Abstract The number of ovarian follicles declines with age resulting in a significant decrease of fertility by the age of 40. However, the age when follicle loss starts to affect ovarian endocrine function is not well recognized. The purpose of the present study was to investigate age-related ovarian/adrenal androgen secretion, which is crucial for estrogen biosynthesis in healthy women and in women with polycystic ovarian syndrome (PCOS). Another aim of the study was to compare the usefulness of different serum markers in assessing ovarian aging and in diagnosing polycystic ovaries (PCOs) and PCOS. The human chorionic gonadotropin (hCG) test was used to study the endocrine potential of ovaries/adrenals. The ovarian capacity to secrete and synthesize androgens was found to be decreased as early as at the age of 30 in regularly menstruating women. In women with PCOS, both basal and hCG-stimulated androgen levels were about 50% higher than in healthy women and they remained high until late reproductive age. Similarly to regularly menstruating women, the androgen secretion capacity in PCOS subjects decreased with age, and estradiol concentrations remained unchanged until the age of 44 years. Adrenal androgen synthesis was not changed during hCG-tests. Since serum antimüllerian hormone (AMH) and follicle stimulating hormone (FSH) levels were changed significantly after the age of 25 years in regularly menstruating women, they may be considered as useful serum markers reflecting the ovarian aging process. In women with PCOS, AMH levels were continuously 2- to 3-fold higher than in healthy women possibly reflecting high follicle number in these women. A decline in ovarian endocrine function before the age of 30 is one of the first signs of ovarian aging. However, in women with PCOS ovarian androgen secretion capacity is markedly increased and remain high throughout the reproductive years. The results of the present studies also indicate that LH/hCG does not play a role in adrenal androgen synthesis, since LH/hCG did not stimulate adrenal androgen synthesis. The measurement of AMH is a useful tool to estimate ovarian aging process as well as to diagnose PCOs/PCOS.

Shigella est une bactérie pathogène à Gram-négatif qui cause la shigellose ou dysenterie bacillaire. Cette maladie est une cause importante de morbidité chez les jeunes enfants et les adultes dans les pays en développement, avec presqu’un million de morts par an. Jusqu'à ce jour, aucun vaccin efficace contre Shigella n’est disponible et la prévalence croissante de souches multirésistantes fait de Shigella une menace importante en termes de santé publique. La virulence de Shigella est médiée par la sécrétion d'un arsenal de protéines (effecteurs) capables d'interférer avec diverses voies de signalisations de la cellule hôte. La sécrétion de effecteurs dépend d'un système de sécrétion de type 3 (SST3), véritable aiguille moléculaire assemblée à la surface bactérienne permettant l’injection les protéines de virulence dans le cytoplasme de la cellule hôte. Le SST3 est assemblé à 37 °C et est composé de trois parties distinctes: i) un bulbe cytoplasmique, ii) un corps basal et ii) une aiguille extracellulaire. Cette dernière est constituée de l’assemblage d’une centaine de copies de la protéine MxiH pour la partie extracellulaire et de plusieurs copies de la protéine MxiI pour la partie périplasmique. Avant le contact cellulaire, le SST3 est maintenu dans un état "OFF" d’une part, par un complexe de protéines (IpaD et IpaB) localisé au sommet de l’aiguille et, d’autre part par une protéine (MxiC) probablement située à la base du SST3 via son interaction avec MxiI. Suite au contact cellulaire, le SST3 est activé et permet la sécrétion d’IpaC, protéine hydrophobe formant, avec IpaB, un pore dans la membrane de la cellule hôte pour permettre la translocation des effecteurs. Suite à la détection du contact cellulaire par le complexe d’extrémité (IpaB et IpaD), un signal est transmit jusqu’à la base de l’aiguille afin d’activer la sécrétion de MxiC et finalement celle des effecteurs. Des études mutationnelles ont montré que la transmission du signal d'activation du haut vers la base de l’aiguille implique les sous-unités de l’aiguille MxiH et MxiI. Ce signal permet la dissociation de MxiC et MxiI et donc la libération des effecteurs. Dans ce travail, nous avons caractérisé le rôle de MxiI dans la transmission du signal d'activation de la sécrétion via une série de mutations dirigées et aléatoires et déterminé le domaine responsable de son interaction avec MxiC. D'autre part, nous avons mis en évidence un lien entre la sécrétion de MxiC par le SST3 et celle d'un effecteur, IcsB. IcsB nécessite une protéine chaperon, IpgA, pour sa stabilité et sa sécrétion. Une fois sécrétée, elle joue différentes fonctions dans la cellule hôte et notamment, permet l’échappement de Shigella à l’autophagie. Dans ce travail, nous avons montré que IcsB est capable de lier le cholestérol et que cette liaison est nécessaire pour l’échappement de de la bactérie à l’autophagie. Nous avons montré, en utilisant des approches biochimiques, qu’IcsB pourrait avoir une activité de type cystéine protéase via une diade catalytique conservée (C306 et H145). Nous avons identifié de nouveaux partenaires d'interaction d’IcsB, tels que OspB et VirA, ce qui pourrait permettre de mieux comprendre la fonction d’IcsB dans la cellule. En conclusion, ce travail a permis de mieux comprendre le mécanisme d'activation du SS T3 ainsi que la fonction de l'effecteur IcsB dans la virulence de Shigella. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

La sécrétion d'hormones et de neuropeptides par les cellules neuroendocrines est assurée par un processus d'exocytose, contrôlé notamment par les GTPases Rho. La compréhension des mécanismes moléculaires régulant la sécrétion neuroendocrine est primordiale. En effet, la majorité des cancers neuroendocrines tels que les phéochromocytomes, sont associés à une perturbation du processus de sécrétion. Actuellement, les mécanismes moléculaires qui induisent de telles perturbations de la sécrétion ainsi que l’impact de l’activité sécrétrice sur le développement des tumeurs neuroendocrines ne sont pas élucidés. Mes travaux de thèse proposent pour la première fois un lien fonctionnel direct entre l'activité sécrétrice des cellules et la vitesse de développement des phéochromocytomes ainsi qu’une altération des voies moléculaires impliquant certaines protéines Rho, en démontrant un lien entre la baisse de l’activité de Rac1 et Cdc42 observée dans les phéochromocytomes et la diminution de l’expression de leurs régulateurs ARHGEF1 et FARP1. / Neuroendocrine cells secrete hormones and neuropeptides through calcium-regulated exocytosis, controlled especially by Rho GTPases. Neuroendocrine tumours, such pheochromocytomas, are generally associated with a dysfunction of secretion. Although this aspect is well known by clinicians, it has never been explored at the molecular level. Moreover, the potential link between secretion and tumour development remains uninvestigated. Altogether, our results demonstrate for the first time the importance of secretion in tumor development of pheochromocytomas and an alteration of the Rho GTPase pathway, by demonstrating a link between the inhibition of Rac1 and Cdc42 activity observed in pheochromocytomas and the decrease of their activators ARHGEF1 and FARP1 expression.

Recent bioinformatic analyses identified an operon encoding a potential Type I Secretion System (T1SS) in Treponema pallidum that we hypothesize functions to export key treponemal virulence factors that may contribute to the unique invasiveness and pathogenesis of this spirochete. The membrane fusion protein component (MFP) of T1SSs in other organisms has been shown to play a role in substrate recognition. Hence, the objective of this project is to use the putative MFP, Tp0965, of the potential T. pallidum T1SS to investigate protein-protein interactions with the T. pallidum virulence factor pallilysin (Tp0751) and assess the possibility of the latter being a T1SS substrate. Moreover, protein-protein interactions between Tp0965 and a Treponema phagedenis lysate are investigated with the goal of identifying putative T1SS substrates in this spirochete that could result in the discovery of novel T. pallidum virulence factors via amino acid sequence similarity. Plate-based binding studies and pull-down assays showed a low level of interaction between recombinant Tp0965 and the previously characterized host-component-binding protease, pallilysin, suggesting that the export of this virulence factor could occur via the putative T1SS. Additionally, bioinformatic analyses of the related but cultivable model spirochete T. phagedenis predicted the presence of a potential T1SS homologous to the putative T1SS in T. pallidum. Thus, a more global and unbiased pull-down assay using “bait” Tp0965 and a “prey” T. phagedenis lysate was carried out, followed by mass spectrometric analysis to identify putative novel T1SS substrates with potential homologs in T. pallidum. We successfully identified a T. phagedenis protein, TphBIg, that showed evidence of an interaction with Tp0965. TphBIg seems to possess characteristics of a T1SS substrate suggesting it may be secreted via this system in T. phagedenis. Upon bioinformatic analysis, it was found that TphBIg showed weak amino acid sequence similarity as well as some structural similarity to the T. pallidum protein, Tp0854. Tp0854 is predicted to contain a sialidase and a phosphatase domain with an RTX motif, which is characteristic of some T1SS substrates. Thus, it was hypothesized that if Tp0854 had characteristics of a T1SS, it may interact with Tp0965. Therefore, the phosphatase domain containing the RTX motif was produced recombinantly and plate-based binding studies indeed suggested an interaction with Tp0965, confirming the in silico-predicted interaction. Future experiments to characterize the potential T1SS and substrates in T. pallidum could comprise the functional and structural characterization of the novel putative T1SS substrate, Tp0854. This would include assays to investigate the putative sialidase and phosphatase activities of Tp0854, as well as the identification of Tp0854-Tp0965 interacting sites. Moreover, as a more definite test for T1SS substrate secretion, T. pallidum pallilysin and/or Tp0854 could be expressed heterologously in an E. coli strain harbouring an endogenous T1SS and test for secretion. Similarly, the reconstitution of the T. pallidum putative T1SS in liposomes could be used to further investigate the secretion of pallilysin and/or Tp0854 via this system. Additionally, the optimized unbiased pull-down technique could be further applied to detect more protein-protein interactions within T. pallidum and potentially lead to the identification of more virulence factors that may be secreted via the T1SS. These studies constitute the first investigation of a putative T1SS and substrates within T. pallidum. Thus, insight gained will lead to a better understanding of the mechanisms facilitating T. pallidum host invasion and may reveal new potential vaccine targets to prevent bacterial dissemination and chronic infection. / Graduate

Les syndromes lipodystrophiques sont des maladies rares caractérisées par une perte sélective et en quantité variable de tissu adipeux (TA). Ils constituent un groupe hétérogène de pathologies du TA, de par des étiologies multiples, la quantité de perte du tissu adipeux (généralisée ou partielle), le caractère congénital ou acquis, l'intégration dans des pathologies systémiques complexes ou non. Sur le plan physiopathologique, la limitation des capacités d'expansion du TA dans ces syndromes conduit aux troubles métaboliques dominés par l'insulinorésistance majeure avec notamment diabète et hypertriglycéridémie. La leptine, hormone aux effets pléiotropes sécrétée par le tissu adipeux, est déficiente dans ces syndromes. Son utilisation thérapeutique dans les syndromes lipodystrophiques permet une amélioration de l'insulinorésistance et des paramètres glucido-lipidiques. Afin de mieux caractériser les mécanismes d'amélioration de ces paramètres, nous avons traité la première cohorte française de patients diabétiques atteints de lipodystrophies génétique par leptine recombinante. Nous avons montré une amélioration de l'insulinosécrétion par des mesures de référence ainsi qu'une diminution des concentrations sériques de l'enzyme PSCK9 sous traitement, qui pourraient contribuer à l'amélioration des paramètres lipidiques. Nous avons également étudié des patients traités par glucocorticoïdes et développant une lipodystrophie glucocorticoinduite et nous avons montré que la concentration de leptine avant traitement est prédictive du risque de survenue de ces lipodystrophies. Ainsi la leptine semble jouer un rôle clé dans la physiopathologie de différents syndromes lipodystrophique. / Lipodystrophic syndromes are very rare diseases characterized by selective loss of varying amounts of adipose tissue. They constitute a heterogeneous group of adipose tissue diseases, by multiple etiologies, the amount of loss of adipose tissue (generalized or partial), congenital or acquired character, integration in complex or non-complex systemic pathologies. On the physiopathological level, the limitation of the capacities of expansion of adipose tissue in these syndromes leads to metabolic disorders dominated by major insulin resistance with especially diabetes and severe hypertriglyceridemia.Leptin, a hormone secreted by adipose tissue, is deficient in these syndromes. It has pleiotropic effects and its therapeutic use in these syndromes allows an improvement of insulin resistance and of glucose and lipid parameters. In order to better characterize the metabolic improvement mechanisms, we treated the first French cohort of diabetic lipodystrophic patients with recombinant leptin and we were able to demonstrate an improvement of insulin secretion by the reference measures calculated during hyperinsulinemic euglycemic clamps and hyperglycaemic clamps. We also showed a decrease in the serum concentration of PSCK9 under treatment, which could contribute to the improvement of lipid parameters. We also studied patients treated with glucocorticoids and with glucocorticoinduced lipodystrophy and we were able to show that the concentration of leptin prior to treatment is predictive of the occurrence of lipodystrophies. Thus leptin appears to play a key role in the pathophysiology of different types of lipodystrophies.

Abstract Orexins, or hypocretins, are peptides originally found in the hypothalamus, and have been shown to be involved in the stabilization and maintenance of sleep and wakefulness. In addition, these peptides are known for their actions on energy homeostasis by increased heat production or physical activity. Previous results suggest them to be also involved in peripheral actions on the regulation of intestinal secretion, depending on the subject’s nutritional status (fasted-fed). Orexin-A and Orexin-B peptides, are derived from the prepro-orexin precursor protein. These ligands bind to two G-protein-coupled receptors, orexin-1 and -2 -receptors. Despite intensive research, the role of orexins has not yet been clarified. The aim of the present study was to investigate the role of orexins and their receptors on sleep and wake patterns, energy homeostasis and intestinal secretion. The effects of orexins on sleep and wakefulness, and energy homeostasis were studied in a novel transgenic mouse line, overexpressing the human prepro-orexin gene. The overexpression of prepro-orexin and orexin-A was confirmed in the hypothalami of transgenic mice. The transgenic mice showed a significant reduction in their REM sleep during day and night time, and differences in their vigilance states in the light/dark transition periods. In addition, the mice demonstrated a significantly elevated day time food intake at room temperature, and an increased metabolic heat production independent of uncoupling protein 1 mediated thermogenesis in brown adipose tissue. Instead, transgenic mice showed increased levels of uncoupling protein 2 in white adipose tissue. Furthermore, transgenic mice significantly decreased their total locomotor activity during the first two nights in response to cold exposure (+4°C). The effect of orexins and their receptors on duodenal HCO3– secretion were studied after an overnight (16 h) food deprivation in an in situ perfused organ. Fasting decreased the expression of orexin receptors in rat duodenal mucosa and in acutely isolated duodenal enterocytes. Furthermore, food deprivation abolished OXA induced duodenal mucosal HCO3– secretion in rats, and intracellular calcium signalling in isolated rat and human duodenal enterocytes. In conclusion, the present thesis demonstrates that orexins inhibit REM sleep. In addition, peptides affect increasingly on metabolic heat production, independent of uncoupling protein 1 mediated thermogenesis. Furthermore, the orexin system has a significant role in duodenal bicarbonate secretion, which is regulated by the presence of food in the intestine.

Les neurotoxines botuliques (BoNTs), agents responsables du botulisme, sont les substances les plus toxiques actuellement connues. L'utilisation d'anticorps neutralisants pour l'immunothérapie passive est la seule solution retenue et accréditée par la FDA à l'heure actuelle. L'immunisation génétique par électroporation intramusculaire d'ADN s'est révélée être une alternative crédible à l'immunisation protéique pour produire des anticorps dirigés contre les sérotypes A et E des BoNTs avec un pouvoir neutralisant suffisamment élevé pour satisfaire aux exigences de la Pharmacopée Européenne. Cependant, les résultats se sont avérés décevants dans le cas du sérotype B, troisième type antigénique associé au botulisme humain. Comprendre la raison de la faible immunogénicité de BoNT/B en vaccination ADN et l'optimiser pour produire des anticorps neutralisants dirigés contre cette dernière ont constitué les deux objectifs de mon travail. En combinant différentes approches, nous avons montré que, contrairement à BoNT/A, la sécrétion de l'antigène BoNT/B est inhibée. Nous avons identifié le réticulum endoplasmique (RE) comme étant l'organite de rétention. Nous avons étudié un lien empirique entre la séquence protéique antigénique de BoNT/B et son accumulation intracellulaire et avons conclu que le mauvais repliement de la protéine antigénique était responsable de sa rétention dans le RE. Parmi les stratégies testées afin d'augmenter l'immunogénicité de BoNT/B en vaccination ADN, l'ajout de sites de N-glycosylation a permis de multiplier par 10 le pouvoir neutralisant des anticorps et l'utilisation des dérivés d'acides biliaires comme adjuvants de formulation est prometteuse. / Botulinum neurotoxins (BoNT) have been characterized to be the most potent toxic substances identified so far. Passive immunization with antiBoNT antisera is the only treatment for botulism to have gained FDA approval. We have previously shown that genetic immunization by the non-viral intramuscular DNA electroporation technique is an effective alternative to recombinant proteins immunization to raise high titer neutralizing antibodies against BoNT serotype A and E. The neutralizing titers obtained were high enough to fit the European Pharmacopeia while it did not for type B, commonly linked to human disease as well. Finding out why BoNT/B immunogenicity is low after DNA vaccination and how we could improve it for generation of high-titer neutralizing antiBoNT/B antiserum were the two main goals of my work. Combining different approaches, we have first shown that BoNT/B antigen secretion was inhibited, while it did not for BoNT/A. We identified the endoplasmic reticulum to be the organelle of retention. Then, we studied an empirical link between BoNT/B antigenic protein sequence and intracellular accumulation. We concluded that protein misfolding could be the reason for BoNT/B retention in the endoplasmic reticulum. Among different strategies tested to improve BoNT/B immunogenicity in DNA vaccination, addition of N-glycosylation sites yielded 10 fold higher neutralizing antibodies titers. Finally, bile salts could be promising adjuvants for plasmid DNA vaccines.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Les récepteurs purinergiques P2Y ont un rôle modulateur de la sécrétion d'insuline et constituent une cible potentielle pour la recherche de nouveaux antidiabétiques. Dans le modèle du pancréas isolé perfusé de Rat, dans des conditions fonctionnelles proches de la physiologie, nous avons montré que l'activation de ces récepteurs potentialise l'effet insulino-sécrétoire d'une stimulation glucosée. Cet effet requiert le métabolisme du glucose ; il est probablement lié à une augmentation de la concentration intracellulaire de Ca2+, et est indépendant d'un effet direct sur les canaux potassiques ATP dépendants. Nous avons également étudié les effets pancréatiques de nouveaux agonistes des récepteurs P2Y, synthétisés par l'équipe du Pr B. Fischer : parmi ces dérivés, le 2-méthylthio-ATP-a-S (isomère A) est un insulino-sécrétagogue gluco-dépendant, hautement efficace et puissant (CE50 de 28,1 nmol/l), avec une très bonne sélectivité tissulaire. Par ailleurs, nous avons exploré l'implication des récepteurs P2Y dans la prolifération cellulaire en utilisant un modèle de lignée cellulaire insulino-sécrétrice issue d'insulinome de Rat (lignée INS-1E) : aux doses utilisées, l'ATP-a-S (agoniste du récepteur P2Y) et le GLP-1 entraînent une réponse insulinique comparable, mais une augmentation de l'AMPc intracellulaire d'amplitude différente ; de plus, contrairement au GLP-1, l'ATP-a-S est sans effet sur la prolifération des cellules. Enfin, en collaboration avec l'équipe du Pr C. Gachet, nous avons montré l'implication des récepteurs de sous-type P2Y1 dans la réponse insulinique d'îlots pancréatiques de Souris. Nos travaux ont donc permis d'améliorer les connaissances sur les récepteurs purinergiques P2Y des cellules ß pancréatiques en ce qui concerne leurs mécanismes d'action, leurs effets sur l'insulino-sécrétion et sur la prolifération cellulaire ; nous avons également contribué au développement d'agonistes spécifiques, potentiellement intéressants pour le traitement du diabète de type 2. / P2Y purinergic receptors play a role in the modulation of insulin secretion and represent a potential therapeutic target for new antidiabetic drugs. In the model of isolated perfused rat pancreas, in functional conditions close to physiology, we have shown that the activation of these receptors amplify the glucose-induced insulin secretion. This effect requires the metabolism of glucose; it is probably related to a rise in intracellular Ca2+ concentration, and is independent from a direct effect on ATP-dependent potassium channels. We have also studied the pancreatic effects of new P2Y receptor agonists, synthesized by the group of Prof. B. Fischer: among these compounds, 2-methylthio-ATP-a-S (A isomer) is a glucose-dependent insulin secretagogue, with high efficacy and potency (EC50 at 28.1 nmol/l), and with very good tissue selectivity. On the other hand, we have investigated the implication of P2Y receptors in cell proliferation, using a model of insulin secreting cell line from rat insulinoma (INS-1E cell line): at the doses used, ATP-a-S (a P2Y receptor agonist) and GLP-1 induce a similar insulin response, but an increase in intracellular cAMP of different magnitude; moreover, in contrast to GLP-1, ATP-a-S is ineffective on cell proliferation. Finally, in collaboration with the group of Prof. C. Gachet, we have shown that the P2Y1 receptor subtype was involved in the insulin response of mice pancreatic islets. Thus, our studies contributed to the improvement of knowledge on P2Y purinergic receptors of pancreatic ß-cells, as regards their mechanisms of action, their effects on insulin secretion and on cell proliferation; we also contributed to the development of specific agonists, potentially interesting for the treatment of type 2 diabetes.

Fluid secretion by insect Malpighian tubules is controlled by haemolymph-bome factors. Two corticotropin-releasing-factor (CRF)-related diuretic peptides, Tenmo¬DH37 and Tenmo-DH47. previously isolated from Tenebrio molitor, were found to stimulate in vitro tubule preparations of Tenebrio molitor via the second messenger cyclic AMP. The stimulatory effect of Tenmo-DH37 was reversed on addition of endogenous antidiuretic peptides (Tenmo-ADFa and ADFb) and exogenous cardioacceleratory peptide 2b (CAP2b), both acting via the second messenger cyclic GMP. The immunocytochemical localization of Tenmo-DH37 and the second antidiuretic peptide isolated from Tenebrio molitor, Tenmo-ADFb, was investigated using antisera raised against these hormones. Neurosecretory cells immunoreactive to Tenmo-DH37 were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH37 were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-ADFb immunoreactivity was localized in the brain, in two pairs of bilaterally symmetrical cells in the protocerebrum. Tenebrio tubule secretion appears entirely dependent on the surrounding K+ concentration and intracellular measurements of the basolateral (Vbl) and indirectly apical membrane potentials (Vap) indicate an appreciable sensitivity of both membranes to the bath K+ concentration, but not to Na+. Secretion assay and electrophysiological results indicate that K+ uptake across the basolateral membrane is primarily through barium-sensitive K+ channels, but also implicate a bumetanide-sensitive Na+/K+/2CI cotransporter, an ouabain-sensitive Na+/KV+-ATPase and glibenclamide-sensitive KATP channels. Furthermore, electrophysiological evidence suggests that fluid secretion/inhibition by endogenous factors is achieved by influencing at least three parameters simultaneously: the rate of H+ extrusion by the V-ATPase, basolateral K+ conductance, and possibly CI- conductance. The effect of amiloride on fluid secretion and pH indicates the presence of a cationic H+ exchanger in Malpighian tubules of Tenebrio. To our knowledge the mealworm Tenebrio molitor provides the first known example of antagonistic interactions between endogenous neuropeptides acting on Malpighian tubules and this study is the first to demonstrate the presence of KATP channels in an insect epithelium. / Thesis (DPhil (Zoology))--University of Pretoria, 2006. / Zoology and Entomology / unrestricted