Myo-inositol is ubiquitous in nature and is found at the structural core of a diverse range of biologically important derivatives, including phosphatidylinositols, inositol phosphates and mycothiol. The synthesis of myo-inositol derivatives is notoriously difficult due to the need to control both regio- and enantioselectivity. As a result, synthetic routes to derivatives of this type are often lengthy and low yielding. The first biosynthetic step in the production of all myo-inositol metabolites is the isomerisation of D-glucose 6- phosphate to L-myo-inositol 1-phosphate as mediated by L-myo-inositol 1-phosphate synthase (INO1). For the protozoan parasite Trypanosoma brucei, INO1 is essential for survival and its version of the enzyme (TbINO1) has a high turnover. This makes TbINO1 an attractive candidate for the biocatalytic production of L-myo-inositol 1- phosphate, and a potential starting point for drastically shortened syntheses of important myo-inositol derivatives. The production of L-myo-inositol 1-phosphate by TbINO1 has been optimised to achieve complete conversion in reaction conditions that facilitate product isolation. Due to problems with an in-batch process, the TbINO1 enzyme was immobilised and the process was transferred to a flow system. This has allowed for production of significant quantities of L-myo-inositol 1-phosphate with a high level of purity. L-myo-inositol 1- phosphate obtained from the flow system has been used to prepare mycothiol glycosylation acceptor, 1,2,4,5,6-penta-O-acetyl-D-myo-inositol, in a concise synthesis with a greatly improved yield over the literature.

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In the context of fetal alcohol spectrum disorders, we investigated how the nitric oxide (NO) signaling pathway influences intracellular calcium (Ca2+) to mediate alcohol resistance, using a primary cell culture model of cerebellar granule neurons (CGN). Alcohol during fetal brain development triggers abnormally high apoptotic cell death in vulnerable neuronal populations, culminating in serious behavioral and cognitive deficits that persist into adulthood. Prior studies demonstrated that the NO signaling pathway [neuronal nitric oxide synthase → NO → soluble guanylyl cyclase → cyclic guanosine monophosphate → protein kinase G (PKG)] mitigates alcohol toxicity, consequently diminishing neuronal loss both in vivo and in vitro. Endoplasmic reticulum (ER) Ca2+ release, a key apoptotic mechanism, requires the inositol 1,4,5-trisphosphate receptor (IP3R), a known PKG substrate. Our studies focused on this crucial intersection point where the NO signaling cascade can influence Ca2+-mediated apoptotic mechanisms, and exposed a downstream mechanism where NO can moderate alcohol neurotoxicity. We hypothesized that as alcohol disturbs neuronal Ca2+ homeostasis to trigger cell death, the NO signaling pathway counters it by limiting Ca2+ release from the ER. We examined first the role of the phospholipase C (PLC) pathway [PLC → inositol 1,4,5-trisphosphate → IP3R → Ca2+] in developmental neurotoxicity through our in vitro CGN model, extending previous in vivo studies. We found that alcohol terminates developing neurons by eliciting abnormal Ca2+ release from the ER rather than from an extracellular source, via a PLC - IP3R-dependent signaling mechanism. Inhibiting either calcineurin or Ca2+ / calmodulin-dependent protein kinase ii (CaMKii), which participate in parallel Ca2+-activated apoptotic cascades, shielded CGN cultures from alcohol. Blocking the mitochondrial Ca2+ uniporter or the mitochondrial permeability transition pore also provided neuroprotection. That the activated pathways must interact to generate cell death likely explains why inhibiting one of multiple parallel signaling cascades limits alcohol toxicity. We next demonstrated that activating the NO pathway downstream at PKG eliminated both alcohol-related neuronal death and the accompanying rapid rise in intracellular Ca2+, an effect that markedly resembled IP3R inhibition. Experiments that temporally manipulated the addition of PKG activators in relation to alcohol exposure linked PKG's obstruction of alcohol-induced Ca2+ elevations to alcohol resistance. In contrast, brain-derived neurotrophic factor (BDNF), which does not rely on PKG to provide neuroprotection, failed to block alcohol-induced Ca2+ elevations while preventing alcohol toxicity. This indicates that although PKG blocks alcohol-induced Ca2+ elevations, averting these Ca2+ elevations is not necessary for neuroprotection. BDNF may confer alcohol resistance through an as yet unidentified process downstream from the disruption of intracellular Ca2+. In summary, we established that 1) alcohol induces toxic Ca2+ elevations originating from the ER through a PLC - IP3R-dependent pathway, and that 2) PKG-mediated alcohol resistance is linked to preventing the intracellular Ca2+ surges. These findings support the hypothesis that the NO signaling pathway shields developing neurons from alcohol by limiting Ca2+ release from the ER.

Cell membrane receptors help to mediate communication between the cell and its environment. The largest group of these membrane receptors belong to the family of G protein-coupled receptors (GPCRs). GPCRs contain seven transmembrane (TM) helices and signal predominantly through heterotrimeric G proteins in response to diverse extracellular stimuli. Previously, three levels of amino acid conservation were proposed to understand the structure and function of a GPCR. This includes “signature” amino acids, “group –conserved” amino acids and amino acids conserved only within a specific subfamily. The group-conserved residues in class A GPCR family involve amino acid conservation of up to 99% when considered as a group of small and weakly polar residues (Ala, Gly, Ser, Cys and Thr). These group-conserved residues have been proposed as key determinants in helix-helix interactions. Therefore, I selected these residues for structure-function analysis in the amine and the prostanoid receptor sub-families of class A GPCRs. Molecular and biochemical assays clearly demonstrate the importance of group-conserved residues in β2-adrenergic receptor and thromboxane A2 receptor (TP) structure and function. These studies led to the identification of a non-synonymous single nucleotide polymorphic variant (nsSNP) A160T in TP to be a constitutively active mutant (CAM). Further, the TP-CAM was used as a pharmacological tool that enabled classification of well-known TP-blockers, into neutral antagonists and inverse agonists. The role of TP-A160T in prostanoid receptors, TP- Prostacyclin receptor (IP) heterodimerization and signaling was investigated. Activation of a GPCR ultimately leads to structural changes in its intracellular loops (ICLs), which in turn activates G-protein. TP activates its cognate G protein (Gαq), while IP mediates signaling, through Gαs. Using TP-IP chimeric receptors, molecular modelling, and site directed mutagenesis studies I determined the specific ICL regions required for G protein coupling in TP and IP. Significant challenges exist in expressing and purifying GPCR-CAMs in amounts required to pursue biophysical studies. Using tetracycline inducible HEK293S system, A160T was expressed at high-levels and CD spectropolarimetry studies were successfully pursued on the purified A160T. The CD spectra showed that the loss of thermal stability of the A160T mutant is due to the subtle changes in the secondary structure of the A160T protein. These studies involving molecular, biochemical and pharmacological approaches provide novel insights into the structure and function of prostanoid receptors TP and IP.

Generation of Ca 2+ signals in cells involves regulation by multiple components controlling Ca 2+ release from the internal stores, Ca 2+ influx across the plasma membrane (PM), elicitation of Ca 2+ sensitive processes and finally the removal of Ca 2+ from the cell. One such mode of facilitating Ca 2+ entry is called store-operated Ca 2+ entry (SOCE) mediated by the store operated Ca 2+ channels (SOCs). SOCE, wherein the depletion of internal Ca 2+ stores triggers the influx of Ca 2+ across the PM, not only plays a vital role in refilling the Ca 2+ stores, but also regulates a multitude of downstream Ca 2+ regulated signalling events. Despite recent advances in elucidating the entry pathway, its molecular identity, biophysical properties and store-depletion signal remains undefined. The most potent inducer of SOCE, thapsigargin (TG), fails to induce Ca 2+ influx in the NG115-401L (401L) cells. This unusual phenotype of the cell makes it a useful model to study the mechanisms and components underlying the SOCE pathway. Although TG failed to induce SOCE in the 401L cells, we report that the activation of intracellular release channels such as the inositol-1,4,5-trisphosphate (fP3Rs) and ryanodine receptors (RyRs) were able to activate Ca 2+ influx upon store depletion. This is in keeping with mechanisms proposed to explain SOCE, namely the conformational coupling hypothesis, wherein depletion of the ER stores signals the release channels to physically interact with the PM SOCs. We found that disrupting the communication between the ER and the PM channels induced by actin disassembly affected both Ca 2+ release and influx. Our study shows that Ca 2+ release and influx is dependent on cortical actin organization and that the RyR mediated release is less regulated by cortical actin than the IP3R induced Ca 2+ release. Studies conducted using 2-aminoethoxy diphenylborate (2-APB), a commonly used SOC blocker, revealed that 2-APB stimulated Ca 2+ release in the 401L cells. This release of Ca 2+ was also found to be dependent on the conformational coupling between the ER and PM SOCs. We also studied the effect of overexpressing various isoforms of the transient receptor potential (TRP) channels. We found that protein kinase C (PKC) differentially regulated the activity of the TRP channels in the 401L cells. PKC activation prolonged the Ca 2+ influx in the wild type cells while attenuating the same in the TRP transfected cells. We also found that influx of surrogate cations (Ba 2+ ) is augmented in the TRPC transfected cells. Our studies reveal that the activation of ER release channels followed by conformational coupling with the PM channels may be a mechanism by which the 401L cells or neurons in general maintain a rigid control over intracellular Ca 2+ concentrations and thus regulate Ca 2+ homeostasis.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca 2+ three-fold had no effect on the MEPP frequency increase to 25 μM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca 2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP 3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP 3 and cyclic AMP/protein kinase A.

<p>Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺]_i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺]_i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺]_i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺]_i oscillations. The [Ca²⁺]_i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺]_i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺]_i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺]_i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺]_i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺]_i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺]_i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺]_i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.</p>

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4&#945;-phorbol 12,13-didecanoate (4&#945;-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar. / Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4&#945;- phorbol 12,13- idecanoate (4&#945;-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.