Academic literature on the topic 'Potassium'

BackgroundThe basolateral potassium channel in the distal convoluted tubule (DCT), comprising the inwardly rectifying potassium channel Kir4.1/Kir5.1 heterotetramer, plays a key role in mediating the effect of dietary potassium intake on the thiazide-sensitive NaCl cotransporter (NCC). The role of Kir5.1 (encoded by Kcnj16) in mediating effects of dietary potassium intake on the NCC and renal potassium excretion is unknown.MethodsWe used electrophysiology, renal clearance, and immunoblotting to study Kir4.1 in the DCT and NCC in Kir5.1 knockout (Kcnj16−/−) and wild-type (Kcnj16+/+) mice fed with normal, high, or low potassium diets.ResultsWe detected a 40-pS and 20-pS potassium channel in the basolateral membrane of the DCT in wild-type and knockout mice, respectively. Compared with wild-type, Kcnj16−/− mice fed a normal potassium diet had higher basolateral potassium conductance, a more negative DCT membrane potential, higher expression of phosphorylated NCC (pNCC) and total NCC (tNCC), and augmented thiazide-induced natriuresis. Neither high- nor low-potassium diets affected the basolateral DCT’s potassium conductance and membrane potential in Kcnj16−/− mice. Although high potassium reduced and low potassium increased the expression of pNCC and tNCC in wild-type mice, these effects were absent in Kcnj16−/− mice. High potassium intake inhibited and low intake augmented thiazide-induced natriuresis in wild-type but not in Kcnj16−/− mice. Compared with wild-type, Kcnj16−/− mice with normal potassium intake had slightly lower plasma potassium but were more hyperkalemic with prolonged high potassium intake and more hypokalemic during potassium restriction.ConclusionsKir5.1 is essential for dietary potassium’s effect on NCC and for maintaining potassium homeostasis.

Aim: To investigate the possibility of substitution of inorganic inputs through organics and to evaluate the effects of bioorganic inputs on microbial population of soil. Methodology: A field experiment of RBD with 13 treatments and 3 replication using combination of organic manures viz., compost/vermicompost, potassic mobilising inoculants along with graded (100, 75 and 50%) levels of inorganic potassium were tried for partial substitution of inorganic potassium with organic source in turmeric cv. Suguna. Results: Considering the yield, the vermicompost + inorganic K (100%) + K mobiliser (32.35 t ha-1) was the best treatment, followed by Compost + K 100% + KM (31.13 t ha-1). Interpretation: These results indicate that soils were less fertile due to deficit in soil nutrients. Recommended doses of organic bio-fertilizer and inorganic fertilizer should be practiced for reclamation of soil status. Application of potassic mobilizer as biofertilizer in turmeric can reduce the application of inorganic potassium and support eco-friendly crop production. Key words: Compost, Curcuma longa, Organic manures, Potassic mobilizer, Turmeric, Vermicompost

Dairy and beef pastures in the high (>800 mm annual average) rainfall areas of south-western Australia, based on subterranean clover (Trifolium subterraneum) and annual ryegrass (Lolium rigidum), grow on acidic to neutral deep (>40 cm) sands, up to 40 cm sand over loam or clay, or where loam or clay occur at the surface. Potassium deficiency is common, particularly for the sandy soils, requiring regular applications of fertiliser potassium for profitable pasture production. A large study was undertaken to assess 6 soil-test procedures, and tissue testing of dried herbage, as predictors of when fertiliser potassium was required for these pastures. The 100 field experiments, each conducted for 1 year, measured dried-herbage production separately for clover and ryegrass in response to applied fertiliser potassium (potassium chloride). Significant (P<0.05) increases in yield to applied potassium (yield response) were obtained in 42 experiments for clover and 6 experiments for ryegrass, indicating that grass roots were more able to access potassium from the soil than clover roots. When percentage of the maximum (relative) yield was related to soil-test potassium values for the top 10 cm of soil, the best relationships were obtained for the exchangeable (1 mol/L NH4Cl) and Colwell (0.5 mol/L NaHCO3-extracted) soil-test procedures for potassium. Both procedures accounted for about 42% of the variation for clover, 15% for ryegrass, and 32% for clover + grass. The Colwell procedure for the top 10 cm of soil is now the standard soil-test method for potassium used in Western Australia. No increases in clover yields to applied potassium were obtained for Colwell potassium at >100 mg/kg soil. There was always a clover-yield increase to applied potassium for Colwell potassium at <30 mg/kg soil. Corresponding potassium concentrations for ryegrass were >50 and <30 mg/kg soil. At potassium concentrations 30–100 mg/kg soil for clover and 30–50 mg/kg soil for ryegrass, the Colwell procedure did not reliably predict yield response, because from nil to large yield responses to applied potassium occurred. The Colwell procedure appears to extract the most labile potassium in the soil, including soluble potassium in soil solution and potassium balancing negative charge sites on soil constituents. In some soils, Colwell potassium was low indicating deficiency, yet plant roots may have accessed potassum deeper in the soil profile. Where the Colwell procedure does not reliably predict soil potassium status, tissue testing may help. The relationship between relative yield and tissue-test potassium varied markedly for different harvests in each year of the experiments, and for different experiments. For clover, the concentration of potassium in dried herbage that was related to 90% of the maximum, potassium non-limiting yield (critical potassium) was at the concentration of about 15 g/kg dried herbage for plants up to 8 weeks old, and at <10 g/kg dried herbage for plants older than 10–12 weeks. For ryegrass, there were insufficient data to provide reliable estimates of critical potassium.

Forty years have transpired since tetraethylammonium was first used to selectively inhibit the potassiuin conductance in squid axons. Since then, a large body of work has emerged describing inhibitors of voltage-gated potassium currents in a variety of cells. The advent of molecular cloning techniques and the cloning of the potassium channel encoded by the Shaker locus in Drosophila has enabled detailed structure­ function studies of several potassium channel subunits. These breakthroughs have also recently enabled studies of the "toxinology" and pharmacology of specific potassium channel subunits expressed heterologously in Xenopus oocytes and other cells. Here we describe the results of some of those efforts, focusing in particular on our work with four members of the Shaker subfamily of potassium channel a-subunits: Kvl.1 through Kvl.4. These subunits are expressed in the central nervous system and other tissues of rodents, and are highly homologous to corresponding subunits expressed in humans. We provide a profile of potency and selectivity for.five snake dendrotoxins as well as several scorpion toxins for these potassium channel subunits expressed in Xenopus oocytes. We also provide similar data for four other peptide toxins and several nonpeptide compounds that had previously been shown to inhibit potassium currents. We discuss several potential clinical applications of potassium channel inhibitors, including demyelinating diseases such as multiple sclerosis, immunosuppression, cardiac arrhythmias, neurodegenerative and psychiatric diseases. Further progress will require, among other things, a greater understanding of the expression patterns of potassium channel subunits in the CNS and elsewhere as well as knowledge of the specific subunit composition of heteromultimeric channels.

Mantenir la homeòstasis de cations és essencial per la supervivència dels éssers vius, i en especial pels organismes unicel·lulars. El llevat Saccharomyces cerevisiae s’ha utilitzat al llarg dels anys com un organisme model per l’estudi del conjunt de processos que controlen els nivells intracel·lulars de cations. El potassi és el principal catió intracel·lular del llevat i està involucrat en diversos processos de la fisiologia d'aquest organisme. Per aquest motiu la seva homeòstasis està minuciosament controlada per un seguit de transportadors que permeten la seva captació, distribució intracel·lular i eliminació de la cèl·lula i per un conjunt de proteïnes reguladores d’aquests processos. Encara que es té constància de la rellevància del potassi en el llevat, les seves dianes específiques i les bases moleculars d’algunes de les seves funcions són poc conegudes. En aquest treball, mitjançant aproximacions d’eliminació del potassi del medi de cultiu, s’han pogut determinar els mecanismes d’algunes de les funcions conegudes del potassi i descobrir-ne de noves. S’ha demostrat que la manca de potassi provoca profundes alteracions en el perfil transcripcional del llevat. Entre elles destaquen l’accentuada repressió del gens que codifiquen proteïnes ribosomals i elements necessaris per la síntesi del ribosoma, dotant d’explicació molecular al ja conegut bloqueig en la síntesi de proteïnes provocat per la manca de potassi. L’eliminació del potassi del medi també provoca una caiguda dels nivells dels aminoàcids cisteïna i metionina que condueix a una activació del gens relacionats amb el metabolisme del sulfat i la síntesi d’aminoàcids sulfurats. Igualment, la privació del potassi comporta una acumulació d’espècies reactives de l’oxigen que produeixen un estat d’estrès oxidatiu a la cèl·lula. Aquesta respon amb l’activació transcripcional dels gens necessaris per combatre l’estrès oxidatiu, eliminar els oxidants i retornar la cèl·lula a un correcte estat redox. El llevat creixent en un medi sense potassi i en presència d’amoni acumula grans quantitats d’amoni a l’interior cel·lular a través del transportador de potassi Trk1 aprofitant la similitud química d’ambdós cations. Aquesta acumulació d’amoni activa vies per la seva fixació i eliminació en forma d’aminoàcids com és la via retrograda mitocondrial. L’eliminació del potassi del medi atura la proliferació cel·lular. Els nostres resultats han demostrat que aquesta aturada podria ser deguda a la disminució de les ciclines i d’elements reguladors de la formació dels anells de septines que afecten la progressió del cicle cel·lular. El potassi també l’hem relacionat amb la homeòstasi d’un nutrient essencial com és el fosfat. L’absència de potassi o la pertorbació de la entrada normal d’aquest catió indueix els gens involucrats en la obtenció i mobilització del fosfat, d’igual manera com ho faria la depleció o limitació del fosfat. En aquetes condicions, la resposta transcripcional d’aquets gens està regulada pels diferents elements que composen la via PHO. L’afectació en l’obtenció del potassi impacte en la normal homeòstasis del fosfat provocant la mobilització de les reserves emmagatzemades en forma de polifosfats. La limitació del potassi, però, no modifica els nivells de fosfat lliure intracel·lular però sí que provoca una caiguda del nivells d’ATP i d’ADP, que podrien ser el senyal d’activació de la via PHO. A més, la pertorbació de la homeòstasis del potassi afecta el creixement dels llevats en medis amb baixos nivells de fosfat. El conjunt de dades obtingudes en aquest treball ha permès descobrir nous vincles entre la homeòstasis del potassi i diversos processos cel·lulars, a més de la connexió d’aquest catió amb la homeòstasis de nutrients com el nitrogen, el sulfat i el fosfat.
The maintenance of cation homeostasis is essential for the survival of all living organisms and especially for microorganisms. The yeast Saccharomyces cerevisiae has been used over the years as a model organism for study the processes that control intracellular cation levels. Potassium is the major intracellular cation in yeast and it has been associated with various relevant cellular processes. For this reason, potassium homeostasis is tightly controlled by several transporters, that allow the cation uptake, intracellular traffic and efflux, and by a set of proteins regulating these processes. In spite of the importance of potassium for yeast physiology, not all relevant functional potassium-related targets have been identified. In this work, potassium starvation conditions are used to determine the mechanisms of some of the known potassium functions and to discover new ones. We show that lack of potassium causes major alterations in the transcriptional profile of yeast cells. These transcriptional changes include the marked repression of genes encoding ribosomal proteins and elements necessary for the synthesis and assembly of ribosomes, providing the molecular basis for previously observed halt in protein synthesis caused by potassium deprivation. The elimination of potassium from the medium also causes a drop in cysteine and methionine levels which lead to transcriptional activation of genes related to metabolism of sulfate and biosynthesis of sulfur-contain amino acids. Similarly, cells deprived for potassium accumulate reactive oxygen species which results in oxidative stress. Concomitantly, cells trigger the transcriptional activation of genes necessary to combat oxidative stress, eliminate oxidants and return cells to the proper redox state. Yeast cells growing on ammonium as nitrogen source but lacking potassium accumulate large amounts of intracellular ammonium, which is transported through Trk1 taking advantage of the chemical similarity of both cations. Ammonium accumulation activates the retrograde mitochondrial pathway, resulting in detoxification of ammonium by its integration into amino acids. The complete removal of potassium from the medium leads to growth arrest. Our results show that this arrest could be due to the decrease in cyclins levels and in proteins involved in the assembly of septin rings, elements that are necessary for cell cycle progression. We also have related potassium to the homeostasis of other essential nutrients such as phosphate. Depletion of potassium from the medium or disturbance of normal potassium uptake induces genes involved in the acquisition and release of phosphate, as it is usually observed in a situation of phosphate starvation. Under these conditions, the transcription of PHO-controlled genes is activated by different regulatory elements of the PHO pathway. Situations that impact on normal potassium homeostasis also cause mobilization of the phosphate reserves stored in form of polyphosphates. Potassium restrictions, however, does not alter the levels of intracellular free phosphate but it causes a drop in the levels of ATP and ADP, which could be the signal for the activation of the PHO pathway. In addition, on media with low levels of phosphate, disruption of normal potassium homeostasis effects yeast growth. The results obtained in this work have been crucial to uncover new links between potassium homeostasis and many important cellular processes, in particular establishing the link between the homeostasis of this cation with that of other essential nutrients such as nitrogen, sulfate, and phosphate.

Orientador: Ricardo de Lima Isaac
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo
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Resumo: Este trabalho consistiu em avaliar a capacidade de remoção da toxina Microcistina-Lr utilizando oxidação por cloro, dióxido de cloro e permanganato de potássio. Os ensaios foram realizados em escala de bancada em jar test simulando o processo de tratamento de água nas ETA's 3 e 4 da SANASA de Campinas. Foram realizados estudos com concentrações de 2.5, 5.0 e 10 µg/L de Mc-Lr quando se utilizou oxidação com cloro e dióxido de cloro e com concentrações de 2.5 e 5.0 µg/ L quando foi utilizado permanganato de potássio como oxidante. Foram feitos testes para verificação da influência da aplicação do oxidante antes e depois do alcalinizante com dosagens de 10µg/L de Mc-Lr. As utilizações do cloro e do permanganato se mostraram eficientes para a remoção da Mc-Lr, enquanto que com a utilização do dióxido de cloro, nenhuma remoção foi alcançada dentro das três horas de testes. Nos testes com aplicação do oxidante antes e depois do alcalinizante, não houve alterações significativas. Quando os testes foram feitos utilizando o tempo, os resultados foram similares para o cloro e permanganato de potássio, com a oxidação ocorrendo durante a primeira hora. Com o dióxido de cloro, a oxidação aconteceu somente durante a sexta hora.
Abstract: This work consisted in evaluate the capacity of removal of Microcystin-Lr toxin using oxidation with chlorine, chlorine dioxide and potassium permanganate. The tests were did using bench -scale tests in jar test equipment to simulated water treatment process of 3 and 4 WTP of SANASA Campinas. Study were did using 2.5, 5.0 and 10 µg/L Mc-Lr concentration when were used chlorine and chlorine dioxide oxidation and 2.5 and 5.0 µg/L Mc-Lr concentration when was used potassium permanganate oxidation. Were did tests to verify the influence of before and after alkali oxidant dosing with 10µg/L Mc-Lr. The chlorine and permanganate uses were efficient to Mc-Lr removal while chlorine dioxide no one removal was obtained with normal quantity used in Convencional Water Drinking Treatment Plant. When the tests were did using time, the results were silimary to chlorine and permanganate with the oxidation happened during the first hour. With chlorine dioxide oxidation, the oxidation happened only during the sixth hour. When the tests were did dosing.
Mestrado
Saneamento e Ambiente
Mestre em Engenharia Civil

Banca: José Roberto de Oliveira e Silva
Banca:Wilma Pereira Bastos Ramos
Banca: Gerhard Malnic
Banca: Sonia Malheiros Lopes Sanioto
Banca: Frida Zaladek Gil
Resumo: Estudamos o efeito da Angiotensina II sobre a secreção de potásssio em túbulo distal final (segmento conector e duto coletor cortical) através da técnica de microperfusão estacionária in vivo e mensuração da atividade catiônica por meio de microeletrodos contendo resina de troca iônica sensível a K+. A perfusão luminal com ANG II reduziu o fluxo secretório de potássio (JK+) observado no grupo controle de 0.900.19 nmol/cm2.s, n=12, para 0.51±0.05, n=9, (ANG II 10-12M), 0.700.22, n=27 (ANG II 10-11M) e 0.630.08 nmol/cm2.s, n=12 (ANG II 10-9M); (p<0.05 teste t pareado). Entretanto, na presença de dose elevada de ANG II (10-6M) não observamos efeito significante sobre o JK+. A presença de Losartan (10-6M), um bloqueador não peptídico do receptor AT1 reverteu o efeito inibitório da ANG II. No intuito de se avaliar a possibilidade da via PLA2/ácido araquidônico/PGE2 participar deste processo de regulação celular, uma vez que tais agentes participam da inibição de outros mecanismos de transporte que envolve a ativação da sinalização celular mediada por Angiotensina II, perfundimos luminalmente PGE2 o qual inibiu o fluxo secretório de K+ em ambas doses utilizadas no presente trabalho; Jk+ controle = 0.930.08 nmol/cm2.s, n=12 para 0.550.05 nmol/cm2.s, n=12 (PGE2 10-9M) e 0.470.04 nmol/cm2.s, (PGE2 10-6M), n=12, (p<0.01). A perfusão com Indometacina (10-5M), bloqueador inespecífico da via PLA2/Ácido Araquidônico/PGE2 associado a Angiotensina II (10-9M) aumentou o JK+ (0.95±0.12 nmol.cm-2.s-1, n = 13) quando comparado a perfusão isolada de ANG II (10-9M) (Jk+ = 0.630.05 nmol/cm2.s, n = 10); (p<0.05). Concluímos que a ANG II inibiu luminalmente a secreção distal de K+ provavelmente acoplado ao receptor AT1 e este efeito pode ser mediado pela via PLA2/Acido Araquidônico/ /PGE2
Abstract: The effect of luminal ANGII on K+ secretion by late distal tubule (connecting segment and initial cortical collecting duct) was studied using "in vivo" stationary microperfusion and K-sensitive microelectrode techniques. Luminal perfusion of ANG II reduced Jk+ from a control value of 0.900.19 (n=12) nmol/cm2.s to 0.51±0.05, n=9, (10-12M), 0.700.22, n=27 (10-11M) and 0.630.08, n=12 (10-9M) nmol/cm2.s (p<0.05 by paired t-test). However, high doses of ANGII (10-6M) had no significant effect on Jk+. Losartan 10-6M, a non-peptide AT1 receptor blocker, reverted the inhibitory effect of ANGII. To test the possibility that the PLA2/arachidonic acid/PGE2 pathway, which had been shown to inhibit other transport mechanisms, is involved in ANGII-mediated cellular signaling cascades, PGE2 was perfused luminally (Jk+ control = 0.930.08, n=12 nmol/cm2.s; 10-9M PGE2, Jk+ = 0.550.05, n-12; 10-6M PGE2, Jk+ = 0.470.04), n=12; both doses reduced K+ secretion significantly (p<0.01). Perfusing with Indomethacin, an unspecific blocker of the PLA2/arachidonic acid/PGE2 path, (10-5M), plus ANG II (10-9M), JK+ increased to 0.95±0.12 (n=13) nmol.cm-2.s-1 compared to ANG II alone (Jk+ = 0.630.05, (n=13) nmol/cm2.s, p<0.05). During luminal perfusion with Indomethacin alone, no significant effect on K+ secretion was seen (Jk+ control = 0.730.05 (n=10) nmol/cm2.s, 10-6M INDO Jk+ = 0.630.07 (n=10), P>0.19. In conclusion, ANG II is able to regulate distal K+ secretion when applied to the tubule lumen, probably via AT1 receptors; it is suggested that the signalling path of the inhibitory effect of ANG II may involve PLA2/arachidonic acid/PGE2

During my Ph.D., my research focused on the involvement and the role of mitochondrial potassium homeostasis in the context of pathophysiological processes. I have been working on three related projects, for which the common aspect is the study of the mitochondrial potassium homeostasis and its modulation by pharmacological tools. My thesis contains a general introduction, in order to give a general up-to-date overview covering all the topics treated during my Ph.D., followed by a collection of the papers where I gave my contribution. Concerning the first project, my studies provided new insights into the mechanism of action of an emerging pro-apoptotic, oncologically relevant molecule, namely salinomycin. This molecule was considered a valinomycin-like K+ ionophore. Its recent identification as a selective inducer of apoptosis in cancer stem cells (CSCs) and different types of non-stem cancer cells, together with the ability to spare healthy cells, led to an increased interest in unravelling its mechanism of action, poorly understood so far. Moreover, since salinomycin has been suggested to act as a K+ ionophore, it is expected to impact mitochondrial function; however detailed information on its mitochondrial effects were not available from the literature. Therefore, I explored its early effects on mitochondrial bioenergetics. In order to do this, I compared its activity with that of valinomycin (K+ ionophore) or of nigericin (K+/H+ exchanger), for which the action was already well defined by others in the past. By using different approaches, ranging from classical bioenergetic studies on isolated mitochondria to more innovative measurement of bioenergetic parameters on intact cells, and of course by exploiting different cellular biology techniques, it has been concluded that salinomycin mediates K+/H+ exchange across the inner mitochondrial membrane, similarly to nigericin. It has been observed that salinomycin was also able to induce cell death of cells lacking some crucial actors of the apoptotic pathway (Bax/Bak-less double- knockout MEF cells). These results were compatible with the idea of direct modulation of mitochondrial function. At this point, the specificity of its action on pathologic B cells isolated from patients with chronic lymphocytic leukemia (CLL) versus B cells from healthy subjects has been investigated. The results indicated that salinomycin, when used above μM concentrations, exerts direct, mitochondrial effects, thus compromising cell survival, even in non-tumoral cells. These results were published in Managò et al., Cell Death and Disease, 2015. Having acquired the “know-how” to assess mitochondrial bioenergetic functions, I also actively contributed to a project carried out in collaboration with Prof. Erich Gulbins from University of Essen (Germany), where I also spent five months of my Ph.D. A strict collaboration between the lab where I did my Ph.D. and the lab of Prof. Gulbins led to the discovery of a mitochondrial voltage-gated potassium channel, mtKv1.3 and to the clarification of its important role during apoptosis. Mechanistically, it has been demonstrated that the pro-apoptotic protein Bax directly interacts with and inhibits mtKv1.3, via a toxin-like mechanism. The direct inhibition of mtKv1.3 leads to hyperpolarization, mitochondrial ROS production, opening of the permeability transition pore (PTP), release of cytochrome c and finally to apoptosis. In accordance to this model, direct pharmacological inhibition of mtKv1.3 by using the membrane-permeant Kv1.3 inhibitors Psora-4, PAP-1 and clofazimine leads to cell death in different types of cancer, as demonstrated by our group. The starting point of my second project was the fact that pyocyanin, a membrane-permeant toxin released by the Gram-negative bacterium Pseudomonas aeruginosa shows structural similarity to clofazimine, a membrane-permeant mtKv1.3 inhibitors. P. aeruginosa causes lung infections in immunocompromised patients and it is known that pyocyanin induces death of neutrophils, which plays an important role in the host’s early acute defence against pulmonary P. aeruginosa infections. However the exact mechanism of action of pyocyanin is still unknown therefore we determined whether its effect is related to Kv1.3 expression, given the crucial role of mtKv1.3 in apoptosis and the structural similarity of pyocyanin to mtKv1.3 inhibitors. First of all, it was observed by patch clamp experiments that pyocyanin is able to inhibit Kv1.3 current. At low concentration (up to 10µM), pyocyanin induced cell death preferentially in cells expressing Kv1.3. However, in the literature pyocyanin was mostly used at higher concentrations (50-100 µM), since in the sputum of patients with P. aeruginosa infections it might reach such a high concentration. Moreover, data from the literature suggested that pyocyanin might have a mitochondrial action and is able to produce high amounts of reactive oxygen species (ROS). Therefore, it has been investigated how pyocyanin impacts on mitochondrial function on a short time scale when used at high concentration (at 50µM). Again, intact cells or isolated mitochondria were used to assay the effect of this compound. It has been shown that pyocyanin determines an instantaneous production of superoxide anion at mitochondrial sites and a rapid but incomplete dissipation of the mitochondrial membrane potential. Further, it has been observed that pyocyanin can replace the function of complex III, while it does not directly alter the function of complex I. Moreover, it has been shown that ROS production induced by pyocyanin activates the acid sphyngomyelinase, shown to be present in mitochondria. This event in turn leads to the formation of ceramide, induction of apoptosis and release of cytochrome c. Genetic deficiency of acid sphyngomyelinase or scavenging of ROS induced by pyocyanin prevented cell death in neutrophils, meaning that pyocyanin, at high concentrations, induces cell death via mitochondrial reactive oxygen species and mitochondrial acid sphyngomyelinase, independently of the expression of the voltage gated potassium channel Kv1.3. These results were published in Managò et al., Antioxidant and redox signaling, 2015. During the last period of my Ph.D, I also studied the effect of newly synthesized Psoralen derivatives and clofazimine for the treatment of cancer. Concerning the first group, they are specific inhibitors of the voltage gated potassium channel Kv1.3; the second one is a molecule already in use in clinic to treat pathologies like leprosy, which also inhibits Kv1.3 current. Due to their lipophilic structure, they are all membrane-permeant, therefore able to reach intracellular membranes, like inner mitochondrial membrane where the voltage gated potassium channel Kv1.3 is also expressed and active. As mentioned above, the specific inhibition of mtKv1.3 triggers cell death. It has previously been demonstrated that Psora-4, PAP-1 (Psoralen derivatives) and clofazimine are able to specifically induce apoptosis in cancer cells in vitro on many cancer cell lines, ex vivo on B-CLL (chronic lymphocytic leukaemia) cells obtained from patients and in vivo on melanoma tumour model. Following these promising results, in collaboration with Professor Cristina Paradisi (Department of Chemical Sciences, University of Padova), PAP-1 and clofazimine derivatives have been synthesized in order to make these molecules more soluble and mitochondriotropic (i.e.: increased tendency to target mtKv1.3). I tested the ability of these new compounds to induce cell death in cancer cells first in vitro. Since the results obtained on different cell lines in vitro were promising and death was strictly dependent on Kv1.3 expression, it has been decided to test these compounds also in vivo on a melanoma model and on a pancreatic cancer model. During my 2-month stay at the end of the first year of my Ph.D. at the Institute for Experimental Cancer Research, University of Kiel (Germany), I performed in vivo experiments, using PAP-1 derivatives and clofazimine on SCID mouse injected with Colo357, a human pancreas tumour Kv1.3 expressing cell line, obtaining a significant reduction of the mass of the tumour (Zaccagnino, Managò et al., under submission to Oncotarget). Moreover, PAP-1 derivatives have been tested on a melanoma model in vivo, obtaining relevant results (Leanza, Romio, Becker, Azzolini, Trentin, Managò et al., manuscript in preparation, not included in the present thesis). The treatment exerted an effect on cancer, without significant side effects on healthy tissues. Some of the most promising clofazimine-derivatives have been selected to be tested on cells deriving from acute myeloid leukaemia (AML) patient. I carried out these experiments during my stay in Germany at the Department of Molecular Biology University of Duisburg-Essen. Blood samples came directly from the haematology ward of the university-hospital in Essen. So far, we obtained preliminary results which are however controversial, since different patients were diagnosed at different stage of the disease (i.e. have different levels of pathological cells) and AML itself is a heterogeneous disease at the molecular and cytogenetic level. Therefore this work has not been included in the thesis. Furthermore, I took part in a project of the laboratory of Prof. Holger Kalthoff (University of Kiel, Germany), concerning the study of the mechanism of action of a seaweed extract, for which I performed the experiments of bioenergetics (Geisen , Zenthoefer , Peipp , Kerber , Plenge , Managò et al., Marine drugs, 2015). Beside the laboratory-based projects, I also contributed to the preparation of two reviews, one concerning intracellular ion channels and cancer (Leanza, Biasutto, Managò et al., Frontiers on Physiology, 2013) and the other regarding in vivo pharmacological targeting of ion channels as possible therapeutic tool against cancer (Leanza, Managò et al., BBA Mol. Cell Research, 2015).
Durante il mio dottorato, la mia ricerca si è concentrata sul coinvolgimento e il ruolo dell'omeostasi mitocondriale di potassio (K+) in processi fisiopatologici. Ho lavorato su tre progetti correlati, per cui l'aspetto comune è lo studio dell’omeostasi del potassio mitocondriale e la sua modulazione tramite strumenti farmacologici. La mia tesi contiene un'introduzione generale, al fine di dare una descrizione generale aggiornata di tutti gli argomenti trattati durante il mio dottorato di ricerca, seguiti da una raccolta di pubblicazioni scientifiche in cui ho dato il mio contributo. Per quanto riguarda il primo progetto, i miei studi hanno fornito nuove informazioni sul meccanismo d'azione di una molecola pro-apoptotica emergente, rilevante dal punto di vista oncologico, nota come salinomicina. Questa molecola è stata considerata uno ionoforo di K+ simile alla valinomicina. La sua recente identificazione come induttore selettivo di apoptosi in cellule staminali tumorali (CSCs) e diversi tipi di cellule tumorali non-staminali, insieme alla sua capacità di risparmiare le cellule sane, ha portato a un interesse crescente verso la comprensione del suo meccanismo d'azione, poco noto finora. Inoltre, poiché è stato suggerito che la salinomicina agisca come uno ionoforo di K+, ci si aspetta un suo effetto sulla funzione mitocondriale. Tuttavia non erano disponibili in letteratura informazioni dettagliate sugli effetti mitocondriali di questa molecola. Pertanto, ho esplorato i suoi effetti istantanei sulla bioenergetica mitocondriale. Per fare questo, ho confrontato la sua attività con quella della valinomicina (ionoforo di K+) e della nigericina (scambiatore K+/H+), l'azione delle quali era già stata ben definita da altri in passato. Utilizzando diversi approcci, che vanno dagli studi bioenergetici classici su mitocondri isolati alla misurazione di parametri bioenergetici con strumenti più innovativi su cellule intatte, e, naturalmente, sfruttando diverse tecniche di biologia cellulare, si è concluso che la salinomicina media lo scambio K+/H+ attraverso la membrana mitocondriale interna , analogamente alla nigericina. Inoltre, è stato visto che la salinomicina è stata in grado di indurre la morte cellulare delle cellule prive di alcuni attori cruciali del processo apoptotico (doppio knock-out di Bax/Bak in cellule MEF). Questi risultati sono compatibili con l'idea di una modulazione diretta della funzione mitocondriale da parte della salinomicina. A questo punto, è stata studiata la specificità della sua azione su cellule B patologiche isolate da pazienti con leucemia linfatica cronica (CLL) versus cellule B di soggetti sani. I risultati hanno indicato che la salinomicina, quando usata sopra concentrazioni mM, esercita effetti mitocondriali diretti, compromettendo così la sopravvivenza delle cellule, anche di quelle non tumorali. Questi risultati sono stati pubblicati in Managò et al., Cell Death and Disease, 2015. Avendo acquisito il "know-how" per valutare le funzioni bioenergetiche mitocondriali, ho anche contribuito attivamente a un progetto realizzato in collaborazione con il Prof. Erich Gulbins dell'Università di Essen (Germania), dove ho anche trascorso cinque mesi del mio dottorato di ricerca. Una stretta collaborazione tra il laboratorio dove ho fatto il mio dottorato di ricerca e il laboratorio del Prof. Gulbins ha portato alla scoperta di un canale del potassio voltaggio-dipendente mitocondriale, mtKv1.3 e al chiarimento del suo importante ruolo durante l'apoptosi. Meccanicisticamente, è stato dimostrato che la proteina pro-apoptotica Bax interagisce direttamente con mtKv1.3 e lo inibisce, tramite un meccanismo simile a quello di alcune tossine. L'inibizione diretta di mtKv1.3 porta ad iperpolarizzazione, produzione di ROS a livello mitocondriale, apertura del poro di transizione di permeabilità (PTP), rilascio di citocromo c ed infine all'apoptosi. Secondo questo modello, l'inibizione farmacologica diretta di mtKv1.3 utilizzando inibitori di Kv1.3 permeanti la membrana come Psora-4, PAP-1 e clofazimina, porta alla morte cellulare in diversi tipi di cancro, come dimostrato dal nostro gruppo. Il punto di partenza del mio secondo progetto è stato l’evidenza che la piocianina, una tossina permeante la membrana rilasciata dal batterio Gram-negativo Pseudomonas aeruginosa mostra somiglianza strutturale alla clofazimina, un inibitore di Kv1.3 permeante la membrana. P. aeruginosa provoca infezioni polmonari nei pazienti immunocompromessi ed è noto che la piocianina induce morte cellulare nei neutrofili, che svolgono un ruolo importante nella difesa precoce acuta dell'ospite contro infezioni polmonari da P. aeruginosa. Tuttavia l'esatto meccanismo d'azione della piocianina è ancora sconosciuta quindi abbiamo voluto studiare se il suo effetto è legato all'espressione di Kv1.3, dato il ruolo cruciale di mtKv1.3 nell’apoptosi e l'affinità strutturale della piocianina agli inibitori di Kv1.3. Prima di tutto, è stato osservato dagli esperimenti di patch-clamp che la piocianina è in grado di inibire la corrente di Kv1.3. A basse concentrazioni (fino a 10 µM), la piocianina induce morte cellulare preferenzialmente in cellule esprimenti Kv1.3. Tuttavia, in letteratura la piocianina è stata utilizzata principalmente a concentrazioni più elevate (50-100 µM), poiché nell'espettorato di pazienti con infezioni da P. aeruginosa potrebbe raggiungere tale concentrazioni. Inoltre, diversi dati in letteratura hanno suggerito che la piocianina potrebbe avere un'azione mitocondriale ed è in grado di produrre elevate quantità di specie reattive dell'ossigeno (ROS). Pertanto, è stato esaminato l'impatto della piocianina sulla funzione mitocondriale con cinetiche brevi, quando usata ad alta concentrazione (50 µM). Ancora una volta, cellule intatte o mitocondri isolati sono stati usati per saggiare l'effetto di questo composto. E 'stato osservato che la piocianina determina una produzione istantanea di anione superossido a livello di siti mitocondriali e una dissipazione rapida ma incompleta del potenziale di membrana mitocondriale. Inoltre, è stato visto che la piocianina può sostituire la funzione del complesso III, mentre non altera direttamente la funzione del complesso I. Infine, è stato dimostrato che la produzione di ROS indotta dalla piocianina attiva la sfingomielinasi acida, presente anche in mitocondri. Questo evento a sua volta porta alla formazione di ceramide, induzione di apoptosi e rilascio del citocromo c. La mancanza di espressione della sfingomielinasi acida o lo scavenging di ROS indotta dalla piocianina impedisce la morte cellulare in neutrofili, che indicando che la piocianina, ad alte concentrazioni, induce la morte cellulare attraverso specie reattive dell'ossigeno mitocondriali e sfingomielinasi acida mitocondriale, indipendentemente dall'espressione del canale del potassio voltaggio dipendente Kv1.3. Questi risultati sono stati pubblicati in Managò et al., Antioxidant and redox signaling, 2015. Durante l'ultimo periodo del mio dottorato di ricerca, ho anche studiato l'effetto di alcuni derivati di psoraleni di nuova sintesi e della clofazimina per il trattamento del cancro. Per quanto riguarda il primo gruppo, essi sono inibitori specifici del canale del potassio voltaggio dipendente Kv1.3; la seconda è una molecola già in uso in clinica per il trattamento di patologie come la lebbra, che agisce anche come inibitore della corrente di Kv1.3. Grazie alla loro struttura lipofila, sono tutti permeanti la membrana, quindi in grado di raggiungere le membrane intracellulari, come la membrana mitocondriale interna dove il canale del potassio voltaggio dipendente Kv1.3 è espresso e attivo. Come accennato in precedenza, l'inibizione specifica di mtKv1.3 innesca la morte cellulare. È stato dimostrato che Psora-4, PAP-1 e clofazimina sono in grado di indurre specificamente apoptosi nelle cellule tumorali in vitro su molte linee cellulari tumorali, ex vivo su cellule B ottenute da pazienti con leucemia linfatica cronica e in vivo su modello di melanoma. A seguito di questi risultati promettenti, in collaborazione con la Prof. Cristina Paradisi (Dipartimento di Scienze Chimiche, Università di Padova), sono stati sintetizzati derivati del PAP-1 e della clofazimina in modo da rendere queste molecole più solubili e mitocondriotropiche (i.e.: indirizzate al mitocondrio). Ho testato prima la capacità di questi nuovi composti di indurre morte cellulare nelle cellule tumorali in vitro. Poiché i risultati ottenuti su diverse linee cellulari sono stati promettenti e l’induzione di morte cellulare era strettamente dipendente dalla espressione Kv1.3, si è deciso di testare questi composti anche in vivo in un modello di melanoma e di tumore pancreatico. Durante il mio soggiorno di due mesi al termine del primo anno di mio dottorato presso l'Istituto per la ricerca sperimentale sul cancro, Università di Kiel (Germania), ho eseguito esperimenti in vivo, usando derivati del PAP-1 e clofazimina su topi SCID iniettati con Colo357, una linea di cellule tumorali umane di pancreas esprimenti Kv1.3, ottenendo una riduzione significativa della massa del tumore (Zaccagnino, Managò et al., under submission alla rivista Oncotarget). Inoltre, i derivati del PAP-1 sono stati testati su un modello in vivo di melanoma, dando risultati rilevanti (Leanza, Romio, Becker, Azzolini, Trentin, Managò et al., manoscritto in preparazione, non incluso nella presente tesi). Il trattamento ha esercitato un effetto sul tumore, senza rilevanti effetti collaterali sui tessuti sani. Alcuni dei più promettenti derivati della clofazimina sono stati selezionati per essere testati su cellule derivanti da pazienti con leucemia mieloide acuta (AML). Ho eseguito questi esperimenti durante il mio soggiorno in Germania presso il Dipartimento di Biologia Molecolare dell'Università di Duisburg-Essen. I campioni di sangue provenivano direttamente dal reparto di ematologia della clinica universitaria di Essen. Finora, abbiamo ottenuto risultati preliminari che sono però controversi, poiché i pazienti sono stati diagnosticati a diversi stadi della malattia (cioè hanno livelli differenti di cellule patologiche nel sangue) e poiché la leucemia mieloide acuta è di per sé una malattia eterogenea a livello molecolare e citogenetico. Questi studi non sono stati inclusi nella tesi. Inoltre, ho partecipato a un progetto del laboratorio del Prof. Holger Kalthoff (Università di Kiel, Germania), riguardante lo studio del meccanismo d'azione di un estratto di alghe marine, per il quale ho eseguito gli esperimenti di bioenergetica (Geisen, Zenthoefer, Peipp, Kerber, Plenge, Managò et al., Marine drugs, 2015). Oltre ai progetti basati sull’attività in laboratorio, ho anche contribuito alla preparazione di due review, una in materia di canali ionici intracellulare in relazione al cancro (Leanza, Biasutto, Managò et al., Frontiers on Physiology, 2013) e l'altra riguardante l’utilizzo in vivo di canali ionici come bersaglio farmacologico come possibile strumento terapeutico nella cura del cancro (Leanza, Managò et al., BBA Mol Cell. Research, 2015).

The application of portable XRF spectrometry (pXRF) for determining concentrations of uranium (U), thorium (Th) and potassium (K) was evaluated using a combination of 12 Certified Reference Materials, 17 Standard Reference Materials, and 25 rock samples collected from areas of known U occurrences or mineralization. Samples were analysed by pXRF in Soil, Mining Cu/Zn and Mining Ta/Hf modes. Resulting pXRF data were compared to published recommended values, obtained by total or near total digestion methods with ICP-MS and ICP-OES analysis. Results for pXRF show a linear relationship, for thorium, potassium, and uranium (&amp;lt;5000 ppm U) as compared to the recommended concentrations. However, above 5000 ppm U, pXRF results show an exponential relationship with under reporting of pXRF concentrations compared to recommended values. Accuracy of the data can be improved by post-analysis correction using linear regression equations for potassium and thorium, and samples with &amp;lt;5000 ppm uranium; an exponential correction curve is required at &amp;gt;5000 ppm U. In addition, pXRF analyses of samples with high concentrations of uranium (e.g. &amp;gt;1 wt.% U) significantly over-estimated potassium contents as compared to the published values, indicating interference between the two elements not calibrated by the manufacturer software.