Mitosis, or cell division, is the mechanism by which cells divide and is an intricate process requiring the action and control of numerous proteins. Such proteins serve either as structural entities within the mitotic spindle, or perform the "work" within the apparatus. In particular, Kinesin-5 motor proteins, a subset within the kinesin motor protein superfamily, are primarily responsible for organization of microtubules (MTs) within the mitotic apparatus, and are consequently vital for efficient mitosis. These proteins utilize energy from ATP hydrolysis in order to "walk" along antiparallel MTs, positioning them into the bipolar mitotic spindle. Loss of Kinesin-5 activity results in formation of a monoastral spindle and subsequent cell cycle arrest. Recently, a wide variety of small molecules have been identified that possess the ability to inhibit certain Kinesin-5 motors. Such compounds, including monastrol (the first Kinesin-5 inhibitor identified), have been employed to study Kinesin-5 activity. A thorough understanding of Kinesin-5 function, combined with the ability to specifically target these proteins with small molecules, may provide the capability to control cell division and may therefore have significant implications in anti-cancer therapies. The following dissertation describes research that utilizes small molecules to probe the function (ATPase activity and MT interactions) of various Kinesin-5 proteins and provides information that will lead to a better understanding of exactly how such proteins function in vivo. Further, a greater knowledge of Kinesin-5 protein activity as well as specific interactions with small-molecule compounds, may lead to the development of more potent, less toxic anti-cancer drugs. / Ph. D.

It is generally acknowledged that a variety of problems affecting aesthetics, health, and corrosivity of potable water can arise during installation of building plumbing systems. These include 'blue water', microbial infestation, and rapid loss of disinfectant residual, among other things. Frequently cited causes of the problems include metallic fines left in the plumbing lines from deburring, cutting and product fabrication; solder flux residuals (water soluble and petroleum based flux); and solvents for CPVC. Mechanistically, some materials such as flux contain high chloride, high ammonia and cause low pH, which can increase the corrosivity of water held in the lines. Indirect effects are also suspected to be important. For example, ammonia from flux and organic carbon from flux or PVC solvents can spur microbial growth, which in turn can reduce pH or otherwise increase corrosivity. Recent work has also demonstrated that problems with lead leaching to water from brass in modern plumbing can actually be worse in PVC/plastic than in copper systems, if certain types of microbes such as nitrifiers proliferate and drop pH. Some of the problems initiated by construction practices can persist indefinitely, causing higher levels of lead and copper in water, or longer term, contributing to failures of the plumbing system. Blue water from high copper concentrations is a confounding problem that continues to arise in some locales of the United States. One public elementary school in Miami Dade County is experiencing blue water issues as manifested by blue ice cubes and sink staining. In addition to the aesthetic problems, copper levels are above the EPA's Copper Action Level of 1.3 ppm. Bottled water has been substituted for tap water consumption, which has created a financial burden. The pH of the school's water ranges from 7.15 - 7.5 and the school itself is located 1 ½ miles off the main distribution line resulting in a very low chlorine residual of between 0.06 mg/L Cl2 and 0.18 mg/L Cl2. On site water was shipped to Virginia Tech from Miami to be used in this study. Preliminary testing showed that an increase in the pH of the water would decrease copper leaching. Several pH's were tested which revealed that increasing the pH of the water to 8.5 would drop copper below 1.3 mg/L. When these recommendations were implemented at the school, the high alkalinity and calcium rich water caused calcite scales to form which clogged the chemical feed nozzles. Further bench scale testing indicated that adding 2 mg/L orthophosphate corrosion inhibitor would effectively decrease copper to a level that would comply with the EPA's Copper Action Limit. Orthophosphate corrosion inhibitors are used by utilities to limit lead and copper corrosion from consumer's plumbing. An evaluation comparing the effects of both 100% orthophosphate inhibitor and orthophosphate/polyphosphate inhibitor blends was performed to study the effects they have on galvanic corrosion, metallic corrosion, microbial growth and the decay of chloramine disinfectant. On site water was sent to Virginia Tech from UNC for use in this bench scale study. The results from this study indicated that 100% orthophosphate inhibitor was the most effective corrosion inhibitor at decreasing metallic corrosion. It has long been known that microbial activity can have significant effects on water quality. This study evaluated nitrifying and heterotrophic bacterial growth in water systems containing copper pipes, a common plumbing product, and flux which is used in soldering copper pipes together in new construction. There are several types of commercially available fluxes which are often used when soldering new pipes together. Flux ingredients vary and can include extremely high concentrations of ammonia, zinc, chloride, tin, copper and TOC. Flux containing high amounts of ammonia can be detrimental to water quality because it can accelerate the occurrence of nitrification, thus creating a cascading set of problems including, but not limited to, pH decrease and copper corrosion. The results from this case study indicated that flushing a pipe system can effectively decrease the high concentrations of flux present in a new construction system; however, high levels of ammonia from flux can create an environment in which nitrifiers may proliferate within the system. Many water utilities in the United States are switching disinfection type from chlorine to chloramine due to the increased stability, longer residual time, and overall safety benefits of chloramine. Although chloramines have been found to be a desirable means for disinfection, chloramine decay is an issue of great concern because if the chloramine residual decays, it can leave a water system unprotected against microbial infestation. A preliminary examination of this issue was performed in a laboratory setting to evaluate the many components that effect the stability of chloramine decay, including alkalinity, phosphate, temperature, and various pipe materials. The results from this experiment revealed that temperature increase, pH increase, and aged tygon tubing all accelerated the rate of chloramine decay. / Master of Science

Prohexadione-calcium (P-ca), as ApogeeTM, was evaluated in 2000 and 2001 for impact to grape vegetative and reproductive development. In 2000, P-ca (250 mg/L) was applied to Seyval, Cabernet Sauvignon, and Cabernet franc (125, 250, and 375 mg/L). P-ca reduced primary shoot growth for all cultivars and decreased cane pruning weight of Seyval. P-ca (375 mg/L) increased Cabernet franc canopy gaps but increased Cabernet Sauvignon lateral leaf area and leaf layer number. P-ca reduced components of yield for all cultivars. In 2001, P-ca (250 mg/L) was applied singularly at weekly intervals to Cabernet Sauvignon clusters and pre and post-bloom to Cabernet franc and Chardonnay canopies. Application at E-L stages 21 and 23 decreased Cabernet Sauvignon fruit set whereas application at E-L stages 26, 27, and 29 reduced berry weight without impacting fruit set. Berry weight reduction correlated to higher color intensity (420+520 nm), anthocyanins, total phenols and phenol-free glycosyl-glucose (PFGG). Cabernet franc vegetative and reproductive development was generally not affected yet treatment increased absorbance at 280, 420, and 520 nm, color intensity, anthocyanins and total phenols. Pre-bloom applications inhibited Chardonnay vegetative development, and reduced components of yield, and fruit chemistry values: hydroxycinnamates, total phenols, flavonoids, PPFG and absorbance at 280 and 320 nm. Post-bloom applications did not affect Chardonnay vegetative or reproductive development, yet increased PFGG. Treatment did not affect Chardonnay wine chemistry but two post-bloom applications increased Cabernet franc wine anthocyanins and total phenols. Wine aroma and flavor triangle difference tests did not indicate significant treatment differences. / Master of Science

Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h). / Doctor of Philosophy / Sphingosine-1-phosphate (S1P) is a molecule related to various diseases, such as cancers and inflammatory diseases. Elevated levels of S1P promote the development of these diseases, thus making it necessary to reduce the production of S1P in patients. Since S1P is generated in human body by an enzyme called sphingosine kinase (SphK), inhibiting the activity of SphK can be beneficial for reducing S1P levels. Developing inhibitors for SphK is also a promising strategy for curing such diseases. A very potent inhibitor has been reported, but it could be metabolized quickly into other inactive metabolites in human, which renders it ineffective. To develop better drug candidates, a series of compounds with similar structure has been synthesized and tested for their potency and metabolic stability. Based on analysis of the relationship between the compound structures and activities, several compounds with less potency and different metabolic stability has been prepared and their efficacy in reducing S1P levels has been tested.

Osteoarthritis (OA) is a chronic joint disease characterized by irreversible cartilage degradation. MMP (matrix metalloproteinase) inhibitors represent a new approach to slowing OA progression by addressing cartilage degradation mechanisms. However, the success of preclinical studies failed to be translated into clinical application. One of the possible reasons is that the disease models in preclinical study can't reflect the biological complexity of human disease. Hydrogel-based cartilage constructs as in vitro models have shown promise as preclinical testing platforms due to their enhanced physiological relevance, improved prediction to human response, high-throughput drug screening, and ease of use. Metalloproteinase-13 (MMP-13) is thought to be a major contributor to the degradation of articular cartilage in OA by aggressively breaking down type II collagen. This study focused on testing MMP-13 inhibitors using a GelMA-alginate hydrogel-based OA model induced by cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). The results demonstrated a significant inhibition of type II collagen breakdown by measuring C2C concentration using ELISA after treatment with MMP-13 inhibitors. Therefore, the study highlights the GelMA-alginate hydrogel-based OA model as an alternative to human-sourced cartilage explants for in vitro drug screening, which can improve the predictability and relevance of preclinical evaluations of MMP-13 inhibitors for osteoarthritis, thereby complementing existing 2D culture, cartilage explant, and animal model studies and addressing the translational gap observed in clinical trials.

Yes / Aberrant transforming growth factor-β (TGF-β) signaling activation is linked to pulmonary arterial hypertension (PAH). BMPR2 mutations perturb the balance between bone morphogenetic protein (BMP) and TGF-β pathways, leading to vascular remodeling, narrowing of the lumen of pulmonary vasculature, and clinical symptoms. This forum highlights the association of the TGF-β pathway with pathogenesis and therapeutic approaches. / Research carried out at Nasim laboratories is funded by GrowMedtech, the Royal Society, the Commonwealth Scholarship Commission (CSC) and the University of Bradford (UoB). N.S. is funded by the CSC and C.C.N. is partly funded by the UoB.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25335号 / 農博第2601号 / 新制||農||1106(附属図書館) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 白井 理, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Doctor of Philosophy / Department of Agronomy / Tesfaye T. Tesso / The lack of effective post-emergence weed control options is often highlighted as one of the major factors behind dwindling acreage under sorghum (Sorghum bicolor (L.) Moench) in the United States. The discovery of herbicide resistance sources in wild sorghum population and subsequent efforts to incorporate them into cultivated sorghum was received with much optimism to change weed management practices in sorghum. As the development of the technology advances, especially of the Acetolactate synthase (ALS) resistance, concerns over the temporary interveinal chlorosis and reduced seedling vigor in some of the resistant families became heightened. This thesis research is designed to shed light on the genetic basis of seedling chlorosis and assess its impacts on yield potential. The study has three parts; the first part is focused on identifying the genetic causes and plant mechanisms associated with the chlorotic phenotype. ALS herbicide resistant sister-lines expressing normal and chlorotic phenotypes were analyzed via RNA sequencing at four time points during seedling growth. The study identified several variants of genes coding chloroplast precursors and those that cause epigenetic modifications. Once confirmed, genetic markers can be developed to track these gene variants in the breeding population and eliminate segregates genetically prone to chlorosis/yellowing. The second part of the study focuses on assessing the effect of ALS resistance associated chlorosis on agronomic and nutritional parameters of sorghum inbred lines. A set of ALS resistant lines expressing different levels of the chlorotic phenotype were evaluated in replicated field trials and laboratory methods. Results showed that interveinal chlorosis delays flowering but does not have negative effect on yield and nutritional parameters with and without herbicide treatment. The last part addresses whether there is any yield drag that may be associated with herbicide resistance traits and foliar interveinal chlorosis. For this, we synthesized a large set (182) of hybrids from ALS resistant, ACCase resistant and regular (susceptible) seed and pollinator parents. The hybrids were then evaluated in three sets at multiple locations during the 2014 and 2015 crop seasons along with commercial checks. The results revealed that resistance to both herbicides do not cause any drag to grain yield. The traits also do not have any negative impact on grain and nutritional quality of resistant hybrids.

The objective of this Ph.D. research was to identify new and novel mechanisms of wild radish (Raphanus raphanistrum L.) resistance to photosystem II (PSII) inhibitors, auxinics, and acetohydroxyacid synthase (AHAS) inhibitors. PSIIinhibitor resistance was demonstrated to be target-site based, and conferred by a Ser264 to Gly substitution of the D1 protein. Auxinic resistance was associated with reduced herbicide translocation to the meristematic regions of resistant wild radish plants. Two new resistance mutations of wild radish AHAS were discovered, including one encoding the globally rare Asp376 to Glu substitution, and another encoding an Ala122 to Tyr substitution, which has never been identified or assessed for resistance in plants previously. Characterization of the frequency and distribution of AHAS resistance mutations in wild radish from the WA wheatbelt revealed that Glu376 was widespread, and that some mutations of AHAS are more common than others. Computer simulation was used to examine the molecular basis of resistance-endowing AHAS target-site mutations. Furthermore, through the computer-aided analysis, residues were identified with the potential to confer resistance upon substitution, but which have not previously been assessed for this possibility. Results from this Ph.D. research demonstrate that diverse, unrelated mechanisms of resistance to PSII inhibitors, auxinics, and AHAS inhibitors have evolved in wild radish of the WA wheatbelt, and that these mechanisms have accumulated in some populations.

<p>Bikunin, a chondroitin sulfate-containing protein of 25 kDa, has protease inhibitory activity and occurs in the plasma in free and complexed form. In inter-α-inhibitor (IαI) and pre-a-inhibitor (PαI) it is covalently linked through its chondroitin sulfate (CS) chain to two or one other polypeptide of about 80 kDa – heavy chains 1 and 2 (H1, H2) and heavy chain 3 (H3) – respectively. Bikunin and the heavy chains are synthesized as precursors, which are proteolytically cleaved and assembled into IαI and PαI in the secretory pathway. The C-terminal extension (CTX) of the heavy chains seems to mediate its own cleavage and theassembly of the complexes. The heavy chains of the IαI family become transferred to hyaluronan during ovulation and inflammation.</p><p>In this thesis, the biosynthesis of PαI, the plasma clearance of bikunin and the binding of IαI to collagen were studied. We found that in H3, a short segment on the N-terminal side of the CTX cleavage site is required for cleavage. Furthermore, the H3 could become linked to free CS chains primed by a xyloside, showing that the bikunin protein core is not needed for coupling. We also identified His649 as a residue essential for coupling, but not for cleavage. </p><p>Bikunin labelled with a residualizing agent, 125I-tyramine cellobiose, was injected into mice to identify tissues involved in its uptake. Half of the radioactivity was recovered in the kidneys, 10% in the liver, and the rest distributed in other tissues. We determined the half-life of bikunin in rat plasma using two independent methods: injection of 125I-bikunin, or hepatectomy followed by assessing the rate of disappearance of endogenous bikunin. Both methods yielded half-time values of 5-7 minutes. Removal of the CS chain did not affect the clearance rate of bikunin.</p><p>IαI and its heavy chains were found to bind to collagen with dissociation constants greater than 2 μM and 0.4-0.6 μM, respectively and this binding was independent of divalent metal ions. We suggest that the interaction of IαI with collagen may play a modulatory role in cell migration or in remodelling of the extracellular matrix.</p>