Dissertations / Theses on the topic 'Potassium'

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).

This study uses the structural coordinates of the determined K+ channels to create comparative models of three diverse members of this family, with the aim of enabling a better understanding of the function of these channels. The K+ channel of primary interest is the hERG K+ channel. The pharmacology of this channel is of considerable interest as serendipitous block of K+ conduction pore may result in cardiac arrest. A set of known antagonists have been docked into novel comparative models of hERG to propose how these drugs interact with the channel. The models have also been subjected to molecular dynamics simulations to investigate the drug binding in more detail and to gain a structural understanding of two critical biophysical properties of this channel: activation and inactivation. Additionally, ancillary domains of the channel have been modelled to provide a tool for interpreting detailed structure-function relationships for the hERG channel. The second channel investigated is the TASK-1 channel. Comparative models of this channel have been created to evaluate mutations that alter selectivity and pH sensitivity. The final K+ channel studied is the Kir2.1 channel. A fundamental property of this channel is its block by polyamines, which prevents the efflux of K+. Comparative models have been created, with a series of polyamine analogues docked into the membrane and cytoplasmic pore regions of this channel. Overall, this study has illuminated the structural basis of several biophysical properties that are intrinsic to normal K+ channel function.

The pancreatic ATP-sensitive potassium (KATP) channels couple glucose metabolism to excitability of the pancreatic β-cells to regulate insulin secretion. The channel subunits, Kir6.2 and SUR1, are encoded by the KCNJ11 and ABCC8 genes respectively. Genetic polymorphisms in these genes, which reduce channel activity, cause congenital hyperinsulinism (CHI) characterized by insulin hyper-secretion and hypoglycemia. The hERG (human ether-a-go-go related gene) potassium channels,encoded by the KCNH2 gene, contribute to the rapidly activating delayed rectifier K+ current (IKr), which is responsible for rapid repolarisation of the cardiac action potential. Decreased hERG channel function causes the Long QT syndrome 2 (LQTS2) and life threatening cardiac arrhythmias. Several mutations in these two clinically important potassium ion channels alter their surface density leading to disease. Therefore, it is of fundamental importance to investigate the trafficking mechanisms that regulate the surface density of these channels. Techniques in cell biology, molecular biology and biochemistry were employed to identify the molecular basis of Sar1-GTPase dependent ER exit of the KATP and hERG channels in COPII vesicles. Blocking the cargo binding sites on the Sec24 protein of the COPII coat with membrane-permeable synthetic peptides prevented ER exit of both these channels. While the diacidic 280DLE282 sequence on the Kir6.2 subunit of KATP channels was found to be the ER exit motif required for entry of the channels into COPII vesicles at the ER exit sites, such a motif was found to be absent on hERG Cterminus. Further, endocytic trafficking mechanism of hERG channels was studied in recombinant (HEK MSRII and HeLa) and native (neonatal rat cardiac myocytes) systems using cell biological and pharmacological tools. hERG channels were found to be internalised by a dynamin-independent, raft-mediated, and ARF6-dependent pathway. A prolonged block of this pathway revealed that the channels could also undergo internalisation by an alternate dynamin-mediated pathway. Internalised hERG channels were found to recycle back to the cell surface and undergo lysosomal degradation. Degradation of the channels was enhanced when Rab11a-GTPase function was disrupted leading to reduced surface density indicating that recycling is crucial to maintain cell surface density of the channels. Thus this study investigated and compared the previously unknown mechanisms of biosynthetic and endosomal trafficking of the KATP and hERG potassium channels with a conclusion that these processes play an important role in maintaining surface density and thereby in the function of these channels in physiological and patho-physiological conditions.

Limbic encephalitis (LE) is a central nervous system disorder that is characterised by memory impairments, confusion, agitation and seizures, and associated with hyperintense lesions of the medial temporal lobe, seen with MRI. Anti-voltage-gated potassium channel (VOKC) antibodies have been been detected in the plasma of a subset of LE patients using a 125I-a-dendrotoxin (a-DTX) radioimmunoprecipitation assay, suggesting that the likely antigens are VOKC Kv1.l, 1.2 and 1.6 subunits. Symptoms of the disease improve markedly with immunosuppression, correlating with similarly dramatic falls in the titre of anti-VOKC antibodies, thus implicating anti-VOKC antibodies in the pathogenesis of LE. Circumstantial evidence from studies of inherited channelopathies and animal models of reduced VOKC activity suggests that VOKC dysfunction may contribute to the pathogenesis of LE. This thesis addresses whether anti-VOKC antibodies (i) bind to a-DTX-sensitive subunits and (ii) affect VOKC function. Immunofluorescence data show that binding of LE patient IgO to the surface of primary neurons and Kvl-expressing HEK-293/HEKTSA cells could not be detected with indirect immunofluorescence. Comparison of intracellular labelling with patient and control IgO showed that no additional labelling could be detected with LE patient IgO. Electrophysiological data show first, that a-DTXsensitive currents could not be reliably isolated from primary cultured hippocampal neurons; second, that NMT or LE samples did not affect VOKCs expressed by neuroblastoma-l cells; third, that none of the LE samples affected potassium currents in Kvl-transfected HEK-293 cells. These data suggest that 'anti-VOKC' antibodies may not bind directly to Kv1.111.2/1.6 homomers, or to a range of Kv1.I/1.2/1.6 subunit-containing heteromers in transfected cells. The findings instead suggest that 'anti-VOKC' antibodies in LE patient plasma may bind to a Kvl-associated protein that contributes to a-DTX-sensitive complexes in the radioimmunoprecipitation assay, but is absent from Kv1.111.2/1.6-transfected cells. Future work to characterise whether another antigen is bound by LE patient anti-VOKC antibodies will be important in determining how these antibodies contribute to the pathogenesis of LE.

Effects of potassium supplementation were evaluated in four studies in endurance horses during races and treadmill exercise. In the first and second studies a potassium-free experimental formula was compared to potassium rich commercial formulas. The first study showed that supplementation increased plasma [K+], and that the extra sodium in the potassium-free experimental formulas helped to attenuate acidosis at the end of the ride. In the second study supplementation also increased plasma [K+], however speeds were lower and no increases were observed in plasma concentrations during the race. Supplementation of potassium during recovery helped to restore plasma [K+]. Higher plasma [Ca++] was found in horses supplied with experimental feeds, due to a lower dietary cation anion balance (DCAB). Three eliminated horses had heart rate arrhythmias and labile heart rates accompanied with higher plasma [K+] and lower [Ca++] than finishers. Also horses supplied with the experimental sodium-rich formula were less dehydrated than the ones receiving commercial formulas. The third study involved an 80 km endurance exercise test on the treadmill, and plasma [K+] was affected by potassium supplementation during exercise and recovery. The supply of potassium caused higher plasma [K+] helping to restore body stores. Also chloride supply in the electrolyte formulas maintained plasma [Cl-] levels during exercise and affected plasma concentrations during recovery. The fourth study showed that potassium supply affects plasma concentration, but also increases lactate production and glucose during sub-maximal exercise. A potassium-free electrolyte supply caused higher plasma [Ca++] during exercise. Higher sodium supply in the potassium-free electrolytes improved hydration during exercise. These studies show that potassium should supplemented after exercise and but not be done during exercise because of the risk of increased neuromuscular excitability.
Ph. D.

Potassium (K⁺) channels shape the electrical activities of cells by changing the frequency and duration of action potentials and by setting the resting potential. The huge diversity of K⁺ channels is conserved across species and is thought to be required to uniquely customise both the active excitable and passive properties of different cell types. When this study began, Drosophila voltage-gated K⁺ currents were thought to be encoded by the four cloned members of this family Shaker, Shab, Shaw and Shal. Mutant analysis had only been performed with Shaker. In order to investigate the in vivo function of another member of the family, a number of methods of making Shaw mutants were used. A more accurate genomic structure and location of Shaw was determined, and I attempted to identify P-element inserts in Shaw. However all the candidate inserts identified were located too far from Shaw to be of practical use for generating mutations affecting Shaw. A dominant negative strategy to remove Shaw function was therefore performed. PCR mutagenesis was used to generate both epitope-tagged full-length and truncated mutant versions of Shaw. Additional control constructs were also made: a full-length wildtype Shaw and a dominant negative version of Shaker. Transgenic lines were generated containing the constructs whose expression was GAL4 inducible. Expression of mutant Shaw, using a number of GAL4 lines, caused an ether-sensitive leg-shaking phenotype.

The chemical and physical effects of potassium promotion on prepared nickel-alumina catalysts have been examined using "in-situ" Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS), Temperature Programmed studies and X-ray Powder Diffraction (XRPD). Various preparative methods were explored to obtain catalyst samples with a high metallic surface area. A sequential precipitation technique was employed, yielding a highly loaded nickel alumina catalyst (56 %wt Ni) with the hydrotalcite structure, typical of other industrial catalysts. Subsequent potassium promotion of this initial catalyst was achieved by impregnation. X.R.P.D. studies showed no observable changes in the bulk structure of the catalyst at room temperature or during calcination with low loadings of potassium (≤ 1 %wt). However, at higher loadings, potassium carbonate/oxide peaks became apparent but there was no indication of any other potassium derivatives. The presence of potassium both inhibited the reduction of the catalyst and brought about a reduction of the nickel surface area in the final reduced catalyst. Infrared studies showed definite changes in the surface chemistry of the catalyst with potassium promotion. Adsorption of carbon monoxide showed a downward shift in the wavenumber of the characteristic bands together with an apparent change in the relative proportions of the different carbon monoxide adsorbed species. Temperature dependent adsorption studies further indicated the formation of an activated species which was increasingly stable as a function of potassium promotion. The same species was observed in analogous carbon dioxide experiments. The effect of potassium on the infrared spectra from adsorbed ethene, propene and but-1-ene was more subtle.

To release energy from chemically stored energy in the biomass was the new investigation in recent years. Utilizing of biomass for this purpose occur in two different ways, directly by burning (combustion) the biomass and indirectly by pyrolysis process which will convert the biomass to three main products, bio- tar, bio- char and synthetic gas. Biomass contains different amount of inorganic compound, especially alkali metals which causes some diverse impacts on combustion, pyrolysis and gasification process such as corrosion, agglomeration and fouling problems. This project aims to investigate the effect of K2CO3 on the pyrolysis and gasification processes of three different types of fuel; wood pellets, forest residue pellets and synthetic waste pellets at three different temperatures, 750 °C, 850 °C and 900 °C respectively. The purpose of this work to study and clarify the influence of K2CO3 on char yield, tar yield and tar compositions and the gasification rate and the reactivity of different fuels char. The pyrolysis process was carried out in a fluidized bed reactor during 2 minutes and the products were tar, char and synthetic gas. In this project interested in char and tar only. Char yield calculated and the results shows the char yield increase with increasing of [K2CO3]. While the tar analysis carried on GC- MS instrument at HB to study the tar yield and compositions. The results showed that potassium carbonate has not so much effect on tar yield and its composition. The last part was gasified the char in TGA with steam and CO2 as oxidizing media to study the influence of [K2CO3] on gasification rate and the reactivity of char samples at different temperatures. The result showed the [K2CO3] has inhibitory effect on gasification rate and the reactivity.

Chlamydia trachomatis is an obligate intracellular bacterium, which is the leading cause of acquired blindness and the most prevalent bacterial sexually transmitted infection worldwide. Chlamydiae exist in two distinct forms. The infectious spore-like elementary bodies (EBs) that invade host cells differentiate into non-infectious reticulate bodies (RBs) that replicate intracellularly within a modified membrane-bound vacuole called the inclusion. Under stress, Chlamydiae can enter a persistent state, in which aberrant bodies (ABs) with reduced metabolic activity are formed. Surprisingly little is known about the mechanisms employed by the bacteria to maintain and manipulate their environment within host cells. This thesis investigates the role of inorganic ions in sustaining the inclusion throughout the Chlamydia infection cycle. Potassium starvation of intracellular RBs either after specific ionophore treatment or inhibition of inward rectifying cellular potassium channels induced the formation of ABs, which no longer differentiated into infectious EBs. These data demonstrate an essential role for potassium during C.trachomatis replication. Analysis of live RBs, using a potassium sensitive fluorescent probe, illustrated that potassium is actively scavenged from the host cell. Furthermore, when bacteria undergo RB-EB differentiation accumulated potassium is released prior to inclusion lysis. Experimentally reducing potassium ion concentration at this stage caused cells to expel bacteria in bursts. This event is distinct from previously described extrusion mechanisms, where either the inclusion is released intact or the host cell is lysed. These data show that RBs actively accumulate potassium during replication, with starvation leading to persistence. Loss of potassium ions during re-differentiation into EBs suggests that potassium efflux has a role in triggering inclusion lysis or bacteria exit from the host cell.

NKN doped samples, (100-x)NKN-xSBN (0 ≤ x ≤ 10) were produced using the conventional mixed oxide route with 0.45 wt% Fe2O3 sintering aid (xSBNF). After 20-24 hours mixing, samples were calcined at 850°C and sintered at 1100–1140°C (± 180°C/hour) for 4 hours. By XRD 4 mol% SBN was found to be the solubility limit for single phase structure. By SEM, second phases were visible when 2 ≤ x ≤ 4; their structure was subsequently shown to be tungsten bronze type (TBT). 2-4 SBNF samples were high density, over 96% theoretical. For x = 0, TC = 457°C, TO-T = 234°C, Pr = 22 μC/cm2 and EC = 16.5 kV/cm. TC was found to decrease by 14.7°C and TO-T by 9.0°C per 1 mol% addition SBN. 2SBNF was the optimal formulation in terms of microstructure and electrical properties, with average grain size 3 μm, Pr = 25 μC/cm2 and EC = 8.8 kV/cm, ρ = 4.7 kΩm and Q = 1.16 eV. This material comprised approximately 90% orthorhombic and 10% tetragonal phases coexisting. Pseudo-cubic lattice parameters are a’ = c’ = 3.947180 Å, and b’ = 3.999996 Å for orthorhombic phase; the tetragonal has a’ = c’ = 3.989798 Å, and b’ = 3.975777 Å.Synchrotron XRD studies were undertaken as a function of temperature on 99.5NKN-0.5CuO + 0.6 wt% Nb2O5 solid and powder samples. The data were Rietveld refined. The solid sample underwent two polymorphic phase transitions at 300°C and 515°C; the latter was between two tetragonal phases: lattice parameters for the tetragonal phase (300-520°C) were a’ = c’ = 4.99557 Å, and b’ = 4.0363 Å; high temperature tetragonal (>500°C) exhibited a’ = c’ = 4.9519 Å, and b’ = 4.4941 Å. The powder sample of the same formulation exhibited more, smaller transformations. It was only orthorhombic at temperatures <140°C with a’ = c’ = 4.10680 Å, and b’ = 4.02620 Å. Above 140°C both orthorhombic and tetragonal phases were present. Another significant transformation occurred at 360°C where the structural unit cell parameters changed significantly. Parameter lengths are provided. P-E data was characterised by Pr = 19.9 μC/cm2 and EC = 13.5 kV/cm. Synchrotron X-ray diffraction analysis of 94 NKN-6LiTaO3 showed that tetragonal phase was present at 20-390°C, although an orthorhombic phase was identified at 20-200°C and again at 340-390°C just before the cubic transition temperature at 390°C. This is a new observation for NKN. A new and simple method for tape casting was developed to reduce powder wastage, enabling thick films of 50 μm to be cast. The reactive templated grain growth (RTGG) method was employed to orient 95NKN-5LiNbO3 and 94NKN-6LiNbO3 samples; CuO was utilised as a sintering aid. Pre-cursor BNN and NN template particles were produced using the molten salt synthesis (MSS) method, using a salt to oxide ratio of 1:1. Resulting NN particles were 15 μm wide and 0.5 μm thick. Eight layered 6LN + 0.4 wt% tapes produced using 10 wt% template particles resulted in 210 μm thick tapes with 67% orientation when sintered at 1150°C. Resulting properties include TC = 440ºC and TO-T = 70ºC, 25 kΩ resistance and capacitance 21.6 pF.

Potassium (K) fertility requirements for cotton ( Gossypium spp) have been a matter of concern due to increasing interest and emphasis on fiber quality and numerous reports of K deficiencies in various cotton producing regions. To address this matter appropriately, a thorough understanding of the chemical, physical, and mineralogical composition of the soils in question is in order. Soil samples were collected from ten sites across southern Arizona that are representative of the common agricultural soils of the region. At all locations soils were sampled to a depth of 120 cm in 30 cm increments. All soils were characterized with respect to chemical composition by the following parameters: exchangeable K, total K, cation exchange capacity and particle size analysis. With the exception of one soil (a soil not commonly employed in cotton production), none of the chemically characterized soils contained less than 150 mg K kg⁻¹ of extractable K in the surface 90 cm of soil. All of the soils contained K- bearing mica and none of the soils contained any K- fixing vermiculite. From the initial chemical and mineralogical information, K fertilization is not likely for similar situations in Arizona. Further research is under way to quantify the K-fixing ability of each soil in this survey and additional field studies are also being conducted to evaluate K fertilization in both Upland (G. hirsutum L.) and Pima (G. barbadense L.) cotton.

Monitoring of patients prescribed potassium supplements Zuzana Hovorkova Degree Project 30 hp, Pharmacotherapy Department of Pharmaceutical Biosciences/Division of Pharmacokinetics and Drug Therapy Supervisor: Linden Ashfield, Examiner: Margareta Hammarlund-Udenaes Introduction: In Northern Ireland, clinical technicians have been supporting pharmacist in their work in over five decades. From initially having mainly administrative tasks, the role of pharmacy technicians has progressed to more clinical activities. Following recent incidents caused by inadequate monitoring of potassium blood levels it was suggested pharmacy technicians could be utilized to improve patient care. Aim: Aim of this project was to ensure the appropriate monitoring and compliance with treatment guidelines for patients prescribed potassium supplements by extending the role of pharmacy technicians at Antrim Area Hospital. Methods: Training for extending the roles of clinical technicians was developed, following the structure of  a regional training programme. Literature search was made to find appropriate background about the subject of interest. Data about monitoring of potassium levels in the relevant patients was collected. Daily monitoring of these patients before and after the change was implemented were compared. Results: Clinical technicians contribution to monitoring of patients prescribed potassium replacement therapy improved guidelines compliance from 66.6 %  to 90 % (x2-test, p = 0.028). Percentage of successful treatment raised from 66 % to 89.7 % (x2-test, p = 0.028). Conclusion: By extending roles of the clinical technicians, better and appropriate monitoring of patients prescribed potassium supplements can be reached. In future, roles of the pharmacy technicians could be extended further, thus releasing  pharmacists time to expand their role, leading to a more cost- effective system and ensuring medicines optimization.

Before Pre-Paleozoic, the Earth's atmosphere had a composition different from today; in fact the organisms are not able to live in aerobic condition. The advent of cyanobacteria brought significant innovations, in fact, these bacteria unlike other phototrophs existing at that time, had chlorophyll molecules and protein complexes that allowed the use of water as electron donor to produce oxygen gas. This innovation developed over millions of years to get the current atmosphere. All these changes led to an inevitable biochemical and metabolic evolution of organisms. In Proterozoic or in early Cambrian, cyanobacteria began to reside within certain eukaryote cells. According to the endosymbionthic theory, chloroplasts evolved from a small primitive cyanobacterium settled within eukaryotic cells. Today, cyanobacteria are found throughout the Earth's environment, from oceans to fresh water and soil in the arctic areas, deserts and hot springs. Our attention is focused on cyanobacterium Synechocystis sp. PCC 6803. This strain was isolated for the first time from fresh water in California and now is considered a good model for scientific studies. It is spontaneously transformable, is able to integrate foreign DNA into its genome by homologous recombination (allowing targeted gene replacement) and can grow in the absence of photosynthesis if a suitable fixed-carbon source such as glucose is provided. Moreover it is the first photosynthetic organism for which the complete genome was sequenced (Kaneko et al., 1996). In the proteome of Synechocystis several putative ion channels can be identified (Kuo et al., 2005). However, none of them have been characterized from the functional point of view and their physiological role is still unknown. Ion channels are ubiquitous membrane proteins that control the passage of ions through biological membranes. These proteins, in all prokaryotes and eukaryotes, allow the correct ion distribution necessary to cellular functions. The basic features of the channels are selectivity and gating, the first is the property that controls the kind of ion that flows across the membrane and the second is the process of opening and closing the ion pathway. In fact the passage through the pore is governed by a "gate," which may be opened or closed by chemical, mechanical or electrical signals (Hille, 2001). Potassium (K+) is the most abundant cation in organisms and plays a crucial role in the survival and development of cells, by regulating enzyme activity and tuning membrane potential. This is one of the reasons for which potassium channels are one of the most studied among classes of channels. The field of prokaryotic potassium channels underwent a rapid development over the past years thanks to the application of a combination of bioinformatics and molecular biology, beside electrophysiology and structural studies. Understanding of their structure and actual mechanism of ion conduction allow to obtain more information about the function of potassium channels in general. A bioinformatic screening of Synechocystis sp. PCC 6803 proteome identified two putative proteins on which we focused our attention. The first one was named SynK and it displays sequence homology with KvAP (a voltage gated potassium channel of A. pernix) (Jiang et al., 2003). The second one, SynCaK, displays sequence homology to MthK, a Ca2+-dependent potassium channel from M. thermoautotrophicum (Jiang et al., 2002). Our goal was to understand whether they actually function as ion channels and to reveal their roles in the physiology of cyanobacteria. The characteristics and function of these proteins were studied through an integrated approach involving molecular biology techniques, biochemistry, electrophysiology and microscopy. SynK was initially identified in the genome of Synechocystis sp. PCC 6803 using the selectivity filter amino acid sequence (TMTTVGYGD) as a query sequence. This protein of unknown function shows six membrane spanning segments (S1-S6) and a pore region between S5 and S6 helixes. Before starting my Ph.D, SynK gene has been cloned and expressed in mammalian cells (Chinese Hamster Ovary, CHO) in fusion with EGFP (a fluorescent protein). Subsequent Western-blotting analysis showed that the fusion protein was correctly expressed. Confocal microscopy studies demonstrated its membrane localization and patch-clamp analysis revealed an activity of voltage-gated outwardly rectifying potassium selective channel in CHO cells. In addition, the double location of SynK in plasma and thylakoid membrane of cyanobacteria was shown by immunogold electron microscopy and Western blot on isolated membrane fractions. During my P.h.D, I performed the construction of two different mutants of SynK channel. The first SynK mutant, corresponding to the protein with a single amminoacid mutation in the selectivity filter of the pore (mutation Y181A), was used for expression in CHO cells. In accordance to the literature, this mutant protein loses its potassium channel activity. I also produced a ∆SynK Synechocystis mutant strain. Its functional analysis allowed to understand the physiological role of SynK in cyanobacteria. In order to characterize the function of the SynK protein, we initially verified that the deletion mutant did not express Synk, using Western blot technique. To evaluate the physiological role of the SynK protein, we initially compared growth of the wild type (WT) and mutant strain in different conditions. Characterization of the mutant phenotype was investigated by comparing photosynthetic activity in WT and mutant strains. Using a similar approach we have identified in the genome of Synechocystis sp. PCC 6803 a second protein classified as a putative potassium channel that displays sequence homology with MthK, a calcium dependent potassium channel from the archeon Methanobacterium thermoautophicum. Using several structural prediction programs, we analyzed the primary sequence of the protein translated from sll0993 and we observed that this protein (that we called SynCaK), like MthK, is predicted to contain two membrane spanning segments, a recognizable K+ channel signature sequence, with only conservative substitutions, and a regulatory sequence for K+ conductance (RCK). Also in the case of sll0993, we cloned and expressed the protein in fusion with GFP in CHO cells and studied their activity by patch clamp. Moreover, in order to study the role of SynCaK in cyanobacteria physiology we produced a SynCaK-deficient Synechocystis mutant. To gain further information about the activity of the channel, we have expressed and started the purification of the protein in another heterologous system, E. coli. Purified recombinant channel proteins are often studied by incorporating them into an artificial planar bilayer system (Ruta et al., 2003). During my Ph.D, I also continued the work begun during my thesis in Biotecnology on the study of ion channels in mitochondria of Graminaceae. Classical bioenergetics techniques reveal activities compatible with the presence of a potassium channel in durum wheat mitochondria, but the study of channels in mitochondria of plant systems is a still unexplored field in the world. To this end, we started a study through the parallel use of different techniques, which allowed a more complete characterization of the activity of channels present in wheat mitochondria. In particular, we followed two approaches. First, biochemical studies on isolated mitochondria, through the use of SDS-PAGE and immunoblotting, allowed the evaluation of the sample used in terms of enrichment and purity (data completely absent in the literature to date). Second, preparations of mitochondria from roots of durum wheat were suitable for electrophysiological studies in particular patch clamp technique, applied for the first time on plant mitochondria. Finally, I was involved in collaboration with the laboratory of Professor Nobuyuki Uozumi at Tohoku University in Japan. This group obtained a mutant for Synechocystis aquaporin. Aquaporins are membrane proteins embedded in the cell membrane that regulate the flow of water. I contributed to the characterization of the acquaporin-less mutant by performing experiments measuring photosynthetic activity. In particular, we performed several experiments of oxygen evolution demonstrating that the photosyntetic efficiency is higher in the mutant with respect to the WT when the organisms are incubated in hyperosmotic medium. The next step is to clarify how exactly a hyperosmotic stress and the absence of aquaporin are correlated with the photosynthesis and what is the underlying mechanism.
Prima del Pre-Paleozoico, l'atmosfera terrestre aveva una composizione diversa rispetto a quella quella di oggi, infatti, gli organismi non erano in grado di vivere in condizioni aerobiche. L'avvento dei cianobatteri ha portato rilevanti innovazioni, infatti, questi a differenza di altri batteri fototrofi esistenti a quel tempo, presentavano molecole di clorofilla e complessi proteici che permisero di utilizzare l’acqua come donatore di elettroni per la produzione dell’ossigeno. Questa modificazione ha permesso, lungo milioni di anni, di ottenere l'attuale atmosfera. Tutti questi cambiamenti portarono ad una inevitabile evoluzione biochimica e metabolica degli organismi. Nel Proterozoico o agli inizi del Cambriano, i cianobatteri iniziarono a risiedere all'interno di alcune cellule eucariotiche. Secondo la teoria endosimbiotica, i cloroplasti evolsero da un piccolo cianobatterio primitivo presente all'interno delle cellule eucariotiche. Oggi, i cianobatteri si trovano in diversi ambienti terrestri, da oceani ad acque dolce, in terre artiche, in deserti ed in sorgenti termali. La nostra attenzione è focalizzata sul cianobatterio Synechocystis sp. PCC 6803. Questo ceppo è stato isolato per la prima volta da una sorgente di acqua dolce in California e ora è considerato un buon organismo modello per studi scientifici. È spontaneamente trasformabile, è in grado di integrare DNA estraneo nel suo genoma attraverso ricombinazione omologa (consentendo la sostituzione mirata dei geni) e può crescere in assenza di fotosintesi se viene fornita un'adeguata fonte di carbonio, come il glucosio. Inoltre è il primo organismo fotosintetico per il quale il genoma è stato sequenziato (Kaneko et al., 1996). Nel proteoma di Synechocystis sono stati identificati diversi putativi canali ionici (Kuo et al., 2005). Tuttavia, nessuno di essi è stato caratterizzato da un punto di vista funzionale e il loro ruolo fisiologico rimane ancora sconosciuto. I canali ionici sono proteine di membrana che controllano il passaggio degli ioni attraverso esse. Queste proteine, in tutti i procarioti e gli eucarioti, permettono la corretta distribuzione ionica necessaria per le funzioni cellulari. Le caratteristiche base dei canali sono la selettività ed il gating, la prima è la proprietà che controlla il tipo di ioni che attraversa la membrana, la seconda è il processo di apertura e chiusura del percorso degli ioni. In realtà il passaggio attraverso il poro è regolato da un gate, che può essere aperto o chiuso da segnali chimici, meccanici o elettrici (Hille, 2001). Il potassio (K+) è il catione più abbondante negli organismi viventi e svolge un ruolo cruciale per la sopravvivenza e lo sviluppo delle cellule, regolando l'attività enzimatica e il potenziale di membrana. Questo è uno dei motivi per i quali i canali del potassio sono una delle classi di canali più studiate. Il campo dei canali del potassio procariotici ha subito un rapido sviluppo negli ultimi anni grazie all'applicazione di una combinazione di tecniche di bioinformatica e biologia molecolare, affiancate a studi di elettrofisiologia e studi strutturali. La comprensione della loro struttura e del meccanismo di conduzione degli ioni permette di ottenere ulteriori informazioni sulla funzione dei canali di potassio in generale. Uno screening bioinformatico del proteoma di Synechocystis sp. PCC 6803 ha individuato due proteine putative su cui abbiamo concentrato la nostra attenzione. La prima è stata chiamata SynK e mostra omologia di sequenza con KvAP (un canale del potassio voltaggio di A. pernix) (Jiang et al., 2003). La seconda, SynCaK, mostra omologia di sequenza con MthK, un canale del potassio Ca2 +-dipendente di M. thermoautotrophicum (Jiang et al., 2002). Il nostro obiettivo era quello di capire se effettivamente queste proteine funzionano come canali ionici e di comprendere il loro ruolo nella fisiologia dei cianobatteri. Le caratteristiche e la funzione di queste proteine sono state studiate attraverso un approccio integrato comprendente tecniche di biologia molecolare, biochimica, elettrofisiologia e microscopia. Il gene SynK è stato inizialmente identificato nel genoma di Synechocystis sp. PCC 6803 utilizzando la sequenza amminoacidica del filtro di selettività (TMTTVGYGD) come sequenza query. Questa proteina di funzione sconosciuta mostra sei segmenti transmembrana (S1-S6) ed una regione del poro tra le eliche S5 e S6. Prima di iniziare il mio Dottorato, SynK è stato clonato ed espresso in cellule di mammifero (Chinese Hamster ovary, CHO) in fusione con la EGFP (una proteina fluorescente). Una successiva analisi western-blotting ha dimostrato che la proteina di fusione è correttamente espressa. Studi di microscopia confocale hanno dimostrato la sua localizzazione nella membrana di cellule CHO e l'analisi patch-clamp ha rivelato un'attività di canale outwardly rectifying selettivo per il potassio. Inoltre, è stata dimostrata per SynK, in frazioni di membrana isolate da cianobatteri, mediante microscopia elettronica (attraverso la tecnica dell’immunogold) e tecniche di western blot, una doppia localizzazione nella plasmamembrana e nelle membrane tilacoidi.Durante il mio Dottorato, è stata eseguita la costruzione di due diversi mutanti del canale SynK. Il primo mutante corrisponde alla proteina con una mutazione puntiforme nel filtro di selettività del poro (mutazione Y181A) e utilizzato per l'espressione in cellule CHO. In base alla letteratura, questa proteina mutante perde la sua attività di canale del potassio. Inoltre, è stato prodotto un ceppo mutante knock-out (ΔSynK) in Synechocystis. La sua analisi funzionale ha permesso di capire il ruolo fisiologico di SynK nei cianobatteri. Al fine di caratterizzare la funzione della proteina SynK, abbiamo inizialmente verificato, attraverso western blot, che il ceppo mutante effettivamente non esprimesse la proteina. Mentre per valutare il ruolo fisiologico della proteina SynK, abbiamo confrontato la crescita del ceppo wild-type (WT) e mutante in diverse condizioni. La caratterizzazione del fenotipo mutante è stata studiata confrontando l’attività fotosintetica nel WT e nel mutante. Utilizzando un approccio simile abbiamo identificato nel genoma di Synechocystis sp. PCC 6803 una seconda proteina classificata come putativo canale del potassio che mostra omologia di sequenza con MthK, un canale del potassio calcio dipendente di Methanobacterium thermoautophicum. Attraverso l’utilizzo di vari programmi di predizione strutturale, abbiamo analizzato la sequenza primaria della proteina tradotta e abbiamo osservato che questa (che abbiamo chiamato SynCaK), come MthK, contiene due segmenti transmembrana, un filtro di selettività tipico dei canali del K+, con sostituzioni conservative, e un dominio di regolazione della conduttanza del potassio (RCK domain). Anche in questo caso, abbiamo clonato ed espresso la proteina in fusione con EGFP in cellule CHO e studiato la loro attività tramite patch clamp. Inoltre, al fine di studiare il ruolo di SynCaK nella fisiologia dei cianobatteri abbiamo prodotto un mutante knock-out per SynCaK. Per ottenere ulteriori informazioni sull’attività del canale, abbiamo espresso e iniziato la purificazione della proteina in un altro sistema eterologo, E. coli. Le proteine canale-ricombinanti sono spesso studiate mediante la loro integrazione in doppi strati artificiali (Ruta et al., 2003). Durante il mio Dottorato, ho anche continuato il lavoro iniziato durante la mia tesi di laurea in Biotecnologie Industriali sullo studio dei canali ionici nei mitocondri delle Graminaceae. Tecniche classiche di bioenergetica hanno rivelato attività compatibili con la presenza di un canale di potassio nei mitocondri di grano duro, ma lo studio dei canali nei mitocondri di sistemi vegetali è un campo ancora inesplorato nel mondo. A tal fine, è stato iniziato uno studio attraverso l'utilizzo parallelo di diverse tecniche, che hanno consentito una caratterizzazione più completa delle attività dei canali presenti nei mitocondri di grano. In particolare, sono stati seguiti due approcci. In primo luogo, studi biochimici sui mitocondri isolati, attraverso l'uso di SDS-PAGE e immunoblotting, che hanno permesso la valutazione del campione utilizzato in termini di arricchimento e di purezza (dati del tutto assenti in letteratura fino ad oggi). In secondo luogo, sono state definite preparazioni di mitocondri da radici di grano duro adatte per studi elettrofisiologici. In particolare, per la prima volta è stata applicata la tecnica di patch clamp su mitocondri vegetali. Infine, ho svolto una collaborazione con il laboratorio del Professor Nobuyuki Uozumi presso la Tohoku University in Giappone. Questo gruppo ha ottenuto un mutante per l’acquaporina di Synechocystis. Le acquaporine sono proteine di membrana incorporate nelle membrane cellulari che regolano il flusso dell'acqua. Ho contribuito alla caratterizzazione del mutant-less acquaporin attraverso esperimenti di misura dell'attività fotosintetica. In particolare, sono stati eseguiti diversi esperimenti di evoluzione di ossigeno che dimostrano che l'efficienza fotosintetica è più alta nel mutante rispetto al WT quando gli organismi vengono incubati in un mezzo iperosmotico. Il passo successivo sarà quello di chiarire esattamente come uno stress iperosmotico e l'assenza di acquaporina sono correlati con la fotosintesi e quindi il meccanismo sottostante.

Le potassium (K), le calcium (Ca) et le magnésium (Mg) sont en compétition pour saturer la CEC du sol. Le lessivage du K a été peu documenté. Cette étude porte sur l'effet de la fertilisation potassique sur le lessivage du K en lien avec celui du Ca et du Mg sous culture de pomme de terre en sols sableux. De 2012 à 2014, deux sites par année étaient établis sur des sols de texture sable loameux près de la ville de Québec. Les séries de sols étaient Morin, Orléans et Saint-Nicolas. Chaque site comprenait huit traitements potassiques constitués d’une combinaison de doses et de sources de KCl et de Sul-Po-Mag répétées trois fois. En 2012, le K fut appliqué à quatre doses (0, 70, 140 et 210 kg K2O ha-1). En 2013 et 2014, une dose de plus de K fut ajoutée (105 kg K2O ha-1) ainsi que trois doses de calcium (15, 125, 250 kg Ca ha-1) appliquées sous forme de CaSO4. L’échantillonnage de la solution du sol dans des lysimètres à succion a eu lieu toutes les deux semaines de la plantation à la récolte. Les concentrations en K (SSK), Ca (SSCa) et Mg (SSMg) furent dosées dans la solution du sol. De 2012 à 2014, l’ajout de fertilisants potassiques n’a pas influencé significativement la SSK sauf à des dates précises. La perte du K était plus élevée au début de la saison de culture, ensuite elle diminuait progressivement mais augmentait à la sénescence de la plante. Pour les sites en 2013 et 2014, la SSCa et la SSMg, étaient significativement influencées par les traitements de K et la date d'échantillonnage. La dose de K n’a généralement pas eu d’impact sur le lessivage du K en sol sableux mais a augmenté le lessivage de SSCa et de SSMg.
Quantification of nutrient movement from agricultural soils is important for both fertilizer and environmental management. This study evaluated the impact of potassium (K) fertilization on K leaching in relation to calcium (Ca) and magnesium (Mg) leaching under potato crop in sandy soils. From 2012 to 2014, two sites per year were established on loamy-sand textured soils near Quebec City. Soil series were Morin, Orleans and Saint-Nicolas. Each site included eight K treatments as a combination of rates and sources of KCl and Sul-Po-Mag with three replications. In 2012, the K was applied at four rates (0, 70, 140 and 210 kg K2O ha-1). In 2013 and 2014, another K rate was added (105 kg K2O ha-1) as well as three rates of calcium (15, 125, 250 kg Ca ha-1) applied as CaSO4. The soil solution was sampled biweekly from planting to harvest using suction lysimeters and the concentrations of K (SSK), Ca (SSCa) and Mg (SSMg) in the soil solution were measured. From 2012 to 2014, adding K fertilizers did not significantly influence the SSK except on specific dates. K leaching was higher at the beginning of the growing season, then it gradually decreased but increased at the end of the growing season until plant reached senescence stage. For all the sites, SSCa and SSMg were significantly influenced by the K treatments and the sampling period. The rate of K generally had no impact on the leaching of K in sandy soils but increased SSCa and SSMg leaching.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Para avaliação do desempenho de três cultivares de alface, Amanda (grupo Crespa), Karla (grupo Lisa) e Lucy Brown (grupo Americana) foram realizados dois experimentos com aplicação de cinco doses de fósforo e cinco doses de potássio, em um Latossolo com teores altos dos nutrientes. O delineamento experimental foi de blocos casualizados, com quatro repetições, em esquema fatorial 3 x 5. As variáveis analisadas foram: os teores dos nutrientes nas folhas, o número de folhas, a área foliar, massa fresca e massa seca da parte aérea, e os teores de P e K no solo. Após a colheita da alface, nos dois experimentos a cv. Karla apresentou maior número de folhas. A adubação potássica não influenciou significativamente as características da alface, sendo recomendado apenas uma adubação de manutenção em Latossolos com teores altos de potássio disponível. A adubação fosfatada, exceto para o número de folhas, influenciou as características da alface com incrementos lineares na massa fresca e massa seca das plantas. Deve ser utilizada a aplicação de até 300 kg ha-1 de P2O5 mesmo em Latossolo com alto teor do nutriente
To evaluate the performance of three cultivars of lettuce, ‘Amanda’ (crisp group), ‘Karla’ (leaf group) and ‘Lucy Brown’ (crisphead group) were carried out two experiments with application of five doses of phosphorus and different doses of potassium in an Oxisol with levels high nutrients. The experimental design was a randomized block design with four replications in a 3 x 5. The variables were: the levels of nutrients in the leaves, leaf number, leaf area, fresh and dry weight of shoot, and the levels of P and K in the soil. After the harvest of lettuce, in both experiments cv. Karla had a higher number of leaves. Potassium fertilization did not influence significantly the characteristics of lettuce, and recommended only a maintenance fertilization in Oxisol with high levels of available potassium. The phosphorus fertilization, except for the number of leaves influenced the characteristics of lettuce with linear increments in fresh and dry weight of plants. Should be used to apply up to 300 kg ha-1 P2O5 same in an Oxisol with high content of nutrients

Background: Kv2.1 and TASK-1 channels are two main contributors of K+ currents in pulmonary artery smooth muscle cells (PASMC). Dysregulation of these channels has been implicated in the pathogenesis of pulmonary hypertension (PH). This thesis aims to delve deeper into the implications of the regulation of Kv2.1 by Kv9.3 in PH. Another subject of interest would be whether NADPH oxidase type 4 (Nox4), one of the major reactive oxygen species (ROS) producers in the PASMC, modulates Kv2.1, Kv9.3, and TASK-1 channels. The effects of several redox agents are also investigated as potential modulators of Kv2.1, Kv9.3, and TASK-1. In addition, this thesis also examined the effect of a Kv2-channel blocker, stromatoxin, on Kv2.1 and Kv9.3. Finally, since amphoterin-induced gene and open reading frame (AMIGO) proteins have recently been shown as novel Kv2.1-interacting partners, their effects on Kv2.1 and/or Kv9.3 are also explored in this study. Experimental approach: Whole-cell patch clamp electrophysiology was used to measure currents of the ion channels expressed in modified tsA-201 cells, in the absence and presence of Nox4 AMIGO and other regulatory molecules. Immunohistochemistry was deployed to visualize the distribution of Kv2.1 and Kv9.3 proteins in the rat lungs and hearts. Key results and Conclusions: This study supports the findings that Kv9.3 regulates Kv2.1 by increasing the current amplitude, shifting the activation threshold to a more negative voltage range, and prolonging the slow component of time constant of deactivation. These effects could be beneficial in PH as this would mean cells could be brought back to its resting membrane potential faster and the transduction of the next action potential can be delayed. Kv2.1 and Kv9.3 have also been detected at the endothelium and PASMC in rat lungs and hearts, further substantiating the claim that these channels are potential players in regulating PH. AMIGO1 and AMIGO2 proteins are confirmed as regulators of Kv2.1 and Kv9.3 proteins. Nox4 does not regulate Kv2.1, Kv9.3, and TASK-1 channels expressed in tsA-201 cells. While hydrogen peroxide (H2O2) does not have any effect on Kv2.1 and Kv9.3, it abolished the current reduction effect of AMIGO2 on Kv2.1/Kv9.3. Other redox agents used in this study such as dithiothreitol (DTT), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and chloramine T (Ch-T) are not modulators of these channels expressed in tsA-201 cells. The lack of effect from Nox4 and these redox agents could suggest that the redox regulation of different Nox subunit/Kv channels combination varies for different cell types due to the different regulatory proteins present in different heterologous expression systems. As with the case of H2O2 and AMIGO2, it is likely that the regulatory proteins, which could facilitate the hypoxia-sensing properties of Nox4 and the effects of the redox agents on the ion channels, are missing in our heterologous expression system, compared with other host cells.

L'optimisation de la production industrielle de KCl à partir de sel minier examine successivement : - 1) les aspects liés à la production de sels purs. - 2) les aspects liés aux adjuvants destinés à en éviter la prise en masse. - 3) les aspects liés à la diversification de la production, (assurance qualité, nouvelles formulations en fonction du marché. ). La première partie concerne l'optimisation de l'unité de production de sels purs. Après avoir développé des moyens de mesure appropriés, il a été possible de modéliser l'appareillage existant : un dissolveur, un cristalliseur et un filtre à bande. L'optimisation consécutive a permis une importante amélioration du cristalliseur en évitant la formation de plaques de sel sur les éléments réfrigérants Au niveau du filtre à bande, les bilans de matière ont permis de localiser les pertes de saumure, réduisant de 50% les pertes totales en sel. La deuxième partie traite des produits anti-mottants. Une étude critique des techniques analytiques existantes (spectrophotométrie infrarouge, ultraviolette, la résonance magnétique nucléaire, le dosage du carbone total) en ont permis de mettre au point une analyse fiable dans la matrice saline. Il a ainsi été possible de comprendre comment ils se fixent aux interfaces et de déterminer l'efficacité relative des agents destinés à les éliminer des sels où ils ne pouvaient être tolérés. Une troisième partie traite des travaux visant à assurer la diversification des produits : l'obtention d'un sel exempt de traces de vanadium a nécessité le développement d'une nouvelle technique analytique d'absorption atomique en four graphite avec prétraitement superficiel ; l'adaptation des produits aux normes de la pharmacopée européenne a fait l'objet d'une étude analytique spécifique ; enfin, l'élaboration d'un produit à usage alimentaire constitué d'un mélange quaternaire chlorure du potassium/glycine/chlorure de magnésium/ eau a été abordée. Son diagramme de phases partiel a été élaboré, ce qui a permis de préciser les domaines d'existence et les cinétiques d'évolution des états métastables des différentes phases susceptibles d'exister dans un produit commercial à température ambiante.

Crystallization of NH₄-struvite (MgNH₄PO4:6H₂O) pellets has proven to be a successful method of recovering nitrogen and phosphorus from wastewaters. Thus far, little work has been done on potassium recovery since it is not considered a water pollutant, nor do we face potassium shortages. However, potassium is an essential plant macronutrient and we are seeing worldwide imbalances in nutrient and fertilizer use, as well as a need for a slow release potassium fertilizer. Development of a full complement NPK fertilizer with NH₄-struvite and K-struvite components may have great potential. Given this, research into potassium recovery through crystallization of K-struvite (MgKPO₄: 6H₂O) is relevant and complements previous work done with NH₄-struvite. The goals of this research were to develop fundamental understanding of K-struvite formation as the first step to recovering potassium, and eventually produce a full complement NPK slow-release fertilizer from wastewaters. This required the determination of new solubility product values for K-struvite at different temperatures, followed by bench-scale experiments to assess K-struvite synthesis under various solution conditions. A model to simulate each batch experiment and to predict optimal supersaturation conditions for K-struvite precipitation was developed using PHREEQC, aqueous equilibrium modelling software. Finally, initial experiments in the UBC fluidized bed reactor (UBC-FBR) were undertaken to assess the pelletization potential of K-struvite. New solubility product values for K-struvite indicate that it is less soluble than previously reported, and the values determined at 10, 25 and 35°C fit the Van’t Hoff model. Optimal Mg:K:P molar ratio for synthesis of pure K-struvite was found to be approximately 3:50:1 in a wastewater matrix with pH 8, P-PO₄ concentration of 8 mM and a Mg:P ratio of 3:1. These concentrations were used in the UBC-FBR to assess the pelletization potential of K-struvite. These initial reactor runs were inconclusive due to an inability to stabilize the reactor without seeding. It would be recommended to seed the reactor during start-up in order to be able to compare process performance with the NH₄-struvite crystallization process in the UBC-FBR. This study showed that formation of pure K-struvite is possible given the right supersaturation conditions in solution, requiring high potassium concentrations.
Applied Science, Faculty of
Civil Engineering, Department of
Graduate

The processing of sensory signals is an important, yet complex task in which a system must extract behaviorally relevant stimulus patterns from a vast array of sensory cues. When a neuron within a major sensory area is presented with a stimulus, one of the important characteristics used to distinguish between types of input is frequency. Often sensory neurons are tuned to narrow stimulus frequency ranges and are thus charged with the processing of subtypes of sensory signals. The weakly electric fish Apteronotus lepthorhynchus senses it's environment through modulations of a self-generated electric field. Two main types of sensory signals can be distinguished based on their frequency patterns. Prey stimuli cause low frequency perturbations of the electric field, while communication signals often result in high frequency signals. Pyramidal neurons in the electrosensory lateral line lobe (ELL) encode the low frequency signals with bursts, while the high frequency signals are relayed with single spikes. This thesis describes how a pyramidal neuron's response patterns can be tuned to specific frequencies by the expression of distinct classes of potassium channels.
I have cloned 3 small conductance (SK) calcium activated potassium channels from cDNA libraries created from the brain of Apteronotus. I have subsequently localized the AptSK channels throughout the brain using both in situ hybridization (AptSK1, 2 & 3) and immunohistochemical (AptSK1 & 2) techniques. The 3 channels showed distinct expression patterns, with the AptSK1 & 2 channels showing a partially overlapping expression pattern, while AptSK3 appears to be expressed in unique areas of the brain. In the ELL AptSK1 & 2 show a partially overlapping expression pattern, appearing in similar pyramidal neurons. However, their distribution within individual cell is unique, with AptSK1 showing a dendritic localization, while AptSK2 is primarily somatic. We have demonstrated that the unique expression pattern of the somatic AptSK2 channel in the ELL coincides with the functional SK currents evaluated through in vitro electrophysiology. Further we have shown that neurons that encode low frequencies do not possess functional SK channels. It thus appears that the presence of the AptSK2 channel subtype can predispose a neuron to respond to specific types of sensory signals.
In an attempt to evaluate if second messengers could modify the AptSK control of frequency tuning I investigated the consequences of muscarinic acetylcholine receptor (mAChR) activation on a pyramidal neurons response patterns. While it had been shown in vivo that mAChR activation increased a pyramidal neuron's response to low frequencies, I have found that this was not due to a decrease in AptSK current, but rather appears to be the result of a down-regulation of an A-type potassium channel.
Taken together the studies that comprise this thesis show how the selective expression of a single potassium channel subtype can control a sensory neurons response to specific environmental cues. The secondary modulation of the A-type current highlights the potential for a second messenger to control a neuron's sensory response through the down-regulation of constitutively expressed potassium current.