Thesis (M. Sc)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 87-100). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ59208.

In this study the mechanisms of Ca²⁺-induced-Ca²⁺-release, effects of membrane depolarizations and the actions of pharmacological intracellular Ca²⁺-modulators were examined in cultured rat dorsal root ganglion (DRG) neurones. The whole cell configuration of the patch clamp technique was used to record action potentials, action potential after-potentials and voltage-activated calcium currents, (I_Ca), calcium-activated chloride currents, (I_CI(Ca)), and non-selective cation currents, (I_CAN), under current and voltage clamp recording conditions, respectively. A sub population of DRG neurones expressed action potential after-depolarizations and I_CI(Ca)tail currents which were due to activation of Ca²⁺-activated Cl^- channels as a result of Ca²⁺ entry. I_CANwas dominantly activated due to Ca²⁺ release from intracellular stores evoked by pharmacological Ca²⁺-releasing agents such as caffeine, ryanodine and dihydrosphingosine. Calcium-activated conductances were identified by estimating reversal potentials of the activated currents, using selective pharmacological blockers and extracellular ionic replacement studies. Calcium-dependence of activated currents was also examined by using high concentration of intracellular Ca²⁺ buffer, EGTA, to prevent elevation of intracellular Ca²⁺-levels and by rapidly buffering raised intracellular Ca²⁺ using intracellular 'caged Ca²⁺ chelator', diazo-2. The involvement of intracellular Ca²⁺- stores was examined by performing experiments in Ca²⁺-free extracellular recording medium and pharmacologically inhibiting release of Ca²⁺ from intracellular stores, using dantrolene. Ryanodine had complex actions on DRG neurones, which reflected its ability to mobilize Ca²⁺, deplete Ca²⁺ stores, and inhibit Ca²⁺ release channels. Ryanodine inhibited action potential after-depolarizations and I_CI(Ca)tail currents by interacting with intracellular stores and preventing amplification of Ca²⁺ signalling by CICR. It was found that CICR observed under physiological conditions in rat DRG neurones involves intracellular Ca²⁺stores which were sensitive to ryanodine. In addition to ryanodine sensitivity these intracellular Ca²⁺ stores could be mobilized by caffeine and dihydrosphingosine.

Thesis (Ph.D.)--Georgia State University, 2006. / Title from file title page. Jenny J. Yang, committee chair; Alfons Baumstark, Giovanni Gadda, committee members. Electronic text (405 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Aug. 20, 2008. Includes bibliographical references (p. 380-405).

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Ulhas P. Naik, Dept. of Biological Sciences. Includes bibliographical references.

La fécondation dépend de la capacité de deux cellules uniques à s’unir :l’ovocyte et le spermatozoïde. Alors que le stock d’ovocytes est établi dès la puberté, les spermatozoïdes sont produits de façon continue à partir d’une réserve de cellules germinales souches (spermatogonies). Immobile et inapte à la fécondation, le spermatozoïde testiculaire doit subir un processus de maturation qui débute dans l’épididyme et se poursuit dans les voies génitales femelles. Bien qu’une première forme de mobilité soit acquise au contact du fluide épididymaire, le spermatozoïde subit, tout au long de son parcours dans le tractus femelle, de nombreuses modifications structurelles et biochimiques appelées « capacitation » et qui s’avèrent indispensables à l’acquisition de son pouvoir fécondant. La production de spermatozoïdes fertiles est un processus hautement régulé, notamment via l’ion calcium ;second messager crucial de nombreux processus de signalisation intracellulaire. Les modifications de la concentration intracellulaire de calcium sont indispensables pour le développement du potentiel de fécondation du sperme via la modification du profil de mobilité vers l’hyperactivation ou encore la réalisation de la réaction acrosomiale. Différents mécanismes de régulation de la concentration intracellulaire calcique ont déjà été mis en évidence dans les spermatozoïdes :canaux calciques, réserves intracellulaires de calcium… Toutefois, peu d’informations relatent la présence et/ou l’implication de protéines liant le calcium (Calcium-Binding Proteins - CaBP) au sein de ce type cellulaire.Notre étude a pour but de caractériser la présence et l’implication potentielle de EF-Hand CaBP dans l’acquisition du pouvoir fécondant des spermatozoïdes murins. Nous nous sommes principalement focalisés sur l’étude de trois membres de cette famille protéique :la calrétinine, la calbindine D-28k et la parvalbumine. Nos investigations ont été menées de manière comparative entre des spermatozoïdes de type Wild-type (WT) et des spermatozoïdes n’exprimant pas une ou plusieurs CaBP (calrétinine knock-out - CR-/-, calbindine D-28k - knock-out CB-/-, triple knock-out pour la calrétinine, la calbindine D-28k et la parvalbumine - CR-/-CB-/-PV-/-). La première partie de notre projet décrit la localisation et l’expression de la calrétinine, la calbindine D-28k et la parvalbumine dans les spermatozoïdes de souris et de rats par immunofluorescence et Western blotting. Ces protéines ont été localisées au niveau de la pièce principale du flagelle et au niveau de la Redundant Nuclear Envelope dans le cas de la parvalbumine. Au cours de la seconde partie du projet, des études de mobilité ainsi que des quantifications du taux de réaction acrosomiale ont été menées en comparant les spermatozoïdes WT aux cellules CR-/- et CR-/-CB-/-PV-/-. Alors qu’aucune différence n’a été constatée entre les taux de réaction acrosomiale WT versus knock-out tant en condition basale qu’induite, l’absence d’une ou plusieurs protéines apparaît influencer certains paramètres de mobilité des spermatozoïdes. En effet, les spermatozoïdes CR-/- et CR-/-CB-/-PV-/- ont présenté une fréquence du battement de flagelle (BCF) plus élevée par rapport à la fréquence mesurée chez les spermatozoïdes WT, paramètre modifié notamment au cours de l’hyperactivation. Cette différence pourrait être liée à une modification du tamponnage calcique ou à une régulation différente du canal calcique CatSper, acteur majeur du développement de l’hyperactivation. Les résultats préliminaires des mesures électrophysiologiques suggèrent un rôle potentiel de la calrétinine dans la régulation de l’activité de ce canal. Par ailleurs, la comparaison entre le nombre de petits par portées WT par rapport aux portées CR-/- ou CR-/-CB-/-PV-/- a indiqué que l’absence d’expression d’une ou plusieurs EF-Hand CaBP réduisait le nombre de naissances par portée au sein des génotypes knock-out.En conclusion, l’ensemble de nos résultats expérimentaux ont démontré la présence de trois EF-Hand CaBP au niveau des spermatozoïdes murins et permis d’apporter de nouvelles informations quant à l’implication potentielle de celles-ci dans l’acquisition du pouvoir fécondant des spermatozoïdes. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / The pathogenesis associated with toxoplasmosis is attributed to repeated rounds of the parasite lytic cycle, which has been shown to be regulated by calcium fluxes. However, little is known about the calcium homeostatic mechanisms utilized by T. gondii. Recently, our lab has identified a novel protein-TgEFP1 (TGGT1_255660), which is predicted to bind Ca2+ through its two EF-hand domains. Interestingly, TgEFP1 showed a unique dual localization at the PLV/ELC and the PV of the parasite. Previous work showed that the PLV/ELC harbors other ion binding and conducting proteins that are important for parasite survival and propagation. However, the function of this compartment in the parasite is unknown. Therefore, I hypothesize that the PLV/ELC, through the function of TgEFP1, plays a key role in calcium homeostasis of T. gondii. To test this hypothesis, we sought to characterize the function of TgEFP1 during the parasite lytic cycle and determine TgEFP1 interacting proteins that also localize to the PLV/ELC. Partial permeabilization and ultrastructure expansion microscopy techniques confirmed the dual localization of TgEFP1 at the PLV/ELC and the PV. TgEFP1 knockout parasites exhibited several phenotypic defects including a faster lytic rate, shorter intracellular cycle, and were more sensitive to calcium ionophore treatment. Signal peptide deletion led to a mislocalization of TgEFP1 as cytosolic puncta, while mutations at key calcium coordinating residues lead to exclusive localization of TgEFP1 at the PV. Lastly, immunoprecipitation assays followed by LC-MS/MS identified a novel lectin-like protein- TgLectin (TGGT1_258950) as a direct interactor of TgEFP1-HA. Collectively, these findings support that through the function of TgEFP1, the PLV/ELC, plays a key role in calcium-dependent processes during the lytic cycle of the parasite.

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

The pedunculopontine nucleus (PPN) is a brainstem structure involved in motor control, sleep and arousal. The boundaries of the PPN are defined by its cholinergic neurons, but it also contains GABAergic, glutamatergic and calcium-binding protein- positive neurons. To further understand the physiological roles of the PPN it is necessary to understand which neuronal subtypes are present in the PPN and how they are connected with other regions of the brain in normal and pathological conditions. In order to address these issues, the total numbers, distributions and neurochemical phenotypes of neurons, positive for the calcium-binding proteins calbindin and calretinin, were studied in the rat PPN. Sagittal, perfuse-fixed rat brain sections were double or triple-immunolabelled to reveal the cholinergic marker choline acetyltransferase (ChAT) with calbindin and/or calretinin. A stereological approach revealed that calbindin- and calretinin-positive neurons account for a large proportion of PPN neurons, but they rarely eo-express ChAT. A combination of immunolabelling for calbindin or calretinin with in situ hybridisation for GAD65/67 or VGluT2 mRNAs revealed that about one third of the calbindin- and calretinin-expressing neurons are GABAergic and preferentially located in the rostral PPN, whereas approximately two thirds are glutamatergic and principally located in the caudal PPN. Additionally, retrograde tracer injections in the subthalamic nucleus (STN) and the gigantocellular nucleus (GiN) showed that the majority of PPN neurons, projecting to one or both of these nuclei, were not cholinergic (70-90%). Less than 10% of STN-projecting neurons expressed calbindin or calretinin and 5% of the GiN-projecting neurons contained calretinin but none contained calbindin. Finally, the expression of the immediate early gene, Egrl, a marker of neuronal activation, was evaluated in STN- and GiN-projecting neurons of the PPN in control and 6-0HDA lesioned animals. No statistically significant differences, in the number of Egr l-positive neurons, were observed between control and 6- OHDA lesioned animals. These findings show that calbindin- and calretinin-positive neurons are abundant in the PPN, heterogeneously distributed and display a GABAergic or glutamatergic phenotype. Additionally, calbindin- and calretinin-positive neurons represent only a minority of the PPN neurons projecting to either the STN, GiN or both nuclei. Results also suggest that the hyperactivity seen in the PPN in the 6-0HDA model of Parkinson's disease may not necessarily be due to the neurons projecting to the STN and/or GiN. Overall, this thesis supports the notion that the PPN is composed of a rich diversity of neuronal cell-types, which are heterogeneously distributed along its rostro-caudal axis. The heterogeneous neurochemistry, connectivity and physiology of these neurons allow the PPN to influence a wide range of brain regions through a variety of pathways presumably underlying its various functional roles.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.