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1
Stephen, John R. "pH regulation in enteric bacteria." Thesis, University of Aberdeen, 1997. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=130919.
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<I>Escherichia coli</I> mutants impaired in growth and survival at low external pH in minimal medium were selected and attempts made to identify the disrupted genes. This study suggested that <I>clpX</I>, encoding a heat-shock induced protease and molecular chaperone, was functional in survival of <I>E. coli </I>at pH 3.3. Promoter probe plasmid libraries of <I>Salmonella typhimurium </I>LT2 DNA were created in <I>E. coli </I>and screened for acid-inducible transcriptional elements, and transcriptionally active fragments of degradative amino-acid decarboxylase genes recovered. Chromosomal gene fusions to the reporter gene <I>lacZ </I>in <I>E. coli</I> generated by Mu DII 1734 insertion were screened in a similar way and suggested that the gene encoding adenylate cyclase (<I>cya</I>) could be induced by mild cytoplasmic acidification. The sequence of a gene known to be inducible by cytoplasmic acidification, <I>inaA, </I>became available during the course of this study. The 5' region of this gene was used to generate a set of plasmids carrying fragments of the acid-inducible promoter transcriptionally fused to a luciferase based reporter system. Elements of the sequence required for induction by cytoplasmic acidification were identified. One of these reporter constructs was used to screen an <I>E. coli </I>Tn<I>10</I> chromosomal insertional mutant library for genes involved in the regulation of <I>inaA. </I>One such mutant had a multiple antibiotic resistant (<I>mar</I>) phenotype. The disrupted loci in 2 other mutants were identified by inverse PCR, sequence analysis and database searches. Both were known only as open reading frames (ORFs) discovered during the sequencing of the entire <I>E. coli</I> genome, and were tentatively identified as <I>yddB </I>(closely linked to <I>gadB </I>and <I>gadC</I>; required for glutamate dependent acid resistance) and <I>f300</I> (closely linked to <I>pldA; </I>required for detergent resistance). The promoter of <I>f300</I> was shown to be sensitive to cytoplasmic acidification. The <I>inaA</I> promoter was also demonstrated to be induced at the onset of stationary phase, and to be independent of the stationary phase and weak-acid inducible σ factor RpoS and also of cAMP levels.
2
Witzel, Dirk. "Pharmakologische Beeinflussung der pH-Regulation kardialer Fibroblasten." [S.l.] : [s.n.], 2003. http://archiv.ub.uni-marburg.de/diss/z2003/0424/.
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3
Rudnicka, Joanna Dorota. "Aspects of pH regulation in Aspergillus nidulans." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415560.
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4
Huertas, Tatiana Munera. "Aspects of pH regulation in Aspergillus nidulans." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526381.
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5
Rivinoja, A. (Antti). "Golgi pH and glycosylation." Doctoral thesis, University of Oulu, 2009. http://urn.fi/urn:isbn:9789514292699.
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Abstract
Glycans, as a part of glycoproteins, glycolipids and other glycoconjugates, are involved in many vital intra- and inter-cellular tasks, such as protein folding and sorting, protein quality
control, vesicular trafficking, cell signalling, immunological defence, cell motility and adhesion. Therefore, their correct construction is crucial for the normal functioning of eukaryotic cells and
organisms they form. Most cellular glycans are constructed in the Golgi, and abnormalities in their structure may derive, for instance, from alkalinization of the Golgi lumen.
In this work we show that Golgi pH is generally higher and more variable in abnormally glycosylating, i.e. strongly T-antigen (Gal-β1,3-GalNAc-ser/thr) expressing cancer cells, than in
non-T-antigen expressing cells. We also confirmed that the Golgi pH alterations detected in cancer cells have the potential to induce glycosylation changes. A mere 0.2 pH unit increase in Golgi pH is
able to induce T-antigen expression and inhibit terminal N-glycosylation in normally glycosylating cells. The mechanism of inhibition involves mislocalization of the corresponding
glycosyltransferases.
We also studied potential factors that can promote Golgi pH misregulation in health and disease, and found that cultured cancer cells, despite variation and elevation in Golgi pH, are fully
capable of acidifying the Golgi lumen under the normal Golgi pH. Moreover, we introduce a Golgi localized Cl-/HCO3- exchanger, AE2a, that participates in Golgi pH regulation by
altering luminal bicarbonate concentration and thus also buffering capacity. Participation of AE2a in Golgi pH regulation is especially intriguing, because it also provides a novel mechanism for
expelling protons from the Golgi lumen.
6
Furimsky, Marosh. "Intracellular pH regulation in hepatocytes of teleost fish." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ58273.pdf.
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7
Grace, Andrew Ashley. "The regulation of intracellular pH and cardiac contraction." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319389.
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8
Taylor, Caroline Joanne. "Intracellular pH regulation in rat brain endothelial cells." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616055.
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9
Lundin, Karl, and Oscar Olli. "Automated hydroponics greenhouse : Regulation of pH and nutrients." Thesis, KTH, Maskinkonstruktion (Inst.), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-226662.
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The purpose of this project is to create a fully automatedgreenhouse that can produce year-round crops, using sensorsand actuators. Temperature in both water and air,relative humidity, water level, nutrient level and pH are allmeasured with different sensors. Though only water level,pH and nutrients will be regulated. The greenhouse will berelying on a hydroponic growing technique, meaning thatthe growing is soil-less and will be done in water. Thismakes measuring and controlling said levels easier and alsominimizes water waste and makes for a more environmentalsystem. The main focus of this project is on regulating pHand nutrient levels of the water. The system has shown tobe stable and self regulating within the desired intervals fornutrient concentration and pH for growing basil.<br>Syftet med det här projektet är att skapa ett automatiserat växthus som kan producera grödor året runt med hjälp av sensorer och aktuatorer. Med olika sensorer mäts temperaturen i både vattnet och luften, relativa luftfuktigheten, vattennivå, pH- och näringsvärden. Dock kommer endast vattennivå, pH- och näringsvärden regleras. Växthuset använder sig av så kallad hydroponisk odling, vilket innebär att odlingen inte sker i jord utan i vatten. Detta underlättar bland annat mätningar och kontrollering av systemet men minimerar även vattenkonsumptionen och bidrar till ett mera miljövänligt system. Projektet kommer i huvudsak inrikta sig på reglering av pH och näringsnivåer av vattnet. Systemet har visats stabilt och har förmågan att reglera sig självt inom önskat intervall för näringskoncentration och pH för att odla basilika.
10
Müller, Matthias. "pH-Regulation und Glukosestoffwechsel - Wirkung von Glut1-Überexpression, Serum und Dexamethason auf den zytosolischen pH." [S.l. : s.n.], 2003. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10733068.
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11
Warner, Christian. "Die pH-Wert-Regulation in kultivierten MCF-7-Mammakarzinomzellen." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=967457246.
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12
Shartau, Ryan Brady. "Vertebrate preferential intracellular pH regulation during severe acute hypercarbia." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/63848.
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Environmental CO2 tensions reach >8 kPa (ca. 79,000 μatm; hypercarbia) in some habitats and create severe acid-base challenges for vertebrates. Typically, during a hypercarbic-induced respiratory acidosis, changes in blood pH are compensated for, which returns pH to its normal value, and this is coupled to tissue pH (pHi) regulation. However, during acute environmental CO₂ exposure, this process may be limited to <2 kPa PCO₂. Some fishes fully protect tissue pH (pHi) (preferential pHi regulation) despite large sustained reductions of pHe (>1 pH unit) and can tolerate PCO₂ >3 kPa. I hypothesized that preferential pHi regulation is used by adult fishes and embryonic amniotes during severe acute acid-base disturbances. This was investigated by examining (1) whether preferential pHi regulation is a general response to various types of acid-base disturbances, (2) surveying fishes for the presence or absence of preferential pHi regulation, and (3) whether preferential pHi regulation is used during development in reptiles.
Using white sturgeon, I found that preferential pHi regulation is not a general response to both respiratory and metabolic acidoses. Despite a robust capacity for preferential pHi regulation during respiratory acidoses, not all tissues were protected during metabolic acidoses to the same degree. Preferential pHi regulation was observed to be a common pattern of acid-base regulation amongst fishes in response to severe acute hypercarbia. A total of 20 species, ranging from basal (“primitive”) to derived, were examined and 18 were observed to use preferential pHi regulation. Finally, developing amniotes (snapping turtle and American alligator) used preferential pHi regulation during severe acute respiratory acidosis, but the capacity for pHi regulation was progressively reduced throughout development.
This thesis demonstrates that preferential pHi regulation is likely a common strategy of acid-base regulation occurring in response to severe acute hypercarbia in adult fishes and possibly amniotes. I propose that preferential pHi regulation is an embryonic vertebrate strategy, that has been retained or lost in adults depending on the environmental acid-base challenges they face.<br>Science, Faculty of<br>Zoology, Department of<br>Graduate
13
Sarkar, Sovan. "Aspects of pH regulation in the fungus Aspergillus nidulans." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321614.
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14
Chen, Jeng-Haur. "The molecular mechanisms of CFTR regulation by intracellular pH." Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422608.
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15
Nicola, Pieris Andrew. "Regulation of intracellular pH in rat brain endothelial cells." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612803.
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16
Schewe, Bettina. "Räumliche und zeitliche Aspekte der intrazellulären pH-Regulation in Epithelien." Phd thesis, Universität Potsdam, 2008. http://opus.kobv.de/ubp/volltexte/2008/2687/.
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Die Speicheldrüsen der Schmeißfliege Calliphora vicina produzieren bei Stimulierung mit dem Neurohormon Serotonin (5-Hydroxytryptamine, 5-HT) einen KCl-reichen Primärspeichel. Der transepitheliale K+-Transport wird durch eine apikal lokalisierte vakuoläre H+-ATPase (V-ATPase) energetisiert. Stimulierung der Speicheldrüsen mit 5-HT aktiviert die apikale V-ATPase, die Protonen aus der Zelle in das Drüsenlumen transportiert. Trotz des auswärts gerichteten Protonentransportes führt die 5-HT-Stimulierung kurioserweise zu einer intrazellulären Ansäuerung. Die Ursachen dieser 5-HT-induzierten Ansäuerung waren unzureichend untersucht. Deshalb war das Ziel dieser Arbeit die Identifikation aller Transporter, die an der intrazellulären pH-(pHi)-Regulation in unstimulierten Speicheldrüsen von Calliphora vicina beteiligt sind und an der Entstehung und Regulation der 5-HT-induzierten pHi-Änderungen mitwirken. Von besonderem Interesse war hierbei die funktionelle Mitwirkung der V-ATPase, deren Beteiligung an der pHi-Regulation in tierischen Zellen bisher wenig untersucht war.
Wesentliche Ergebnisse dieser Arbeit waren:
• Messungen des pHi-Wertes in der unstimulierten Drüse zeigten, dass vor allem
die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter an der Aufrechterhaltung des Ruhe-pHi beteiligt sind.
• Zur Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung
(NH4Cl-Vorpuls) tragen ebenfalls im Wesentlichen die V-ATPase und mindestens
ein Na+-abhängiger HCO3--Transporter bei. Der Na+/H+-Antiporter hat in der
unstimulierten Drüse keinen messbaren Einfluss auf den Ruhe-pHi.
• Die Wiederherstellung des Ruhe-pHi nach einer intrazellulären Alkalisierung
(Na-acetat-Vorpuls) ist Cl--abhängig, aber auch unter extremen Bedingungen
waren die Zellen noch in der Lage sich vollständig von einer intrazellullären
Alkalisierung zu erholen. Einen entscheidenden Anteil daran hat offenbar die hohe intrazelluläre Puerkapazität.
• Ein Na+-abhängiger Glutamat-Transporter ist per se kein pHi-regulierender
Transporter, seine Aktivität hat jedoch Einfluss auf den Ruhe-pHi in der
unstimulierten Speicheldrüse von Calliphora vicina.
• 10 nM 5-HT induzieren in den Calliphora Speicheldrüsen eine intrazelluläre
Ansäuerung. An dieser Ansäuerung ist der Na+/H+-Antiporter entscheidend
beteiligt. Auch eine klare Cl--Abhängigkeit der 5-HT-induzierten Ansäuerung konnte beobachtet werden. Wahrscheinlich ist eine gekoppelte Aktivität von Na+/H+-Antiporter und Cl-/HCO3--Antiporter.
• Messungen mit einem O2-empndlichen Fluoreszenzfarbstoff zeigten, dass Stimulierung der Speicheldrüsen mit 5-HT die Zellatmung aktivierte. Der cAMP- und der IP3/Ca2+-Weg tragen auf komplexe Weise zu der 5-HT-induzierten Aktivierung der Zellatmung und damit auch zu den 5-HT-induzierten pHi-Änderungen bei.
• Mit molekularbiologischen Untersuchungen ist es gelungen den Na+-abhängigen
Glutamat-Transporter, den Na+/H+-Antiporter, die Carboanhydrase und die
Untereinheit C der V-ATPase in den Calliphora Speicheldrüsen direkt
nachzuweisen. Zudem konnte erstmals der direkte Nachweis für die Expression
eines nH+/K+-Antiporters in den Speicheldrüsen von Calliphora vicina erbracht
werden.
Diese Arbeit trug ganz wesentlich zum Verständnis der pHi-Regulation in der
unstimulierten und stimulierten Speicheldrüse von Calliphora vicina bei. Mechanismen die zur Aufrechterhaltung und Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung bzw. Alkalisierung beitragen, konnten mit pHi-Messungen und auch molekularbiologisch nachgewiesen werden. Die Mechanismen, welche die 5-HT-induzierte intrazelluläre Ansäuerung verursachen, konnten ebenfalls aufgeklärt werden. Zudem wurde an den Calliphora Speicheldrüsen eine neue optische Methode zur Messung des O2-Verbrauchs in tierischen Geweben etabliert.<br>The tubular salivary glands of the blowfly Calliphora vicina consist of a single layer of epithelial cells. Stimulation with the neurohormone serotonin (5-hydroxytryptamine,5-HT) induces the secretion of a KCl-rich primary saliva. Transepithelial K+-transport is energized by a vacuolar-type H+-ATPase (V-ATPase) which is located in the apical membrane. 5-HT stimulates the apical V-ATPase which transports protons out of the cells into the lumen of the glands. Despite this outward directed proton transport, 5-HT stimulation leads to an intracellular acidication. The causes of this intracellular acidication were poorly understood. Therefore the aim of this thesis was the identication of all pHi regulating transporters which are involved in pHi regulation
in the unstimulated salivary glands of Calliphora vicina and which contribute to the 5-HT-induced pHi changes. Of special interest was the functional role of the V-ATPase,whose contribution to pHi regulation in animal cells is, as yet, not well studied.
Key results were:
• pHi measurements in unstimulated glands showed that mainly the V-ATPase and
at least one Na+-dependent HCO3--transporter are involved in maintenance of
resting pHi.
• V-ATPase and at least one Na+-dependent HCO3--transporter are also necessary
for the recovery from an intracellular acidication (NH4Cl prepulse).
• Recovery from an intracellular alkali load (Na-acetate prepulse) is partially
Cl--dependent.
• A Na+ dependent gluatamate-transporter is present in Calliphora salivary glands and its activity aects the resting pHi.
• 10 nM 5-HT induce an intracellular acidication. This acidication is
Na+-dependent, EIPA-sensitive and also Cl--dependent. No DIDS-sensitivity
was observed. A coupled activity of a Na+/H+-antiporter and a Cl-/HCO3-
-antiporter was suggested.
• Using O2-sensitive fluorescent microbeads I could show that 5-HT stimulation
of the Calliphora salivary glands activates cellular respiration. The cAMP and Ca2+-signalling pathways contribute in a complex manner to the 5-HT-induced activation of cellular respiration and consequently, also to the 5-HT-induced
intracellular acidication.
• The expression of a Na+ dependent glutamate-transporter, a Na+/H+-antiporter,
a carbonic anhydrase, subunit C of the V-ATPase and a nH+/K+-antiporter were
determined on mRNA level by RT-PCR.
This thesis contributes signicantly to the understanding of pHi regulation in
unstimulated and stimulated salivary glands of Calliphora vicina. Mechanisms which contribute to the maintenance and recovery of resting pHi were identied by
using pHi measurements and molecular biological techniques. Mechanisms which
are responsible for the 5-HT-induced intracellular acidication were also clarified.
Furthermore a new optical method for measuring O2 consumption in animals cells
was established by using the Calliphora salivary glands as a model.
17
Hasselblatt, Peter Werner. "Die pH-Regulation isoliert in-vitro-perfundierter Kolonkrypten der Ratte." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961011122.
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18
Turk, Boris. "Papain-like cysteine proteinases : regulation by proteinase inhibitors and pH /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1996. http://epsilon.slu.se/avh/1996/91-576-5227-9.gif.
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19
Maddox, Angela D. M. "Mechanisms of intracellular pH regulation in ciliated tracheal epithelial cells." Thesis, University of Aberdeen, 1999. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU112227.
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Regulation of intracellular pH (pH<sub>i</sub>) was studied in ciliated epithelial cells isolated from sheep tracheae. Microspectrofluorimetry of the pH -sensitive dye 2'; 7' - biscarboxyethyl -5(6) -carboxyfluorescein (BCECF) was used to measure pH<sub>i</sub> in single cells plated on coverslips and mounted in a temperature regulated superfusion chamber on a microscope stage. In both HCO<sub>3</sub><sup>-</sup>containing and HCO<sub>3</sub><sup>-</sup> -free conditions, pH<sub>i</sub> was ~ 7.20 and buffering capacity was found to be inversely related to pH<sub>i</sub>. Recovery from an acid load (induced by NH<sub>4</sub>Cl prepulse) was Na<sup>+</sup>-dependent. In HCO<sub>3</sub><sup>-</sup> -free conditions, inhibition of pH<sub>i</sub> recovery by hexamethylene amiloride (HMA) identified Na<sup>+</sup>/H<sup>+</sup> exchange as the principal mechanism of acid extrusion. In contrast, in HCO<sub>3</sub><sup>- </sup>- role of Na<sup>+</sup>/H<sup>+</sup> exchange in pH<sub>i</sub> regulation under more physiological conditions. However, dihydro- 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (H<sub>2</sub>DIDS) affected this Na<sup>+</sup> and HCO<sub>3</sub><sup>-</sup> -dependent pH<sub>i</sub> recovery and suggested that Na<sup>+</sup>-dependent recovery from intracellular acidification in HCO<sub>3</sub><sup>-</sup>- containing conditions was mediated primarily by an H<sub>2</sub>DIDS-sensitive component. Inhibition of pH<sub>i</sub> recovery by bafilomycin A<sub>1</sub> in HCO<sub>3</sub><sup>-</sup> -containing conditions also suggested that a proton pump was present in these cells. The failure of inwardly and outwardly - directed Cl<sup>-</sup> gradients to affect pH<sub>i</sub> recovery suggested that Cl<sup>-</sup> -dependent mechanisms were not present. The effects of HMA and H<sub>2</sub>DIDS on Na<sup>2+</sup> -dependent alkalinisation in cells preincubated in Na<sup>+ </sup>-free media were also investigated. Surprisingly, only HMA affected the rate of Na<sup>+</sup> -dependent alkalinisation. This directly contradicted the data reported above and implied that Na<sup>+</sup>/H<sup>+</sup> exchange was the primary pH<sub>i</sub> regulatory mechanism in HCO<sub>3</sub><sup>-</sup> - containing conditions in these cells. In conclusion, pH<sub>i </sub>in isolated sheep ciliated tracheal cells appears to be regulated by three acid-extruding mechanisms.
20
Milner, Peter Ian. "Physiology and pathology of intracellular pH regulation in equine articular chondrocytes." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614332.
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21
Meyer, Johanna Charlotte [Verfasser]. "Regulation of µ-opioid receptor function by pH / Johanna Charlotte Meyer." Berlin : Freie Universität Berlin, 2020. http://d-nb.info/1215099681/34.
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22
Meehan, James. "Inhibition of pH regulation as a therapeutic strategy in breast cancer." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28875.
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The abnormal regulation of H+ ions, leading to a reversed pH gradient in cancer cells when compared to normal cells, is considered to be one of the most distinctive features of cancer. However, this characteristic has yet to be fully exploited as a therapeutic target in cancer. This project assessed whether targeting pH regulating proteins, which permit cancer cells to survive in the hostile hypoxic and acidic tumour microenvironment, could produce an effective therapeutic response in breast cancer. The pH regulating proteins carbonic anhydrase IX (CAIX), Na+/H+ exchanger 1 (NHE1) and vacuolar (H+)-ATPase (V-ATPase) were the focus of this thesis. Initial experiments were conducted in 2D tissue culture before progressing into 3D, using models that more faithfully re-create the in vivo tumour microenvironment. Expression analysis conducted with MCF-7, MDA-MB-231 and HBL-100 human breast cancer cell lines cultured in 2D, and in 3D as multicellular tumour spheroids, showed that protein and mRNA levels of CAIX were very responsive to lower O2 concentrations. Both MDA-MB-231 and HBL-100 cells displayed large increases in CAIX expression levels in hypoxia, with the HBL-100 cell line exhibiting the largest change in CAIX mRNA (42-fold increase) and protein (78-fold increase) levels in 0.5% O2 conditions. Hypoxia inducible factor 1-α (HIF-1α) controls the expression of CAIX, but the induction of CAIX in hypoxic MCF-7 cells was lower in comparison to the other cell lines, despite the presence of similar levels of HIF-1α. The differences in CAIX expression observed between the cell lines was consistent with a varying activity of factor inhibiting HIF-1 (FIH-1), an oxygen sensor that controls signalling through HIF-1α, as siRNA targeting FIH-1 led to increased levels of CAIX in hypoxic MCF-7 cells. While NHE1 protein levels did increase in hypoxic conditions in MCF-7 cells in 3D, overall, the expression levels of both NHE1 and V-ATPase were not as responsive to changes in O2 concentrations when compared to CAIX across differing O2 conditions in each of the cell lines. Inhibitors targeting CAIX, NHE1 and V-ATPase were all shown to reduce cancer cell number in 2D culture conditions. Differing O2 conditions changed the sensitivity of these cell lines to CAIX inhibition. Cells cultured in 20% O2 conditions were responsive to CAIX inhibition, while acute hypoxic cells cultured in 0.5% O2 displayed an increased resistance to drug treatment. Chronically hypoxic cells, which had spent over 10 weeks in 0.5% O2 before treatment, exhibited a re-sensitisation to CAIX inhibition. 3D invasion assays demonstrated that CAIX inhibition significantly reduced the invasion of cells from MDA-MB-231 spheroids into collagen type 1 in both 20% O2 and 0.5% O2 conditions, while drugs targeting either NHE1 or V-ATPase had no such inhibitory effects. Preliminary clonogenic assays, used to assess radiation sensitivity and performed with MDA-MB-231 spheroids, indicated that inhibitors targeting CAIX and NHE1 led to a significant decrease in colony formation when combined with irradiation, compared to either drug treatment or irradiation alone. Further invasion assays, carried out with primary tissue derived from human patients, showed that drugs targeting CAIX retained their inhibitory effects when tested on heterogeneous cancer material of varying tumour subtypes. CAIX inhibition also exhibited anti-cancer effects in vivo on mouse MDA-MB-231 xenografts, significantly reducing the proliferation and growth of tumours within mice. Together, this work demonstrates that inhibitors targeting the pH regulation mechanisms of cancer cells have potential in the treatment of breast cancer, highlighted by their capacity to affect cancer cell number, cancer cell invasion, and their ability to combine with irradiation. Of the 3 pH regulatory molecules studied, CAIX appears to be the target with the most therapeutic potential.
23
Siyanov, Violetta. "Intracellular pH Regulation by Sodium-Hydrogen Exchanger Isoforms in Preimplantation Mouse Embryos." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32251.
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Intracellular pH (pHi) impacts many cellular mechanisms including cellular metabolism, gene expression, cell volume regulation, cell survival and proliferation. Most cells use two general pHi regulatory mechanisms: HCO3-/Cl- antiporters (AE, Slc4a family) to reduce internal alkaline load, and Na+/H+ exchangers (NHE, Slc9a family) that protect cells from acidosis. Previous studies with preimplantation (PI) embryos have shown robust activity of HCO3-/Cl- exchanger in all stages of development. It was also determined that inhibition of this exchange with the stilbene AE inhibitor 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) was detrimental to embryo development from the 2‐cell stage to blastocyst when cultured at high external pH. In this study I investigated which of the five known plasma membrane NHE isoforms was present and active within mouse PI embryos and their role as pHi regulators throughout preimplantation embryo development. In mouse oocytes and preimplantation embryos, mRNAs were detected encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in fully-grown oocytes through one-cell stage embryos and then generally lower levels after the two-cell stage. No transcripts for NHE2 (SLC9A2) or NHE5 (SLC9A5) were detected. Measurements of intracellular pH during recovery from acidosis, induced by transient ammonium pulse, suggested that recovery occurred and was mediated by NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). This recovery was inhibited by 1 mM amiloride, a general NHE inhibitor. The observed residual recovery was attributed to passive passage of protons across the membrane, rather than the activity of NHE4 (an amiloride-resistant isoform), since no further decrease in recovery rates from acidosis was observed upon amiloride increase to 5 mM. Furthermore, recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over that achieved with cariporide alone, indicating that NHE3 did not play a functional role in pHi regulation at any stage assessed. Another regulator of intracellular pH against acidosis, previously reported to be active in oocytes and 1-cell embryos, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. This indicated that NHE1 is likely the only significant regulator of pHi in preimplantation mouse embryos, at least after the 1-cell stage. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development, as assessed by development to the blastocyst stage and cell lineage allocation. However, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not further decrease embryo development to the blastocyst stage more extensively under conditions of chronic acidosis than at normal pH. This suggests that mouse PI embryos have a restricted ability to counteract chronic acidosis by means of pHi regulatory mechanisms, despite clearly being able to recover from acute acidosis via NHE1 activity.
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Arai, Fumihito, Naoya Inoue, and Hisataka Maruyama. "Optical pH Regulation Using Photochromic Material for Selective Cell Injection of Nanosensors." IEEE, 2010. http://hdl.handle.net/2237/14476.
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Abohamed, Abdulrahman. "Regulation of TASK-2, a pH-sensitive, two pore domain potassium channel." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421414.
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Paradise, Ranjani Krishnan. "Chemomechanical regulation of integrin activation and cellular processes in acidic extracellular pH." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/75840.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.<br>This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Cataloged from student submitted PDF version of thesis.<br>Includes bibliographical references (p. 162-176).<br>It is well established that extracellular pH (pHe) becomes acidic in several important physiological and pathological contexts, including the tumor and wound microenvironments. Although it is known that acidic pHe can have profound effects on cell adhesion and migration processes integral to tumor progression and wound healing, the molecular mechanisms underlying the cellular responses to acidic pHe are largely unknown. Transmembrane integrin receptors form a physical linkage between cells and the extracellular matrix, and are thus capable of modulating cell adhesion and migration in response to extracellular conditions. In this thesis, computational and experimental approaches are used to investigate the role of acidic extracellular pH in regulating activation and binding of integrin [alpha]v[beta]3, and to characterize the consequences for downstream subcellular- and cellular-scale processes. Molecular dynamics simulations demonstrate that opening of the integrin [alpha]v[beta]3 headpiece occurs more frequently in acidic pHe than in normal pHe, and that this increased headpiece opening can be partially attributed to protonation of ASP[beta]127 in acidic pHe. These computational data indicate that acidic pHe can promote activation of integrin [alpha]v[beta]3. This is consistent with flow cytometry and atomic force microscope-enabled molecular force spectroscopy experiments, which demonstrate that there are more activated [alpha]v[beta]3 receptors on live [alpha]v[beta]3 CHO-B2 cell surfaces at acidic pHe than at normal pHe 7.4. Put together, these atomistic- and molecular-level data suggest a novel mechanism of outside-in integrin activation regulation by acidic extracellular pH. Next, the consequences of acid-induced integrin activation for subcellular- and cellular-scale processes are investigated. Kymography experiments show that [alpha]v[beta]3 CHO-B2 cell membrane protrusion lifetime is increased and protrusion velocity is decreased for cells in pHe 6.5, compared to cells in pHe 7.4. Furthermore, [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 form more actin-integrin adhesion complexes than cells in pHe 7.4, and acidic extracellular pH results in increased cell area and decreased cell circularity. Cell migration measurements demonstrate that [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 migrate slower than cells in pHe 7.4, and that the fibronectin ligand density required for peak migration speed is lower for cells in pHe 6.5. Together, these data show that acidic pHe affects subcellular- and cellular-scale processes in a manner that is consistent with increased integrin activation in this condition. Finally, the migration behavior of [alpha]v[beta]3 CHO-B2 cells, bovine retinal microvascular endothelial cells, and NIH-3T3 fibroblasts in an extracellular pH gradient is investigated. Results demonstrate that NIH-3T3 fibroblasts do not exhibit directional preferences in the pHe gradient, but that [alpha]v[beta]3 CHO-B2 cells and bovine retinal microvascular endothelial cells migrate preferentially toward the acidic end of the gradient. These data suggest that acidic extracellular pH may serve as a cue that directs migration of angiogenic endothelial cells to poorly vascularized regions of tumors and wounds. Overall, this thesis research results in multiscale, in-depth understanding of extracellular pH as a critical regulator of cell function, with associated implications for tumor growth, wound healing, and the role of proton pumps in cell migration.<br>by Ranjani Krishnan Paradise.<br>Ph.D.
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Stewart, Andrew Kenneth. "Intracellular pH regulation associated with proton-coupled dipeptide transport in mouse small intestine." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298321.
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Williams, Mark R. "pH and calcium regulation in the lens epithelial cells : a fluorimetric dye study." Thesis, University of East Anglia, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334394.
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Millard, Alisha Jane. "A proteomics based approach to the analysis of pH regulation in Aspergillus nidulans." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442034.
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Martinez-Zaguilan, Raul. "The role of intracellular pH and calcium in the regulation of cellular functions." Diss., The University of Arizona, 1992. http://hdl.handle.net/10150/185889.
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Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes, including turnover of inositol phospholipids, activation of a number of kinases, and changes in intracellular pH (pHⁱⁿ) and calcium ([Ca²⁺]ⁱⁿ). Changes in both pHⁱⁿ and [Ca²⁺](in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e. secretion, neurotransmission, proliferation and differentiation. The intracellular pH changes that follow mitogenic activation are complex and may reflect several different H⁺ transporting mechanisms. There are at least three main systems involved in the regulation of pHⁱⁿ in eukaryotic cells: (a) the mitogen stimulated Na⁺/H⁺ exchange, which electroneutrally raises pHⁱⁿ and can be inhibited by amiloride and its derivatives; (b) a variety of HCO₃⁻-based mechanisms which can alkalinize or acidify the cytosol, and can be inhibited by stilbene disulfonate derivatives; (c) and a plasma membrane H⁺-ATPase, which represents the least understood mechanism of pHⁱⁿ regulation. Under non-pathological conditions, pHⁱⁿ regulation is generally achieved by Na⁺/H⁺ exchange and HCO₃⁻-based mechanisms. Missexpression of a H⁺-ATPase in the plasma membrane can lead to a chronically high pHⁱⁿ in some tumor cells and might contribute to carcinogenesis. Chapter I explains the dissertation format and the relationship of the manuscripts included in three appendices. This chapter also indicates my contribution to each of these manuscripts. Chapter II is a summary of the most important findings in these manuscripts. Appendix I deals with the role of Na⁺/H⁺ exchange and Cl⁻/HCO₃⁻ exchange in the regulation of pHⁱⁿ. Appendix II deals with the role of H⁺-ATPase in the maintenance of a chronically high pHⁱⁿ and its possible involvement in tumorigenesis. Appendix III describes a technique to simultaneously measure pHⁱⁿ and [Ca²⁺]ⁱⁿ by fIuorescence spectroscopy. This appendix also describes the application to study the role of pHⁱⁿ and Ca²⁺ in the regulation of cell growth and progesterone secretion.
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Arai, Fumihito, Tatsuro Takahata, Ayae Honda, Naoya Inoue, Taisuke Masuda, and Hisataka Maruyama. "Optical pH Regulation Using Functional Nanotool Impregnating with Photo-Responsive Chemical for Intracellular Measurement." IEEE, 2010. http://hdl.handle.net/2237/14484.
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ASTARIE, CATHERINE. "Contribution a l'etude du ph intracellulaire, sa regulation et ses anomalies dans l'hypertension arterielle." Paris 6, 1990. http://www.theses.fr/1990PA066021.
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Le ph cytosolique (phc) en modulant l'activite metabolique des cellules et en participant aux mecanismes de proliferation et de croissance cellulaires, pourrait jouer un role dans l'hypertension arterielle. Dans la plaquette sanguine humaine non stimulee ex vivo, nous avons montre par l'indicateur fluorescent de ph, bcecf, que l'homeostasie du phc est controlee par le gradient transmembranaire de na#+, independamment de l'echangeur na#+/h#+ et des transporteurs de hco#3# et par des mouvements intracellulaires de ca#2#+. Lors d'une acidification intracellulaire, l'echange na#+/h#+ caracterise par la mesure directe de phc, presente des proprietes cinetiques comparables a celles decrites dans d'autres types cellulaires. Dans l'hypertension essentielle, nous avons observe une alcalinisation du cytosol plaquettaire, qui est independante des ions na#+ et d'une activation de l'echangeur na#+/h#+ et des transporteurs de hco#3#. Elle est associee chez 30% des patients a une augmentation de ca#2#+i. Une alcalinisation intracellulaire a ete retrouvee dans les plaquettes de rats spontanement hypertendus adultes (shr) et pas dans celles de rats wistar rendus hypertendus par administration chronique d'un glucocorticoide de synthese. Chez des rats shr ages de 3 jours, une alcalinisation cytosolique est presente dans les cardiomyocytes en culture et pas dans les fibroblastes. Dans l'hypertension arterielle du rat shr, si l'anomalie de phc des cellules excitables, qui preexiste a l'elevation de la pression arterielle, soutend leur hyperreactivite et l'hypertrophie et/ou l'hyperplasie, elle pourrait participer a la genese de l'hypertension arterielle
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GRENIER, AMY. "REGULATION OF APOPTOTIC ALKALINIZATION THROUGH PHOSPHORYLATION OF SODIUM HYDROGEN EXCHANGER VIA P38 MITOGEN ACTIVATED PROTEIN KI." Master's thesis, University of Central Florida, 2006. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4316.
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Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus of NHE1. To identify the p38 MAPK phosphorylation sites on NHE1, in vitro p38 MAP kinase assays coupled to deletion analysis of NHE1 and mass spectrometry, identified four possible p38 MAPK phosphorylation sites. To establish that NHE1 causes apoptotic alkalinization and determine whether the identified phosphorylation sites on NHE1 are functionally significant, we used PCR site directed mutagenesis to mutate T717, S722, S725, and S728 on the C-terminus of NHE1. Stable NHE1 deficient cell lines, expressing wild type (WT) NHE or the four mutated sites (F4MUTNHE), were assessed for apoptotic alkalinization using the pH-sensitive fluorescent protein, destabilized YFP. Our results show that NHE1 is required for apoptotic alkalinization, since expression of WT NHE restored alkalinization in an NHE deficient cell line, and that this process requires the phosphorylation of the p38 MAPK target sites, since mutation of all four sites prevented the apoptotic alkalinization response.<br>M.S.<br>Department of Molecular Biology and Microbiology<br>Burnett College of Biomedical Sciences<br>Molecular Biology and Microbiology
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Perry, Leslie M. "Regulation of Alternative Sigma Factors During Oxidative and Ph Stresses in the Phototroph Rhodopseudomonas Palustris." Thesis, University of North Texas, 2014. https://digital.library.unt.edu/ark:/67531/metadc700009/.
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Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.
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Touati, Eliette. "La phosphatase acide de ph optimum 2,5 d'escherichia coli : un systeme pour l'etude des effets negatifs de l'amp cyclique." Paris 6, 1986. http://www.theses.fr/1986PA066531.
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Hong, Meng Yew. "Intracellular pH Regulation in H+-ATPase-rich Ionocytes in zebrafish larvae Using in vivo Ratiometric Imaging." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35749.
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The H+-ATPase rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. In the present study, a ratiometric imaging technique using the pH indicator dye BCECF was developed to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo. Initial validation experiments demonstrated that HR cells subjected to respiratory acidosis (1% CO2) or metabolic alkalosis (20 mM NH4Cl) exhibited changes in BCECF 513/438 emission ratios which were consistent with the expected effects of these treatments on pHi. Subsequent experiments focussed on the involvement of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (isoform NHE3b; zslc9a3.2) and the H+-ATPase (atpv1aa) in pHi regulation. Additionally, the role of HR cell carbonic anhydrase (“CA2-like a”) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. To do so, relative HR cell pHi changes were monitored during acid-base challenges in shams and in fish experiencing morpholino gene knockdown of either NHE3b, H+-ATPase or “CA2-like a”. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalization were altered markedly in fish experiencing knockdown of “CA2-like a”, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. Knockdown of “CA2-like a”, although presumed to limit H+ availability to NHE3b and H+-ATPase, yielded qualitatively similar results to knockdown of either single H+ excretory pathway. The metabolic alkalosis and subsequent acidification associated with NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.
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Strecker, Kristina [Verfasser]. "Untersuchungen zur pH-Regulation am Blättermagenepithel des Schafes mit H+-sensitiven Mikroelektroden / Kristina Strecker, geb. Kosmis." Berlin : Freie Universität Berlin, 2011. http://d-nb.info/1025239989/34.
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LE, PRIGENT KARINE. "Regulation du ph intracellulaire dans les myocytes cardiaques normaux et pathologiques : systemes membranaires dependants du sodium." Paris 11, 1997. http://www.theses.fr/1997PA112201.
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Les travaux de recherche, rapportes dans ce memoire de these, concernent la modulation des mecanismes membranaires de regulation du ph intracellulaire (ph#i), dependants du sodium, dans les myocytes cardiaques normaux et diabetiques. La premiere partie de ce travail a ete consacree a l'analyse de facteurs responsables de la diminution d'activite de l'echangeur na#+/h#+, observee au cours du diabete. Elle a permis de demontrer que, en l'absence de modification du pouvoir tampon intrinseque cellulaire pour les ions h#+, le diabete reduit significativement l'efflux acide via l'echangeur na#+/h#+ ( 42% a un ph#i de 6,9). Nos resultats indiquent que cette diminution d'activite n'est pas due a une sensibilite differente de l'echange na#+/h#+ au ph extracellulaire chez les diabetiques. En revanche, elle est vraisemblablement reliee a la diminution du calcium libre cytosolique (ca#2#+#i) observee dans les myocytes ventriculaires de rats diabetiques. De plus, les resultats obtenus en presence d'un inhibiteur de la proteine kinase dependante de la calcium-calmoduline (pkii), sont en faveur d'une alteration de la modulation de l'echangeur par la pkii chez les diabetiques. A partir de l'ensemble de ces resultats, notre conclusion est la suivante : en raison d'une diminution de la ca#2#+#i, la voie de signalisation impliquant la pkii et controlee par le calcium cytosolique, pourrait etre affectee dans les cellules de diabetiques. Ceci entrainerait une moindre phosphorylation de l'echangeur na#+/h#+ ou de proteines regulatrices de l'echangeur, elles-memes substrats de proteines kinases, et par consequent, une diminution d'activation de l'echangeur. Dans la deuxieme partie de notre travail, nous avons caracterise, dans les myocytes cardiaques de rat adulte, les proprietes physiologiques du mecanisme membranaire alcalinisant dependant du bicarbonate. Il s'agit d'un transporteur mettant en jeu l'ion na#+ mais pas l'ion cl#-, autrement dit, d'un cotransport na#+-hco#3#-. Nos travaux suggerent en outre que la modulation de son activite ne fait intervenir ni le calcium intracellulaire, ni une phosphorylation dependante de la calcium-calmoduline. En revanche, son activite apparait modulee par le metabolisme glycolytique, et l'inhibition de cette voie entraine une diminution d'activite de ce cotransport. La contribution respective de l'echangeur na#+/h#+ et du cotransporteur na#+-hco#3#- a l'extrusion acide est de 67% et 33% a un ph#i de 6,75. Toutefois, dans les myocytes cardiaques de diabetiques, ou l'activite de l'echangeur na#+/h#+ est diminuee et celle du cotransporteur inchangee, la regulation du ph#i apres une acidification intracellulaire peut dependre majoritairement de l'activite du cotransporteur.
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Feldhaus, Heike Verfasser], Stefan [Akademischer Betreuer] Gründer, and Marc [Akademischer Betreuer] [Spehr. "ASIC-vermittelte Regulation des intrazellulären pH in Neuronen des murinen Kortex / Heike Feldhaus ; Stefan Gründer, Marc Spehr." Aachen : Universitätsbibliothek der RWTH Aachen, 2017. http://d-nb.info/1162451106/34.
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Feldhaus, Heike [Verfasser], Stefan Akademischer Betreuer] Gründer, and Marc [Akademischer Betreuer] [Spehr. "ASIC-vermittelte Regulation des intrazellulären pH in Neuronen des murinen Kortex / Heike Feldhaus ; Stefan Gründer, Marc Spehr." Aachen : Universitätsbibliothek der RWTH Aachen, 2017. http://d-nb.info/1162451106/34.
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May, Oliver [Verfasser]. "Regulation des elektrogenen Natrium Bikarbonat Kotransporters NBCe1 und der pH-Homöostase im Kolon der Maus / Oliver May." Hannover : Technische Informationsbibliothek (TIB), 2016. http://d-nb.info/1100314105/34.
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Heipertz, Katrin Sophie [Verfasser]. "Die vH+-ATPase und ihre Bedeutung für die pH-Regulation bei ovinen Pansenepithelzellen / Katrin Sophie Heipertz." Berlin : Freie Universität Berlin, 2007. http://d-nb.info/1022414917/34.
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Wilkins, Robert James. "The relation between cartilage acidity, the synthesis of its matrix and intracellular pH regulation in bovine articular chondrocytes." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334927.
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Zhu, Wen-hui, and 朱文輝. "Regulation of apotosis in human leukemic HL-60 cells: roles of caluium, protein kinase C and intacellular pH." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1995. http://hub.hku.hk/bib/B31235499.
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Ng, Fu Liang. "The impact of the blood pressure-associated genetic locus at SLC4A7 on gene expression and intracellular pH regulation." Thesis, Queen Mary, University of London, 2017. http://qmro.qmul.ac.uk/xmlui/handle/123456789/24732.
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Genome-wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/HCO3 - co-transporter NBCn1 which regulates intracellular pH (pHi) in a range of tissues, including vascular smooth muscle and endothelium. Notably, the SLC4A7 knockout mouse has been shown to have an altered blood pressure phenotype. This thesis presents a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. There were genotype-dependent differences in DNA-nuclear protein interactions by formaldehyde-assisted isolation of regulatory elements, electrophoretic mobility shift assays and DNA pulldown assays. Subsequently, there were also genotypedependent differences in SLC4A7 expression level and NBCn1 availability at the plasma membrane. In turn, SLC4A7 genotype is associated with Na+/HCO3 --dependent steady-state pHi and recovery from intracellular acidosis. The genotypic effect on pHi regulation was independent of the calcineurin activity, or the amino acid substitution E326K resulting from a missense polymorphism. However, in the presence of Na+/H+ exchange activity, the SLC4A7 genotypic effect on net base uptake and steady-state pHi was detected only in vascular smooth muscle cells but not endothelial cells. The finding of a genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells provide an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus with blood pressure.
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Parton, Richard M. "Confocal microscopy analysis of the roles of intracellular pH in the regulation of polarised growth of Dryopteris protonemata." Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/15585.
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The roles of pH and [Ca<SUP>2+</SUP>] were investigated in relation to the maintenance and regulation of polarity in tip-growing <I>cells of Dryopteris affinis </I>protonemata. Intracellular [ion] was analysed by confocal microscopy of ion-sensitive dyes and ratio imaging. Initially, physiological and cytological aspects of tip growth in apical chlorocytes and rhizoid cells were examined. Findings are discussed in relation to the suitability of protonemata for the study of tip growth and with respect to other commonly studied cell types exhibiting polarised growth. Secondly, the suitability of different methods of introducing ion-sensitive dyes into cells were assessed. Calcium-sensitive dyes failed to load by any method except microinjection, which could not be used routinely because of the poor recovery of cells after injection and rapid vacuolar internalisation of injected dye free-acids. The pH sensitive dyes BCECF and carboxySNARF-1 could both be loaded into cells as their cell permeant AM esters without detrimental effects on cell health. However, sequestration within organelles, particularly the vacuole, proved to be a significant limitation. The problems of dye localisation were compensated for to a certain degree by the use of confocal microscopy and ratiometric analysis. Finally, cytoplasmic pH was examined in growth rhizoids by confocal ratio imaging of AM ester-loaded cSNARF-1. The limits of spatial resolution and precision of pH measurement by this method were estimated at ~ 1μm<SUP>2</SUP> and ~ 0.1 pH unit, respectively (over the pH range 6.9 to 7.3). For rhizoids, average cytoplasmic pH was estimated at 7.1 - 7.3, based on <I>in vitro</I> calibration. No significant cytoplasmic pH gradient (ΔpH of >0.1 unit) was found to be associated with tip growth.
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Ladoux, Annie. "Etude du ph intracellulaire et de sa regulation au cours de la differentiation des cellules leucemiques myelomonocytaires humaines." Paris 7, 1988. http://www.theses.fr/1988PA077092.
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Zhu, Wen-hui. "Regulation of apotosis in human leukemic HL-60 cells : roles of caluium, protein kinase C and intacellular pH /." Hong Kong : University of Hong Kong, 1995. http://sunzi.lib.hku.hk/hkuto/record.jsp?B16553226.
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Antoniel, Manuela. "The unique histidine of F-ATP synthase subunit OSCP mediates regulation of the permeability transition by matrix pH." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3427232.
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The “Permeability transition” (PT) is one of the most studied events that may trigger cell death and is due to a Ca2+- and ROS-dependent opening of a nonspecific pore, called PTP, whose molecular nature has been long debated. Recently, our research group has demonstrated that PTP forms from FoF1 ATP synthase dimers, demonstrating the ability of this complex to switch from the key enzyme for the aerobic synthesis of ATP into a potential cell death mediator.
The goal of this PhD thesis has been to define which structural changes of ATP synthase are responsible for the pH modulation of PTP. Indeed, it is well known from nineties that the optimum matrix pH for PT occurrence is about 7.3, and a decrease leads to decreased probability of PTP opening. The pH effect has been ascribed to reversible protonation of His residues located on the PTP that can be blocked by the histidine modifying reagent diethyl pyrocarbonate (DPC). Moreover, in mammalian cells, similarly to the drug Cyclosporin A (CsA), acidic pH also promotes release from the inner membrane of the matrix protein Cyclophilin D (CyPD), which is a well-known PTP activator.
As our group demonstrated that CyPD binds to the ATP Synthase OSCP subunit, mainly through electrostatic interactions and resulting in partial enzyme inhibition, the hypothesis has been advanced that the unique histidine located on OSCP, His112 according to bovine numbering, may be responsible for both the pH effects on CyPD (un)binding to ATP synthase and on PTP/ATP synthase opening. OSCPHis112 is exposed to the solvent and is located in the flexible linker region between the structured N- and C-terminal domains of OSCP.
The results obtained by ATP synthase immunoprecipitation from bovine heart mitochondria showed that acidic pH induces CyPD release that is prevented by DPC, perfectly matching the effect of DPC on CyPD-PTP interaction. DPC also prevented the binding at low pH of the inhibitor protein IF1 to ATP synthase, but this effect is probably not relevant to PTP modulation. ESI-MS and ESI-MS/MS analyses of the OSCP isolated from DPC-treated mitochondria revealed that the 95-113 peptide shows a mass shift of +72 Da, which is indicative of carbethoxylation of the unique His112. These data therefore strongly support the hypothesis that OSCP His112 is part of the binding site of CyPD on the protein, so that its protonation by lowering pH favors CyPD release. Of note, this region contains several residues of glutamic acid conferring a low potential surface, which is complementary to the mainly high potential surface of CyPD. Consistently to this model, DPC inhibits the ATPase activity of ATP synthase only when CyPD is released from OSCP, i.e. in the presence of CsA and in mitochondria from CyPD-null mice. Replacement of OSCPHis112 with a Gln in HEK cells, by the CRISPR/Cas9 system, showed its involvement even in the effect of low pH on PTP opening. Indeed, the PTP open probability is not affected by acidic pH only in mutated cells, while DPC reverts the pH inhibition exclusively in wild type cells. Finally, evaluation of the structural stability of the ATP Synthase dimers at low pH by Blue-native PAGE excluded their destabilization, which could affect PTP formation.
In summary, these data provide a convincing model for the pH modulation of PTP, as well as a compelling evidence that ATP synthase and PTP are the same molecular entity.<br>La transizione di permeabilità (PT) mitocondriale è uno degli eventi coinvolti nella morte cellulare tra i più studiati e implica l’apertura nella membrana mitocondriale interna di un poro aspecifico, definito PTP, indotta da ioni calcio in presenza di stress ossidativo, la cui natura molecolare è stata a lungo dibattuta. Recentemente, il gruppo di ricerca presso cui è stata svolta la presente tesi di dottorato ha dimostrato che il PTP si forma da complessi dimerici dell’enzima FoF1 ATP Sintetasi, trasformando l’enzima essenziale per la sintesi aerobica di ATP in mediatore di morte cellulare.
L’obiettivo della presente tesi è stato quello di definire le basi molecolari della modulazione del PTP da parte del pH di matrice rispetto alle proprietà strutturali di ATP Sintetasi. E’ infatti noto dagli anni ’90 che le probabilità di apertura del PTP sono massime a pH 7.3 e diminuiscono rapidamente al diminuire del pH. Tale effetto è stato imputato alla protonazione di residui di istidine del PTP, in quanto l’inibizione veniva bloccata dal reagente specifico per le istidine dietilpirocarbonato (DPC). Inoltre, era stato dimostrato che in cellule di mammifero un pH acido favorisce, similmente alla Ciclosporina A (CsA), il rilascio dal PTP della proteina di matrice Ciclofilina D (CyPD), che, quando legata al PTP, è un ben noto attivatore del poro.
Poiché studi recenti dello stesso gruppo avevano dimostrato che in mammifero la ciclofilina D interagisce, prevalentemente mediante interazioni elettrostatiche, con la subunità OSCP di ATP Sintetasi, inducendo un’inibizione parziale dell’attività catalitica dell’enzima, si è ipotizzato che l’unico residuo di istidina di OSCP, His112 in base alla numerazione in bovino, fosse coinvolto nella modulazione pH-dipendente sia del legame della CyPD che della probabilità di apertura del PTP/ATP Sintetasi. OSCPHis112 è esposto al solvente ed è localizzato nella parte non strutturata della proteina, legante i domini ad α-elica N- e C-terminali.
I risultati ottenuti mediante immunoprecipitazione di ATP Sintetasi da mitocondri di cuore bovino hanno dimostrato che pH acidi inducono il rilascio della CyP e che tale rilascio viene bloccato dalla presenza di DPC, in perfetto accordo con quanto precedentemente osservato rispetto al PTP. L’analisi ESI-MS e ESI-MS/MS della subunità OSCP isolata da mitocondri trattati con DPC ha stabilito che il peptide 95-113 di OSCP subisce un aumento della sua massa molecolare corrispondente a 72 Da, indicativo della carbetossilazione dell’unica istidina. Questi dati suggeriscono quindi che OSCPHis112 sia coinvolta nel sito di legame della CyPD, in quanto l’interazione viene sfavorita dalla sua protonazione. His112 è infatti localizzato in una regione caratterizzata dalla presenza di numerosi residui di acido glutammico avente un potenziale di superficie negativo, che è complementare al potenziale di superficie della CyPD prevalentemente positivo. In accordo con questo modello, il DPC è risultato un inibitore dell’attività catalitica di ATP Sintetasi esclusivamente quando la CyPD non è legata ad OSCP, come in presenza di CsA o nei topi KO per la CyPD (Ppif-/-). Inoltre, la sostituzione di OSCPHis112 con un residuo di Gln mediante il sistema di mutagenesi CRISPR/Cas9 in una linea cellulare umana (HEK293T) ha permesso di verificare che la stessa istidina è coinvolta nell’effetto del pH sull’apertura del PTP, in quanto solo nelle cellule mutate la probabilità di apertura del PTP non diminuisce a pH acido, e, in accordo, il DPC reverte l’inibizione a pH acido esclusivamente nelle cellule wild type. Infine, l’analisi della stabilità strutturale dei dimeri di ATP Sintetasi in funzione del pH mediante Blue-native PAGE ha consentito di escludere una loro destabilizzazione a pH acidi, che avrebbe potuto influire sulla formazione del PTP.
In conclusione, questi risultati, oltre a fornire un modello convincente per la modulazione pH-dipendente del PTP, costituiscono altresì una prova molecolare importante che ATP Sintetasi e il PTP sono la stessa entità molecolare.
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Veguilla, Rosa Angelica. "Transcriptional Regulation of OCA2 and POMC by a cAMP-Dependent Mechanism and Implications in Skin Pigmentation." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10095.
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Skin Pigmentation represents the major natural protection against the deleterious effects of Ultraviolet light and involves a crosstalk between keratinocytes and melanocytes. Pigment synthesis or melanogenesis is initiated by the binding of \(\alpha\)-Melanocyte Stimulating Hormone \((\alpha-MSH)\) to the Melanocortin 1 Receptor (MC1R), expressed by the melanocytes. α-MSH is generated by cleavage of pro-opiomelanocortin
hormone (POMC), produced by both melanocytes and keratinocytes. Activation of MC1R leads to an increase in cAMP levels, causing the expression of the transcription factor MITF. MITF regulates the expression of the enzymes involved in melanogenesis as well as genes important for the survival and proliferation of melanocytes. Pigment synthesis, which occurs in specialized organelles called melanosomes, involves the regulation of different proteins as well as fine homeostatic tuning such as melanosomal pH regulation. The POMC derivative, \(\alpha-MSH\), begins the pigmentation pathway by activating the MC1R signaling pathway, and OCA2 regulates the end of this pathway by controlling tyrosinase activity. The OCA2 gene has been shown to be important in the control of intra-melanosomal pH to allow optimal conditions for the activity of Tyrosinase, the limiting enzyme of pigment (melanin) production. OCA2 polymorphisms have been linked to oculocutaneous albinism type 2 and to blue eye color, demonstrating the importance of this gene in fine pH regulation on pigment production. Polymorphisms in POMC have also been linked to red-haired/fair-skin color in humans. Despite the effort to dissect the mechanisms involved in the control of pigmentation, the transcriptional regulation of POMC and OCA2 are still not fully understood. In this study, we investigate the relevance of the cAMP/CREB pathway in the transcriptional regulation of these two proteins. Our data shows that both POMC and OCA2 expression increases after stimulation of the cAMP/CREB pathway. We demonstrate that MITF transcriptionally regulates OCA2: the cAMP/CREB pathway therefore induces OCA2 in a MITF-dependent manner. On the other hand, our data reveals that POMC may be regulated by cAMP in a MITF-independent fashion but consistent with the hypothesis of a positive feedback loop within the MC1R signaling pathway.