Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-163) Also available online.

DISSERTATION (PH.D.)--THE UNIVERSITY OF MICHIGAN

Thesis (doctoral)--Universiteit van Amsterdam, 1990. / Includes bibliographical references.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 85-110.

Abnormal thyroid cell proliferation causes human diseases, such as goiter, thyroid adenoma or carcinoma and primary hypothyroidism resulting from hypoplasia. Thyrotropin (TSH), mainly acting through cAMP and cAMP-dependent protein kinases (PKA), is considered the main regulator of thyrocyte proliferation and differentiation. The general aim of this thesis was to get new mechanistic insights in the regulatory action of the cAMP pathway on various functions of thyrocytes, including proliferation.<p><p>During the first part of this thesis work, we have collaborated to a study of the effects of the TSH/cAMP pathway on small G proteins of the Rho family and their impact on the actin cytoskeleton and thyroid cell function. This study, performed in canine thyrocytes, showed for the first time, that the TSH/cAMP/PKA pathway inactivates the three small G proteins RhoA, Rac1 and Cdc42 and the RhoA/ROCK/LIMK/cofilin pathway. Inactivation of the latter appeared both necessary and sufficient to mediate the action of TSH and PKA on the reorganization of actin microfilaments and its morphological impact. Moreover, this inactivation by PKA of Rho-mediated actin polymerization also played an important role in the cAMP-dependent expression of thyroid differentiation genes. On the other hand, a residual RhoA activity appeared to be required for mitogenesis. This dependence of DNA synthesis on RhoA activity was not mediated by ROCK-dependent events nor by the integrity of the actin cytoskeleton. Indeed, DNA synthesis induction was unexpectedly resistant to actin depolymerisation in canine thyrocytes, which explains how it could be compatible with the cAMP-dependent microfilament disruption. <p><p>This first study did not provide new insights on how the cAMP/PKA-dependent mitogenic stimulus can trigger cell cycle progression, which, in thyrocytes, depends on the phosphorylation of pRb by the cyclin D3-CDK4 complex. In the various in vitro thyroid models, the only convergent early signaling event found in response to TSH/cAMP, insulin and growth factors was the phosphorylation and activation of p70 S6K1 which largely depends on mTOR (mammalian Target Of Rapamycin). The main part of the work in this thesis has been devoted to investigation of the action of TSH/cAMP on the mTOR pathway. mTOR is a therapeutic target for a wide variety proliferative disorders and rapamycin derivates are now considered in anti-cancer treatments.<p><p>We have shown for the first time in PC Cl3 rat thyroid cells that TSH, through cAMP, activates mTORC1, leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMPK and GSK3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate PKB, nor induce TSC2 phosphorylation at Thr1462 and Tyr1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity associated with augmented 4E-BP1 binding to raptor. This could be caused in part by Thr246-phosphorylation of PRAS40, which was found as an in vitro substrate of PKA, but other regulatory events likely remain to be uncovered. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and pRb phosphorylation induced by TSH and insulin. Rapamycin reduced cyclin D3 accumulation but not the abundance of cyclin D3-CDK4 complexes. However, rapamycin inhibited the activity of these complexes and the activating Thr172-phosphorylation of CDK4 stimulated by both TSH and insulin. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 Thr172-phosphorylation. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

In Escherichia coli, the speC gene encodes biosynthetic ornithine decarboxylase (ODC), an enzyme that catalyzes the decarboxylation of ornithine to produce putrescine. The two polyamines, putrescine and spermidine, and the cyclic AMP (CAMP) - cAMP receptor protein (CRP) are known to inhibit the expression of ODC via undefined mechanisms. A single copy of the speC’-lacZ fusion plasmid pOL-1, containing an 843 base pair fragment including the spec promoter, was transferred to the E. coli CB806 chromosome to create E. coli λCBOL. In cell-free extracts prepared from E. coli λCBOL supplemented with cAMP, putrescine, or spermidine, the B-galactosidase activity encoded by the speC’-lacZ fusion was compared to the ODC activity encoded by spec. Only cyclic AMP and putrescine repressed the speC’-lacZ fusion. Cyclic AMP, putrescine, and spermidine all repressed the spec gene. A 444 bp AluI restriction fragment, containing a putative CRP binding site and a downstream open reading frame (ORF2) present on the strand complementary to speC, was fused to lacZ to create a transcriptional fusion, pCC2L. Analysis of E. coli CB806/pCC2L revealed that there was no detectable β8- galactosidase activity from the ORF2-lacZ fusion. However, promoter activity was detected in the opposite direction (3’ to 5’) of ORF2 as alkaline phosphatase activity, encoded on the same plasmid, increased in the presence of CAMP. A 678 bp DraI-AatII fragment, containing the CRP binding site and an adjacent open reading frame (ORF3) present on the speC coding strand, was subcloned into plasmid pBR322 to create pBCR. In the presence of 10 mM cAMP, E. coli CB806/pBCR exhibited an 18% inhibition in ODC activity encoded by spec. It is proposed that ORF3 encodes a protein that represses speC in the presence of CAMP. / Master of Science

Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Ischaemic preconditioning (PC) is the phenomenon whereby a short episode of coronary occlusion followed by reperfusion protects the myocardium against a subsequent period of prolonged (also called index or sustained) ischaemia. Even though the exact mechanism of PC remains to be established, it implies that the heart has an endogenous protective mechanism against ischaemia which, if identified, may have important clinical implications. The importance of establishing the mechanism of PC lies in the potential to convert this biological phenomenon into a therapeutic modality to be used clinically. If mediated by certain components of a signal transduction pathway, such a goal will be achievable. Several triggers and signal transduction pathways have been implicated in the mechanism of protection induced by PC: for example, receptor-dependent endogenous triggers (such as adenosine and opioids) and receptor-independent endogenous triggers (such as free radicals and calcium). However, the involvement of both the ~-adrenergic signalling pathway as well as nitric oxide (NO) in PC has not been defined. It has been suggested that all triggers are linked to a common final pathway, for example, activation of protein kinase C (PKC) and/or the mitogen-activated kinases (MAPKs), in particular p38 MAPK. However, the role of the latter is still controversial. The aim of this study was to: (A) characterize changes in the cyclic nucleotides, cAMP and cGMP, and p38 MAPK occurring during the entire experimental procedure in an attempt to gain insights into the possible mechanisms involved in ischaemie PC (Chapter 3); (8) establish the significance of the changes observed in cAMP and cGMP by pharmacological manipulation of their respective pathways (Chapters 4 and 5); (C) establish the role of p38 MAPK in ischaemie PC: trigger or mediator involvement (Chapter 6). Isolated perfused working rat hearts were preconditioned by 3 x 5 min global ischaemia, interspersed by 5 min reperfusion, followed by 25 min global ischaemia and 30 min reperfusion. Functional recovery during reperfusion was used as end-point. Hearts were freeze-clamped at different times during the PC protocol, sustained ischaemia, as well as during reperfusion. Tissue cyclic nucleotides (cAMP and cGMP), cyclic nucleotide phosphodiesterase (cAMP- and cGMP-PDE) activities, adenylyl cyclase and protein kinase A activities and p-adrenergic receptor characteristics were determined. p38 MAPK activation was also assessed by Western blotting, using dual phospho-p38 MAPK (Thr180ITyr182) antibody as well as activating transcription factor 2 (ATF2) activation. In addition, to evaluate the role of p38 MAPK in PC protection, the effect of inhibition of p38 MAPK activation, by 8B203580, was determined in adult isolated rat cardiomyocytes as well as in isolated perfused rat hearts. Based on the results obtained, it is proposed that during a multi-cycle ischaemie PC protocol triggers (presumably endogenous catecholamines and NO) are released which induce cyclic changes in cyclic nucleotides, cAMP and cGMP. Both these cyclic nucleotides transiently activate the downstream stress kinase, p38 MAPK, which may trigger further downstream adaptive processes. Furthermore, the sustained ischaemic period of PC hearts was characterized by attenuated cAMP and elevated cGMP levels, as well as attenuated activation of p38 MAPK, which was associated with cardioprotection. In addition, pharmacological attenuation of p38 MAPK activation during sustained ischaemia led to functional recovery. It is concluded that the cardioprotection of PC is due to attenuation of ischaemia-induced p38 MAPK activation. Pharmacological manipulation of this kinase should be considered as a therapeutic modality in the future. / AFRIKAANSE OPSOMMING: Isgemiese prekondisionering (PK) verwys na die verskynsel waardeur 'n kort, verbygaande episode van isgemie gevolg deur herperfusie, die miokardium teen 'n daaropvolgende langdurige periode van isgemie beskerm. Die presiese meganisme van beskerming van PK moet nog opgeklaar word, maar dit impliseer dat die hart oor 'n endogene beskermingsmeganisme beskik wat, indien geïdentifiseer, belangrike kliniese implikasies mag hê. Die belang van opklaring van die meganisme van PK lê daarin dat 'n biologiese verskynsel in 'n terapeutiese modaliteit vir kliniese gebruik, omgeskakel kan word. Sou dit deur bepaalde komponente van 'n seintransduksiepad gemedieër word, is so 'n doel bereikbaar. Verskeie stimuli en seintransduksiepaaie is in PK betrokke: byvoorbeeld, reseptorafhanklike endogene stimuli (soos adenosien en opioïde), asook reseptor-onafhanklike endogene stimuli (soos vrye radikale en kalsium). Die betrokkenheid van die padrenerge seintransduksiepad asook stikstofoksied (NO) in PK egter nog nie behoorlik evalueer nie. Dit is voorgestel dat alle stimuli op 'n finale algemene pad uitloop, soos byvoorbeeld die aktivering van protein kinase C (PKC) en/of die mitogeen-geaktiveerde kinases (MAPKs), spesifiek die p38 MAPKs. Laasgenoemde se rol in PK is steeds kontroversieël. Die doel van die studie was dus: (A) karakterisering van die veranderinge in die sikliese nukleotiede, cAMP en cGMP, en p38 MAPK wat tydens die hele eksperimentele prosedure plaasvind, in 'n poging om meer insig te verkry aangaande moontlike meganismes betrokke in isgemiese PK (Hoofstuk 3); (8) bepaling van die belang van die waargenome veranderinge in cAMP en cGMP deur hulonderskeie paaie farmakologies te manipuleer (Hoofstukke 4 en 5); (C) bepaling van die rol van p38 MAPK in PK: betrokkenheid as stimulus of mediator (Hoofstuk 6). Geïsoleerde, geperfuseerde werkende rotharte is geprekondisioneer deur blootstelling aan 3 x 5 min globale isgemie, afgewissel met 5 min herperfusie, gevolg deur 25 min globale isgemie en 30 min herperfusie. Funksionele herstel tydens herperfusie is as eindpunt gebruik. Harte is op verskillende tye tydens die PK protokol, volgehoue isgemie, asook herperfusie gevriesklamp. Weefsel sikliese nukleotiede (cAMP en cGMP), die aktiwiteit van sikliese nukleotied fosfodiesterases (cAMP- en cGMP-PDE), adeniel siklase en protein kinase A (PKA) asook die eienskappe van die p-adrenerge reseptor is gemeet. p38 MAPK aktivering is met Westerse oordragtegnieke bepaal, deur van dubbel gefosforileerde p38 MAPK (Thr180fTyr182) antiliggame asook geaktiveerde transkripsie faktor 2 (ATF2) gebruik te maak. Die rol van p38 MAPK in PK beskerming is evalueer deur die effek van inhibisie van p38 MAPK aktivering met SB 203580, in volwasse geïsoleerde rot kardiomiosiete asook in geïsoleerde geperfuseerde rotharte, te bepaal. Na aanleiding van die resultate, is voorgestel dat, tydens 'n multi-siklus isgemie PK protokol, stimuli (moontlik endogene katekolamiene en NO) vrygestel word wat die sikliese veranderinge in sikliese nukleotiede, cAMP en cGMP, veroorsaak. Beide hierdie sikliese nukleotiede aktiveer die distale stres kinase, p38 MAPK, op 'n betekenisvolle, maar verbygaande manier. Hierdie kinase mag verdere distale aanpassingsprosesse stimuleer. Die volgehoue isgemiese periode van PK harte is gekenmerk deur verminderde cAMP en verhoogde cGMP vlakke, asook verminderde aktivering van p38 MAPK. Hierdie veranderinge is met beskerming van die hart teen isgemie geassosieer. Daarbenewens, farmakologiese vermindering van p38 MAPK aktivering tydens volgehoue isgemie het tot verbeterde funksionele herstel gelei. Die gevolgtrekking is gemaak dat die beskermende effek van PK die gevolg is van verminderde aktivering van isgemies-geïnduseerde p38 MAPK. Farmakologiese manipulasie van hierdie kinase moet in die toekoms as terapeutiese modaliteit oorweeg word.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 66-80). Also available on the Internet.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains iv, 60 p. : ill. (some col.). Includes abstract. Includes bibliographical references.