing cellular inputs, whereby its regulation and subsequent association with CypD may have implications in overall mitochondrial quality control. Though the inner workings of this signaling cascade are just beginning to be elucidated, they suggest the existence of a previously unappreciated pathway at the mitochondrial level.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

歯科基礎医学会, 金子 知生 = Tomoo Kaneko, 歯に矯正力を加えた際の圧迫側歯周組織の三次元的様相について = Three-dimensional situation of periodontal tissue at pressure side incident to orthodontic tooth movement, 歯科基礎医学会雑誌, 36(2), APR 1994, pp.170-186 / Hokkaido University (北海道大学) / 博士 / 歯学

In this study, Azotobacter vinelandii ATCC13705 (A. vinelandii), which is a free-living, nitrogen-fixing, gram-negative, and aerobic rod bacterium, was need to evaluate its ability to biodegrade tetracyanonickelate (TCN) under different conditions. Results show that A. vinelandii was able to biodegrade various concentrations of TCN (1, 10, and 20 mM) under aerobic conditions. Oxygen consumption and nitrogenase activity were investigated at 1 mM of TCN. Results indicate that the production of ammonia and methane was observed when TCN was consumed. Results suggest that nitrogenase was possibly involved in the enzymatic degradation of TCN. Moreover, higher degradation rate of TCN, higher nitrogenase activity, higher oxygen consumption, and higher specific growth rates were also observed at log growth period. Results suggest that the hypothesis of respiratory protection of nitrogenase is supported. Moreover, the addition of ammonia (1, 5, and 10 mM) would cause the decrease of TCN degradation rate (28%) during a 24-hr incubation period. Inhibition of TCN degradation (degradation rate¡G16% for 24 hrs) was observed when nitrite (5 and 10 mM) was added into the growth medium. Furthermore, the addition of 8% of glucose would significantly enhance the TCN degradation by the resting cells (degradation rate¡G43% for 8 hrs) . Results from this study provide us insight into the characteristics and mechanisms of TCN conversion by A. vinelandii.

Simulating blood flow in microvessels is a major challenge because of the numerous blood cells suspended in the blood. Furthermore, red blood cells (RBCs), which constitute 45% of the total blood volume, are highly deformable. RBCs deformation and RBC-RBC interactions determine the complex rheology of the blood. In this research, we simulate the blood flow in periodic two dimensional channels and conduct numerical experiments on the cell-free layer which appears near the wall. We use the boundary integral method and the smooth particle mesh Ewald method to represent the blood flow, and cells are modeled as deformable capsules. In the numerical experiments, we examine four possible mechanisms that may contribute to the cell-free layer: RBC deformation, RBC aggregation, configuration constraint, and the lubrication mechanism. Our simulations correctly represent hemodynamic phenomena such as the blunt velocity profile and the Fåhræus effect. We observed that more deformable RBCs migrate more away from the wall, and, consequently, the thickness of the cell-free layer increases. However, RBC aggregation increased the cell-free layer thickness by only 5%. In the experiment on the configuration constraint, no cell-free "layer" was detected when we removed cells which intersected an artificial constraint in the microvessel. In the last experiment on the lubrication mechanism, the cell-free layer disappeared at a no-shear stress boundary, and the hematocrit profile was similar to that in the constraint test. Therefore, this research clearly shows that the cell-free layer is generated by the lateral migration of deformable RBCs due to the lubrication mechanism. / text