The enhancing effect of K on alfalfa (Medicago sativa L.) yield has frequently been reported, but the nature of this influence has not been clearly shown. It was assumed that K contributed either to increased leaf expansion, thereby resulting in a larger photosynthetic surface, or to higher CO₂ assimilation rates per unit leaf area. Sand culture technique for growing plants was used in two greenhouse experiments, while field plants were grown in plots with soil differing in available K. Yield increases were consistently obtained with high K. Added K increased plant height and leaves per plant and per plot, The rate of leaf accumulation was higher as K increased. Leaf size and weight per unit area also increased, as did stomatal number and aperture. Larger epidermal cells and greater numbers per leaf were observed with high K nutrition. Percent K in plants was associated with rate of K application. Net photosynthesis rates of excised leaves increased with potassium application, but all K levels had similar light compensation points. Leaves from plants with added K had lower CO₂ compensation points, indicating higher efficiency of CO₂ assimilation. Based on these data, K appears to function both to increase the effective photosynthetic surface through greater leaf initiation and development and to increase the rate of CO₂ utilization per unit leaf area. The latter increase probably results from greater CO₂ diffusion into substomatal cavities. / Ph. D.

In an effort to provide wastewater treatment operators with the capability to be proactive in assessing and solving deflocculation events, this study has tested the components of a biosensor to predict deflocculation and investigated the mechanistic cause of deflocculation relating to chlorination of activated sludge cultures. In order to effectively manage upset events, it is necessary to know the source of an upset and the causative mechanism that the source initiates. The Glutathione-gated potassium efflux (GGKE)induced activated sludge deflocculation biosensor incorporates novel microtechnology with a whole cell biological element to predict deflocculation from electrophilic sources. This sensor utilizes microfluidic channels to conduct influent wastewater across a biofilm of Eschericia coli K 12 and monitors the bacterial response to the influent. The bacterial response, which is efflux of K+ ion from the cytoplasm, is monitored with a fluorescence-based sensor called an optode. The components of the system satisfy the project requirements, which include minimal expense (both operation and manufacture), on-line capability and minimal maintenance. The research conducted to date demonstrates the ability of the components of the biosensor to fulfill the design requirements. The optode K+ detector successfully measured an increase in soluble K+ following the exposure of E. coli K-12 to the electrophile N ethyl malemide. The manufacture of the microfluidic device has been completed and the device has demonstrated the ability to conduct influent under negative pressure across an established biofilm with the optode in place. The establishment of a biofilm under expected hydrodynamic conditions has also been completed. Future research efforts will include integrating the components of the biosensor into a working prototype that will be capable monitoring the reaction of bacteria to the presence of electrophilic compounds in wastewater. Sensors of this nature will provide operators with the early warning necessary to be proactive against toxic upsets rather than reactive. The knowledge needed to create a biosensor resides in the identification of bacterial response mechanisms that cause upset events in wastewater treatment facilities. The biosensor that has been developed relies on the discovery of the link between electrophile-induced GGKE and activated sludge deflocculation. Research has been concluded, which expands the role of GGKE and activated sludge deflocculation to include chlorine-induced GGKE. Through a series of laboratory-scale reactors, a relationship has been established between chlorine addition to control filamentous bulking, increased soluble K+ levels and an increase in effluent suspended solids . The results demonstrate that the addition of chlorine to control filamentous bulking may elicit the GGKE mechanism, initiating activated sludge deflocculation, similar to observations of chlorination at full-scale activated sludge wastewater treatment facilities. Establishing a mechanistic cause of deflocculation related to chlorination will permit operators to apply chlorine in a manner that may avoid deflocculation, rather than reacting to deflocculation after it has occurred. / Master of Science

Corn grain yields can be limited by nitrogen (N) and potassium (K) availability on sandy coastal plain soils when soil moisture is adequate for high yields. This study evaluated irrigated corn grain yield response to late-season (just prior to tassel) N and K fertilizer applications, and enabled the proposal of a method to predict potential for corn to utilize late-season N applications based on soil moisture. In an experiment to evaluate late-season fertilizer application rates, N and K were applied in a complete factorial of five N and K rates ranging from 0 to 112 kg ha-1. Additionally, water use of high yielding corn was measured, and historical weather patterns evaluated in an effort to predict the need for late-season fertilizer applications based on soil moisture. Grain yield was increased significantly by late-season N applications in three of four experiments. Potassium applications did not affect yield, and there were no interactions between N and K. Significant drainage due to high rainfall levels in 2000 prohibited further refinement of corn water use data for Virginia climatic conditions. Historical weather patterns, potential evapotranspiration of corn, and soil water holding properties were evaluated. In order to provide corn with adequate moisture during a two-week moisture-sensitive critical period beginning at tassel, soils must be near field capacity at the start of the period and receive above-average (75th percentile) rainfall during the period. / Master of Science

Les cotransporteurs K+-C1" (KCCs) sont exprimés en abondance dans le système nerveux où leur rôle est de maintenir les neurones hypoexcitables. Ds sont représentés par quatre isoformes dont les mécanismes de régulation et propriétés cinétiques diffèrent de façon appréciable. Nous avons récemment démontré que chacun des KCCs peut s'organiser en homooligomères ou hétérooligomères avec d'autres KCCs sans toutefois vérifier si certaines des associations observées menaient à des entités fonctionnelles et si oui, quel comportement elles engendraient. Dans ce travail, nous avons utilisé les ovocytes de xénopes pour caractériser les propriétés du cotransport K+-C1" quand un seul isoforme (KCC2 ou 4) est exprimé ou quand deux isoformes (KCC2 et 4) le sont et ce, après avoir prouvé que ces transporteurs pouvaient exister dans la même cellule neuronale de façon endogène. Nos résultats ont montré que les constantes cinétiques du transport différaient substantiellement entre les ovocytes exprimant KCC2/KCC4 et KCC2 seul, alors qu'elles différaient aussi entre les ovocytes exprimant KCC2/KCC4 et KCC4 seul mais de façon beaucoup moindre. Fait à noter, ces constantes étaient différentes de celles prédites sur la base d'une activité additive provenant de deux transporteurs indépendants et certaines de ces constantes, le coefficient de Hill entre autres, se sont révélées sensibles au Cl" intracellulaire (Cl'i). Ces résultats suggèrent que l'association KCC2-KCC4 dans les neurones mène à la formation d'une entité fonctionnelle avec des caractéristiques uniques et pourraient jouer un rôle intrinsèque dans le contrôle de l'excitabilité. Ils suggèrent aussi que les KCCs réagissent au Cl"; en changeant leur cinétique et que contrairement à la pensée populaire, ils fonctionnent avec deux sites de liaison pour le Cf.

Yes / Electrophysiological properties and molecular background of the zebrafish (Danio rerio) cardiac inward rectifier current (IK1) were examined. Ventricular myocytes of zebrafish have a robust (−6.7±1.2 pA pF−1 at −120 mV) strongly rectifying and Ba2+-sensitive (IC50=3.8 μM) IK1. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9±1.5 and 6.3±1.5 %) and the atrium (28.9±2.9 and 64.7±3.0 %). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba2+ block, drKir2.4 was the most sensitive (IC50=1.8 μM) and drKir2.1a the least sensitive channel (IC50=132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba2+- sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances.

Slo2, a large conductance potassium channel in the nervous system is important for regulating neuronal function and excitability. Mutations in the gene that encodes the Slo2 channel are associated with neurological disorders, including epilepsy and intellectual disabilities in humans. However, much remains unknown about the genes and proteins that regulate Slo2 channel activity in the physiological system. This study investigates regulation of SLO-2, a homologue of mammalian Slo2 in C. elegans, by thoc-7 in an RNA editing-dependent pathway. Prior research has shown that adr-1, the gene important for RNA editing, promotes SLO-2 function by RNA editing of scyl-1 that encodes a regulator of SLO-2. To gain a better understanding of the regulation of SLO-2, this study employed a forward genetic approach to screen for mutants with a specific phenotype. Through SNP mapping and whole genome sequencing, we identified the gene thoc-7, which is predicted to be involved in mRNA export from nucleus, from the isolated mutants. The identification was further confirmed by CRISPR/Cas9-mediated gene knock-out, which showed a similar phenotype to the mutant strain. Results of electrophysiological recordings suggest that thoc-7 likely contributes to SLO-2 function in a common pathway with scyl-1. A reporter gene revealed strong expression of thoc-7 in most of the cells of C. elegans, particularly muscular and digestive system. Translational fusion with GFP showed the primary localization of the THOC-7 protein in cytoplasm, with some weak expression in the nucleus. RT-qPCR analysis suggests that thoc-7 regulates scyl-1 by through a post-transcriptional mechanism, possibly involving the transport of mRNA from cytoplasm to nucleus. This study highlights thoc-7 as a potential key regulator recruited by adr-1 to control SLO-2 via scyl-1 expression.

Master of Science / Department of Agronomy / Michel D. Ransom / Factors governing potassium fixation and release are poorly understood. This study was conducted to investigate the effects of clay mineralogy and structural iron oxidation state on potassium fixation. Five reference clays and two soil clays were used to capture a range in mineralogical compositions and potassium behaviors. Reference clays used were illite (IMt-1), kaolinite (KGa-1b), montmorillonite (STx-1b), nontronite (NAu-2), and vermiculite (VTx-1). Soil clays used were from the upper 15 cm of a Belvue loam (BEL) and a Cherokee silt (CHE). Potassium fixation capacities were measured on unaltered as well as sodium dithionite reduced forms of each clay. Ferrous and total iron contents were determined photometrically using 1, 10-phenanthroline. Potassium fixation was measured by potassium saturating the clays and washing off exchangeable and solution potassium with solutions of magnesium chloride; samples were then acid digested and the amount fixed was calculated as the amount of potassium in the acid digestion minus the amount originally in the sample. BEL released potassium rather than fixed it while CHE tended to release potassium in the unaltered form and fix potassium in the reduced form. Structural iron reduction significantly impacted the amounts of potassium fixed by VTx-1 and NAu-2, which had the highest total iron contents of all the clays evaluated. NAu-2 and VTx-1 both on average fixed less than 1 mg K g clay[superscript]-1 in the unaltered form and an average of 6 and 11 mg K g clay[superscript]-1, respectively, in the reduced form. Regardless of being in the unaltered or reduced form, KGa-1b fixed essentially no potassium and IMt-1 and STx-1b fixed intermediate amounts of potassium—2 to 4 mg K g clay[superscript]-1 on average. The effects of clay mineralogy and structural iron oxidation state on potassium fixation can largely be explained through an understanding of layer type, layer charge, and charge distribution. In order for potassium fixation to occur, interlayer sites need to be accessible and available. Generally, the greater the negative layer charge the greater the amounts of fixation, with tetrahedral layer charge favoring fixation more than octahedral layer charge, and layer charge being a function of structural iron oxidation state.

強心苷是一類多年普遍用於心力衰竭治療的化合物,包括蟾蜍靈和地高辛。鈉泵（也可稱為鈉鉀ATP酶）是強心苷的受體。最近流行病學研究，體外實驗，動物實驗和臨床試驗表明，強心苷具有癌症治療的強大潛力。 / 大腸癌是全球第三大殺手，約有一半的大腸癌患者需要手術切除後的輔助治療。因此，通過化療殺死腫瘤細胞，是一個可行的辦法來治療大腸癌患者。在本課題的研究中，強心苷抗人結腸癌的作用在HT-29和Caco-2細胞上進行了評價與闡釋。在結腸癌細胞研究模型中，蟾蜍靈誘導caspase非依賴性的細胞死亡，伴隨沒有早期凋亡，沒有聚（ADP-核糖）聚合酶(PARP)與caspase-3裂解，這些發現與強心苷誘發其它類腫瘤細胞凋亡的機製完全不同。相反，蟾蜍靈激活自噬途徑，促進LC3-II積累和自噬流動。此外，其它強心苷如地高辛與烏本苷也促使LC3-II在HT-29細胞內聚集。沉默ATG5和Beclin-1顯著降低蟾蜍靈誘導的LC3- II積累和細胞死亡。蟾蜍靈誘導的自噬與活性氧（ROS）產生和JNK活化相關。我們的研究結果揭示了蟾蜍靈藥物對抗結腸癌細胞的一種新的機制，開闢了強心苷通過自噬途徑來治療大腸癌的可能性。 / 最近的研究表明，強心苷誘導多種癌細胞系的細胞包括促使凋亡與自噬的細胞週期阻滯在G2／M期。然而，沒有詳細的信息闡述強心苷如何阻滯細胞週期進展。在本課題研究中，我們研究了強心苷介導的細胞週期阻滯的分子機制。蟾蜍靈處理的HeLa H2B-YFP細胞被阻滯在前中期，伴隨姐妹染色單體凝聚，染色體未排列在赤道板，未退出有絲分裂期。這一結果被蟾蜍靈誘導的四倍DNA含量細胞既不在四倍體G1期也不在胞質分裂期進一步證明。此後，我們檢測了紡錘體組裝和染色體分離所需的Aurora激酶和Polo-like kinase 1 (Plk1)。結果發現，在HT-29和HeLa細胞上，蟾蜍靈和其它強心苷能顯著降低總蛋白質和磷酸化的Aurora激酶與Plk1。此外，我們還發現，蟾蜍靈通過PI3K下調有絲分裂酶的活性。這些結果已經通過沉默鈉泵α做了驗證。總之，我們的結果表明, 蟾蜍靈和其它強心苷鈉鉀泵抑製劑強有力的抑制細胞在前中期是通過PI3K/HIF-1α/NF-κB途徑下調Aurora激酶的蛋白質和磷酸化水平和Plk1的蛋白質水平。我們的研究發現在了解如何利用強心苷的潛能治療癌症以及認知鈉泵在細胞週期中的功能方面提供了有用的信息。 / The sodium pump (also known as Na+/K+-ATPase) is the receptor for cardiac glycosides, a group of compounds including bufalin and digoxin which have been commonly used for heart failure treatment for many years. Recent epidemiological studies, in vitro studies, animal studies and clinical trials have shown that cardiac glycosides have potential applications for cancer treatment. / Colorectal cancer is the third leading cause of cancer death worldwide and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treat patients with colorectal cancer. In this study, the effects of cardiac glycosides were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to their well documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis, as well as poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. Moreover, other cardiac glycosides digoxin and ouabain could also induce the accumulation of LC3-II in HT-29 cells. The silencing of ATG5 and Beclin-1 significantly reduced bufalin-induced LC3-II accumulation and cell death. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS) and JNK activation. My findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer by cardiac glycosides through an autophagy pathway. / Recent studies have revealed that cardiac glycosides induce G2/M phase arrest in many cancer cells, which include apoptosis- and autophagy-promoting cells. However, no detailed information is available on how cardiac glycosides arrest cell cycle progression. In this study, I studied the molecular mechanisms of cell cycle arrest mediated by cardiac glycosides. Bufalin-treated HeLa H2B-YFP cells were arrested at prometaphase, as characterized by the presence of sister chromatid cohesion, absence of chromosomes alignment on the metaphase plate, and failure to exit mitosis. This result was further confirmed by bufalin-induced cells with 4N DNA content in neither tetraploid G1 phase nor cytokinesis. Thereafter, I detected the Aurora kinases and Polo-like kinase 1 (Plk1), which are required for both spindle assembly and chromosome segregation. It was found that bufalin and other cardiac glycosides could significantly reduce the total protein and phosphorylation of Aurora kinases and Plk1 in HT-29 and HeLa cells. In addition, I found that PI3K was responsible for the bufalin-induced downregulation of the activities of mitotic kinases. This result was validated by silencing of sodium pump alpha. Taken together, my results demonstrate that bufalin and other cardiac glycoside inhibitors of the sodium pump potently arrest cancer cells at prometaphase by downregulating the total protein and phosphorylation of Aurora kinases and the total protein of Plk1 through the PI3K/HIF-1α/NF-κB pathway. My findings provide useful information in understanding how cardiac glycosides could be exploited for their potentials in treating cancer and in identifying the function of sodium pump in cell cycle progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Chuanming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.iii / Abstract --- p.vi / Abstract (in Chinese) --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- The chemical structure of cardiac glycosides --- p.2 / Chapter 1.3 --- The traditional use of cardiac glycosides in cardiology --- p.4 / Chapter 1.4 --- The role of cardiac glycosides in cancer treatment --- p.4 / Chapter 1.5 --- The mechanisms of action by cardiac glycosides in cancer --- p.5 / Chapter 1.5.1 --- The structure and functions of cardiac glycosides receptor sodium pump --- p.5 / Chapter 1.5.2 --- Sodium pump as anticancer target --- p.6 / Chapter 1.5.3 --- The signal pathways involved in anticancer effect of cardiac glycosides --- p.7 / Chapter 1.6 --- The role of cardiac glycosides in apoptosis and autophagy --- p.8 / Chapter 1.7 --- Objectives of this project --- p.12 / Chapter Chapter 2 --- Bufalin induces autophagy but not apoptosis in human colon cancer cells --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Reagents and antibodies --- p.19 / Chapter 2.2.2 --- Cell culture --- p.19 / Chapter 2.2.3 --- Cell viability and cell death assay --- p.20 / Chapter 2.2.4 --- Annexin V and PI staining --- p.20 / Chapter 2.2.5 --- Cell cycle analysis --- p.21 / Chapter 2.2.6 --- Analysis of cleaved caspase-3-positive cells by flow cytometry --- p.21 / Chapter 2.2.7 --- Western blot analysis --- p.21 / Chapter 2.2.8 --- Immunofluorescence analysis of LC3 distribution --- p.22 / Chapter 2.2.9 --- RNA isolation and RT-PCR --- p.22 / Chapter 2.2.10 --- siRNAs transfection and treatments --- p.23 / Chapter 2.2.11 --- Transmission electron microscopy --- p.23 / Chapter 2.2.12 --- Statistical analysis --- p.24 / Chapter 2.3 --- Results --- p.24 / Chapter 2.3.1 --- Bufalin induces cell death and cell cycle arrest at G2/M phase in colon cancer cells --- p.24 / Chapter 2.3.2 --- Bufalin induces caspase-independent cell death in colon cancer cells --- p.28 / Chapter 2.3.3 --- Bufalin induces autophagy in colon cancer cells --- p.30 / Chapter 2.3.4 --- Bufalin-induced autophagy is dependent on ATG5 and Beclin-1 --- p.37 / Chapter 2.3.5 --- Increased autophagy is responsible for bufalin-induced cell death --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Bufalin mediates autophagic cell death through ROS generation and JNK activation --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Reagents and antibodies --- p.46 / Chapter 3.2.2 --- Cell culture --- p.47 / Chapter 3.2.3 --- Cell viability and cell death assay --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.47 / Chapter 3.2.5 --- Quantification of cells with > 5 LC3 punctate staining --- p.47 / Chapter 3.2.6 --- siRNAs transfection and treatments --- p.48 / Chapter 3.2.7 --- RNA isolation and RT-PCR --- p.48 / Chapter 3.2.8 --- ROS analysis --- p.48 / Chapter 3.2.9 --- JC-1 staining --- p.49 / Chapter 3.2.10 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- Bufalin induces autophagy-mediated cell death via ROS generation --- p.50 / Chapter 3.3.2 --- Activation of JNK is required for the upregulation of ATG5 and Beclin-1, and subsequent autophagy-mediated cell death in response to bufalin --- p.54 / Chapter 3.3.3 --- ROS generation is upstream of JNK activation in bufalin-induced cell death --- p.59 / Chapter 3.3.4 --- Bufalin-induced ROS generation is derived from mitochondria --- p.62 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- Bufalin arrests cells at prometaphase --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Reagents and antibodies --- p.70 / Chapter 4.2.2 --- Cell synchronization --- p.70 / Chapter 4.2.3 --- Mitotic index analysis of phosphorylation of MPM2 --- p.71 / Chapter 4.2.4 --- Cell cycle analysis --- p.71 / Chapter 4.2.5 --- Time-lapse experiments --- p.71 / Chapter 4.2.6 --- Immunofluorescence analysis of phospho-histone H3 (Ser10) --- p.72 / Chapter 4.2.7 --- Western blot analysis --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.3.1 --- Bufalin reduces mitotic marker phosphorylation of histone H3 and MPM2 and increases cells with 4N DNA content --- p.73 / Chapter 4.3.2 --- Increased cells with 4N DNA content after bufalin treatment are in neither a tetraploid G1 phase nor a cytokinesis arrest --- p.77 / Chapter 4.3.3 --- Bufalin-treated cells can enter prophase, but fail to pass through metaphase --- p.80 / Chapter 4.4 --- Discussion --- p.83 / Chapter Chapter 5 --- Bufalin induces prometaphase arrest through downregulating mitotic kinases --- p.87 / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.89 / Chapter 5.2.1 --- Reagents and antibodies --- p.89 / Chapter 5.2.2 --- Cell synchronization --- p.90 / Chapter 5.2.3 --- Immunofluorescence staining --- p.90 / Chapter 5.2.4 --- siRNAs transfection and treatments --- p.91 / Chapter 5.2.5 --- Western blot analysis --- p.91 / Chapter 5.2.6 --- Statistic analysis --- p.91 / Chapter 5.3 --- Results --- p.92 / Chapter 5.3.1 --- Bufalin downregulates Aurora A and B in protein and phosphorylation levels --- p.92 / Chapter 5.3.2 --- Bufalin prevents Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores --- p.97 / Chapter 5.3.3 --- Bufalin prevents Plk1 recruitment to mitotic centrosomes and unattached kinetochores through downregulation of protein levels of Plk1 --- p.101 / Chapter 5.3.4 --- Bufalin decreases the activities of Aurora A, Aurora B and Plk1 through PI3K pathway --- p.105 / Chapter 5.3.5 --- HIF-1α and NF-κB pathways are involved in sodium pump-mediated the regulation of mitotic kinases --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General discussion --- p.115 / Chapter 6.1 --- Potential toxicity of bufalin --- p.115 / Chapter 6.2 --- Cardiac glycosides induced programmed cell death --- p.115 / Chapter 6.3 --- Signal pathways involved in cardiac glycosides-mediated autophagy --- p.117 / Chapter 6.4 --- The relationship between ROS and JNK in cardiac glycosides-induced autophagy --- p.120 / Chapter 6.5 --- The role of ROS in apoptosis and autophagy --- p.121 / Chapter 6.6 --- The role of cardiac glycosides in cell cycle arrest --- p.122 / Chapter 6.7 --- Application of cardiac glycosides in combination with chemotherapy and radiotherapy --- p.125 / Chapter Chapter 7 --- Conclusions and future perspectives --- p.127 / References --- p.133 / Appendices --- p.153 / Publication --- p.153

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O influxo de K+ na faixa de alta afinidade à controlado por transportadores dos tipos canal e carreador, os quais diferem na sua sensibilidade ao Ãon NH4 +. A atividade de ambos os componentes, nÃo-sensÃvel (canal) e sensÃvel (carreador) ao Ãon NH4 + dependem da enzima de membrana plasmÃtica, H+-ATPase. Objetivou-se a caracterizaÃÃo fisiolÃgica e molecular da absorÃÃo de K+ de alta afinidade em sorgo forrageiro [Sorghum bicolor (L.) Moench] sob a influÃncia do estado nutricional da planta e de diferentes fontes de nitrogÃnio inorgÃnico na soluÃÃo de crescimento. Sementes do genÃtipo CSF20 foram germinadas e cultivadas em soluÃÃes nutritivas contendo dois nÃveis de K+ (0,2 e 1,4 mM) e trÃs diferentes regimes de nitrogÃnio inorgÃnico (NO3 - e NH4 + a 4 mM, e a mistura NO3 -/NH4 +, ambos a 2 mM). ApÃs 15 dias de cultivo (t0) em soluÃÃes nutritivas completas, as plantas foram submetidas à deficiÃncia de K+ por 1 (t1), 2 (t2) e 3 (t3) dias. No t0, os teores de K+ da parte aÃrea foram reduzidos ignificativamente pela presenÃa do Ãon NH4 +, enquanto que, nas raÃzes a reduÃÃo foi significativa apenas nos cultivos com 0,2 mM de K+. Conforme o aumento do perÃodo de deficiÃncia, os teores de K+ nas raÃzes e na parte aÃrea tenderam a diminuir em razÃo da diluiÃÃo provocada pelo crescimento da planta. Em plantas cultivadas com 0,2 mM de K+ e com NO3 - e NO3 -/NH4 +, as eficiÃncias de absorÃÃo de K+ foram similares. Entretanto, a presenÃa do Ãon NH4 + como Ãnica fonte de nitrogÃnio, afetou severamente esse parÃmetro. O efeito do Ãon NH4 + na eficiÃncia de absorÃÃo foi mais ameno quando as plantas foram cultivadas a 1,4 mM de K+. A eficiÃncia de transporte de K+ nÃo diferiu em nenhum dos tratamentos testados. As curvas de depleÃÃo de K+ mostraram que as plantas cultivadas com NO3 -/NH4 + e NH4 + apresentaram maior capacidade para esgotar o K+ (100 μM) da soluÃÃo de depleÃÃo quando comparadas Ãquelas crescidas com NO3 -. No t2, maiores taxas de Imax foram observadas nas plantas cultivadas com NH4 + como Ãnica fonte de nitrogÃnio. O Km para o K+, das plantas provindas dos cultivos com 0,2 e 1,4 mM de K+, apresentou valores menores nas plantas tratadas com NH4 + e NO3 -/NH4 +. A induÃÃo da absorÃÃo de alta afinidade de K+ foi influenciada pelo conteÃdo de K+ dos tecidos aÃreos. Ensaios com inibidores mostraram que o influxo de K+ foi significativamente inibido pelo Ãon NH4 + a 1 mM em plantas cultivadas com NO3 - como Ãnica fonte de nitrogÃnio. Entretanto, a capacidade de absorÃÃo de K+ foi reduzida pela presenÃa de TEA nas plantas cultivadas em soluÃÃes contendo o Ãon NH4 + como Ãnica fonte de nitrogÃnio. Esses resultados sugerem que em plantas de sorgo sob deficiÃncia de K+ e na presenÃa do Ãon NH4 + no meio de crescimento, canais de K+ podem contribuir significativamente para o influxo de K+. Por outro lado, na ausÃncia do Ãon NH4 + durante o crescimento, um sistema de transporte mediado por carreadores de K+ seria a via principal para o influxo desse nutriente. A atividade de transporte de H+ da H+-ATPase de membrana plasmÃtica isolada de raÃzes mostrou que a deficiÃncia de K+ (t2) estimulou a capacidade de formaÃÃo do gradiente de H+ em presenÃa do Ãon NH4 + nas plantas provindas dos cultivos com 1,4 mM de K+, enquanto que naquelas provindas dos cultivos com 0,2 mM de K+, o Ãon NH4 + teve efeito na velocidade inicial do transporte de H+ e na hidrÃlise do ATP. A deficiÃncia de K+ na presenÃa do NO3 - nÃo estimulou as atividades da bomba de 11 H+. ImunodetecÃÃo com anticorpos especÃficos contra H+-ATPases de membrana plasmÃtica de plantas mostrou a induÃÃo de duas isoformas nas membranas plasmÃticas oriundas de plantas cultivadas com 0,2 mM de K+, independentemente da fonte de nitrogÃnio e dos perÃodos de deficiÃncia. SeqÃencias gÃnicas correspondentes a genes de H+-ATPases de membrana plasmÃtica (SBA1 e SBA2), canais (SbAKT1) e carreadores de K+ (SbHAK1) foram selecionadas no genoma do sorgo e seus nÃveis de expressÃo em raÃzes analisados por PCR em tempo real. Os genes SBA1 e SBA2 pertencem, respectivamente, aos grupos II e I da famÃlia das H+-ATPases. Em raÃzes provindas dos cultivos com 0,2 mM de K+ e na presenÃa do Ãon NH4 + os nÃveis dos transcritos de SBA1 e SBA2 foram significativamente expressos a partir do tempo t2 de deficiÃncia de K+, enquanto que na presenÃa do Ãon NO3 - eles foram reduzidos conforme o aumento do tempo de deficiÃncia. Na dose mais alta de K+, os transcritos de SBA1 tiveram sua expressÃo incrementada pela deficiÃncia de K+ em presenÃa do Ãon NH4 + como Ãnica fonte de nitrogÃnio. Ambos os genes tiveram um incremento transitÃrio dos nÃveis dos transcritos no t1 de deficiÃncia de K+ na presenÃa do Ãon NO3 -. Transcritos dos genes SbAKT1 e SbHAK1 nÃo foram detectados. AnÃlises filogenÃticas mostraram que SbAKT à um canal de K+ da famÃlia Shaker, compartilhando origem evolutiva comum com vÃrios canais de K+ de gramÃneas. Os resultados sugerem que a homeostase iÃnica do K+ à alterada pelo Ãon NH4 + em plantas de sorgo. No entanto, a adaptaÃÃo das plantas à presenÃa do Ãon NH4 + envolve a induÃÃo de um sistema altamente eficiente para a aquisiÃÃo de K+, com a participaÃÃo de canais de K+ e da H+-ATPase de membrana plasmÃtica / K+ influx in the range of high affinity is mediated by K+ carriers and channels, which can be distinguished by its differential sensibility to NH4+. The activity of the NH4+-sensitive component (carrier) and NH4+-insensitive component (channel) depend upon plasma membrane H+-ATPase. This work aimed the physiological and molecular characterization of system mediating K+ uptake in the high-affinity range of concentration in sorghum [(Sorghum bicolor (L.) Moench)]. The effect of K+ starvation and nitrogen inorganic source of the growth solution on high-affinity K+ uptake was also studied. Seeds of sorghum, genotype CSF20, were germinated and placed in modified one-fourth Hoagland solutions, which were formulated to contain 0,2 and 1,4 mM K+ and three nitrogen inorganic source, NO3- and NH4+ (4 mM) and NO3-/NH4+ in combination (2mM/2mM). Plants were grown for 15 days (t0) in complete nutritive solutions and then incubated in a K+-free solutions for one (t1), two (t2) and three (t3)days. At t0, K+ content of shoot was significantly decreased in plants grown in the presence of NH4+. In roots, the presence of NH4+ did altered the K+ content only of plants cultivated in solutions with lowest K+ concentration. The K+ content of the plant tissues was progressively reduced according to increasing of starvation period and increasing of dry matter (dilution). The highest reductions were observed in K+ content of the shoot. At the lowest level of K+ (0,2 mM), plants grown in solutions containing NO3- and NO3-/NH4+ showed similar uptake efficiency of K+. Whereas, the presence of NH4 as sole nitrogen source, reduced severely the absorption rates of K+. The effect of NH4+ on uptake efficiency of K+ was alleviated by increasing the external K+ concentration to 1,4 mM K+. At both levels of K+, the translocation rate of K+ was not altered by the presence of NH4 + and by K+ starvation. Assays of depletion in the external medium of K+ (100 μM) showed that plants growing in the presence of NO3-/NH4+ and NH4+ were more efficient to deplete external K+ than plants grown with NO3- , as sole nitrogen source. Kinetic parameters were significantly different at second day (t2) of deprivation. In the presence of NH4 +, as sole nitrogen source, Imax values were higher than those of plants grown in NO3-/NH4 + and NO3 -. On the other hand, Km values were lower in plants from solutions containing NH4+ and NO3 -/NH4 + than those cultivated in NO3-, as sole nitrogen source. High-affinity uptake of K+ responded to changes in plant K+ status, mainly to K+ content of aerial parts. K+ uptake by plants growing in NO3- was significantly inhibited by inclusion of NH4 at 1 mM in depletion solution, whereas it was not inhibited in plants grown with NH4 +, as sole nitrogen source. TEA (tetraethylammonium) inhibited the K+ influx of plants cultivated in solution containing NH4 +, as sole nitrogen source. The results suggest that factors such as presence of K+ or NH4 + during plant growth determine the relative contribution of each component to high-affinity uptake system of K+. In K+-starved plants grown in the presence of NH4 +, K+ channel could contribute to K+ uptake in the range of high-affinity concentration. On the other hand, in K+-starved plants grown without NH4 +, K+ carrier could constitute the principal route for K+ uptake. Active H+-transport driven by plasma membrane H+-ATPase of sorghum roots was stimulated by K+ deficiency and NH4 + in plants from solutions with high K+ level. However, these factors affected the initial rate 13 of H+-pumping and hydrolytic activity of ATP, but not H+-gradient formation, in plants from solutions containing 0,2 mM K+. These activities were not changed when plants were cultivated in growth solutions containing NO3 - and submitted to K+-starvation. Two specific isoforms of PM H+-ATPase by immuno-detection were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO3 --grown. cDNA sequences corresponding to plasma membrane H+-ATPase (SBA1 and SBA2), K+ channels (SbAKT) and K+ carrier (SbHAK1) from sorghum genome were identified. The expression level of these genes was analyzed by real-time PCR. SBA1 and SBA2 genes were included at subfamily II and I of plasma membrane H+-ATPase, respectively. In plants cultivated in solutions containing 0,2 mM K+ and with NH4+, the accumulation of SBA1 and SBA2 transcripts was observed after 48 h (t2) of K+ deficiency. Whereas, the expression level of these genes was reduced in plants cultivated with NO3 -. In roots came from solutions containing 1,4 mM K+ level, the accumulation of SBA1 transcripts was only observed in the presence of NH4 + and after 48 h (t2) of K+ deficiency. The transcript level of both genes increased only at t1 in roots cultivated in solution containing NO3 -, as sole nitrogen source. Transcipts of SbAKT and SbHAK1 were not detectable by real-time PCR. Phylogenetic analysis revealed that SbAKT belongs to Shaker channel family of plants and is also closely related to members of other gramineous species. The results suggest that K+ homeostasis in NH4+-grown sorghum plants may be regulated by a high capacity for K+ uptake, which is dependent upon the H+ -pumping activity of PM H+-ATPase and K+ channels

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 86-96). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;