published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Stimulation of urine production by the Malpighian (renal) tubules in Rhodnius prolixus is regulated by at least two diuretic hormones, CRF-related peptide and serotonin, that have traditionally been believed to function through the activation of cAMP-mediated intracellular second messenger pathways. In this study I demonstrate that serotonin stimulation triggered, in addition to cAMP, intracellular Ca2+ waves in the Malpighian tubule cells of R. prolixus. Treatment with the intracellular Ca2+ chelator BAPTA-AM blocked the intracellular Ca2+ waves and reduced serotonin-stimulated fluid secretion by 75%. This suggests a role for intracellular Ca2+ signaling in the excretory system of R. prolixus. Serotonin stimulated Malpighian tubules (MTs) exposed to Ca2+-free saline plus BAPTA-AM secreted an abnormal fluid, showing: increased K+ concentration, reduced Na+ concentration and lower pH. These results along with measurement of transepithelial potential (TEP) suggest that the basolateral Na+:K+:2Cl- cotransporter (NKCC) activity is reduced in tubule cells treated with BAPTA-AM, suggesting that Ca2+ is required to modulate the activity of the basolateral NKCC. Treatment with the non-hydrolysable cell-permeable cAMP analog, 8Br-cAMP, produced fluid with the same K+ and Na+ concentration and at the same secretion rate as serotonin-stimulated tubules. In addition, 8Br-cAMP triggered intracellular Ca2+ oscillations similar to those obtained with serotonin. 8Br-cAMP-stimulated tubules treated with BAPTA-AM decreased their fluid secretion by about 40% and increased Na+ concentration, similar to the effect observed on serotonin-stimulated tubules. Therefore, I conclude that the intracellular Ca2+ waves triggered by serotonin are mediated by cAMP. The role of inositol-3-phospate (InsP3) in Ca2+ release was tested by treating the tubules with the InsP3 receptor blocker xestospongin. The treatment decreased fluid secretion rate as well as the amplitude of Ca2+ waves in serotonin-stimulated tubules. These results suggest that serotonin activates the production of InsP3 and, most likely, diacylglycerol (DAG). Thus, I decided to test whether the protein kinase C (PKC) may be involved in serotonin-stimulated secretion. The PKC inhibitors chelerythrine and bisindolylmaleimide (BIM) decreased secretion fluid rate in serotonin-stimulated tubules by 50% and 70%, respectively. Fluid secreted by tubules treated with BIM showed no differences in K+ and Na+ concentrations compared to controls, however both ion fluxes decreased. The evidence suggests that PKC is involved in serotonin stimulated secretion; the mechanism is still not understood. Taken together, the results suggest that cAMP, Ca2+ and PLC-PKC pathway are involved in serotonin stimulated secretion. However cAMP stimulation is enough for maximal secretion rate. Therefore PLC-PKC must act downstream of cAMP. Based on those results we hypothesize that serotonin binds a GPCR, increasing cAMP by activation of an adenylate cyclase (AC). Subsequently, cAMP is somehow able to activate PLC, which finally produces Ca2+ release, PKC activation and NKCC upregulation.

Thesis (Ph.D.)--Tufts University, 2002. / Adviser: Aurelie Edwards. Submitted to the Dept. of Chemical Engineering. Includes bibliographical references (leaves 210-219). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Oncology. Title from pdf file main screen (viewed on August 1, 2009). Includes bibliographical references.

During excitation-contraction coupling, Ca2+ transient induced by the depolarization of membrane potential is the trigger of mechanical contraction in cardiac myocytes, which is responsible for the pumping function of the heart. However, mechanisms underlying intracellular Ca2+ regulation and the coupling between Ca2+ transient and membrane potential are not completely understood. Abnormalities in intracellular Ca2+ regulation have been observed during heart failure and cardiac arrhythmias, such as intracellular Ca2+ alternans and T-tubule disorganization. In this project, intracellular Ca2+ dynamics in different types of cardiac myocytes were investigated by using computer modelling. For atrial myocytes, a biophysically detailed computer model was developed to describe the observations of Ca2+ alternans and Ca2+ wave propagation in cardiac myocytes lacking T-tubules. The model was validated by its ability to reproduce experimental observed Ca2+ wave propagation under normal condition and the influences on spatial Ca2+ distribution by modifying various aspects of Ca2+ cycling, such as Ca2+ influx, SR Ca2+ uptake and SR Ca2+ release in cardiac myocytes lacking T-tubules. Mechanisms underlying the genesis of Ca2+ alternans in this type of cell were investigated by the model. Furthermore, a spontaneous second Ca2+ release was observed in response to a single voltage stimulus pulse with enhanced Ca2+ influx as well as SR Ca2+ overload. For the ventricular myocytes, an existing canine model was used to study the genesis of APD and intracellular Ca2+ alternans under various conditions. The genesis of Ca2+ alternans was investigated by analyzing the relationship between systolic Ca2+ concentration and SR Ca2+ content. On the other side, the roles of SR Ca2+ regulation and action potential restitution in the genesis of intracellular Ca2+ and APD alternans were also examined under various conditions. In addition, it was shown that spatially discordant Ca2+ alternans was generated when the Ca2+-dependent inactivation of ICa,L was strong. It tended to be concordant for weak Ca2+-dependent inactivation of ICa,L. For the sinoatrial node cells, a mathematical model was developed to simulate stochastic opening of unitary L-type Ca2+ channel and single RyR channel, thereby reproducing experimental observed local Ca2+ release during diastolic depolarization phase of the action potential. Simulation results of ionic channel block and modifications of SR Ca2+ regulation suggested a limited role of intracellular Ca2+ in the automaticity of central SA node cells.

Understanding how a therapy will impact the injured kidney before being administered would be an asset to the clinical world. The work in this thesis advances the field of mathematical modeling of the kidneys to aid in this cause. The objectives of this work are threefold: 1) to develop and personalize a model to specific patients in different diseased states, via parameter estimation, in order to test therapeutic trajectories, 2) to use parameter estimation to understand the cause of different kidney diseases, differentiate between potential kidney diseases, and facilitate targeted therapies, and 3) to push forward the understanding of kidney physiology via physiology-based mathematical modeling techniques. To accomplish these objectives, we have developed two models of the kidneys: 1) a broad, steady-state, closed-loop model of the entire kidney with human physiologic parameters, and 2) a detailed, dynamic model of the proximal tubule, an important part of kidney, with rat physiologic parameters. To readily aid physicians, a human model would easily fit into the clinical workflow. Since there is a lack of invasive human renal data for validation and parameter estimation, we employ a minimal modeling approach. However, to aid in deeper understanding of renal function for future applications, targeted therapy testing, and potentially replace invasive measures, we develop a more detailed model. The development of such a model requires invasive data for validation and parameter estimation, and hence we model for rodents, where such invasive data are more readily available. The kidneys are composed of approximately one million functional units known as nephrons. The glomerular filtration rate (GFR) is the rate at which the kidney filters blood at the start of the nephron. This filtration rate is highly regulated via several control mechanisms and needs to be maintained within a small range in order to maintain a proper water and electrolyte balance. Hence, fluctuations of GFR are indicative of overall kidney health. In developing the human kidney model, we also sought to understand the relationship between blood pressure and GFR since many therapies affect blood pressure and subsequently GFR. This model describes steady-state conditions of the entire kidney, including renal autoregulation. Model validation is performed with experimental data from healthy subjects and severely hypertensive patients. The baseline model’s GFR simulation for normotensive and the manually tuned model’s GFR simulation for hypertensive intensive care unit patients had low root mean squared errors (RMSE) of 13.5 mL/min and 5 mL/min, respectively. These values are both lower than the error of 18 mL/min in GFR estimates, reported in previous studies. It has been shown that vascular resistance and renal autoregulation parameters are altered in severely hypertensive stages, and hence, a sensitivity analysis is conducted to investigate how changes in these parameters affect GFR. The results of the sensitivity analysis reinforce the fact that vascular resistance is inversely related to GFR and show that changes to either vascular resistance or renal autoregulation cause a significant change in sodium concentration in the descending limb of Henle. This is an important conclusion as it quantifies the mapping between hypertension parameters and two important kidney states, GFR and sodium urine levels. Glomerulonephritis is one of the two major intra-renal kidney diseases, characterized as a breakdown at the site of the glomerulus that affects GFR and subsequently other portions of the nephron. This disease accounts for 15% of all kidney injuries and one-fourth of end-stage renal disease patients. The human kidney model is used to estimate renal parameters of patients with glomerulonephritis. The model is an implicit system and in developing an optimization algorithm to use for parameter estimation, we modify in a novel way, the Levenberg-Marquardt optimization using the implicit function theorem in order to calculate the Jacobian and Hessian matrices needed. We further adapt the optimization algorithm to work for constrained optimization since our parameter values must be physiologically feasible within a certain range. The parameter estimation method we use is a three-step process: 1) manually adjusting parameters for the hypertension comorbidity, 2) iteratively estimating parameters that vary from person to person using no-kidney- injury (NKI) data, and 3) iteratively estimating parameters that are affected by glomerulonephritis using labeled diseased data. Such a process generates a model that is personalized to each given patient. This patient-specific model can then be used to simulate and evaluate outcomes of potential therapies (e.g., vasodilators) on the model in lieu of the patient, and observe how alterations in blood pressure or sodium level affect renal function. Parameter estimation in the presence of glomerulonephritis is a challenging task due to the complexity of the kidney physiology and the number of parameters to estimate. This is further complicated by comorbidities such as hypertension, cardiac arrythmia, and valvular disease, because they alter kidney physiology and hence, increase the number of parameters to estimate. We chose to focus on hypertension since it is very prevalent in hospitals and intensive care units. It was found that over all patients, average model estimates of GFR and urine output rate (UO) were within 9.2 mL/min and 0.71 mL/min for NKI data. These results are expectedly better than those achieved from the non-personalized model since the parameters are now specific to each patient. The results also demonstrate our ability to non-invasively estimate GFR with less error than the 18 mL/min currently possible. The estimations were validated by ensuring that the estimated parameter values were physiologically sound and matched the literature in terms of expected values for different demographic groups. It is vital for a properly functioning kidney to maintain solute transport throughout the nephron. Kidney diseases in the nephron can manifest themselves via the solute transport mechanisms. To understand how these diseases affect the kidney and to simulate transporter- targeting therapies, we have developed a detailed model, starting from the human model previously developed, of one portion of the kidneys, the proximal tubule. The proximal tubule is the site of the most active transport within the nephron and the target for several therapies. Our goal is to study and understand the dynamic behavior of the proximal tubule when solute transporters breakdown and to investigate treatment therapies targeting certain solute transporters. The proposed model is dynamic and includes several solutes’ transport mechanisms, with parameters for rats. We chose to investigate diabetic nephropathy and the associated sodium-transporter alteration (knockout) therapy. Diabetic nephropathy is characterized as kidney damage due to diabetes and affects 30% of diabetics. In terms of reducing hyperfiltration, a potential cause of diabetic nephropathy where an overabundance of solutes and fluid are filtered at the glomerulus, the model demonstrates that knockout of this transporter results in a reduction in sodium and chloride reabsorptions in the proximal tubule, thereby preventing hyperfiltration. Further, we conclude that vital flows for maintaining kidney homeostasis, fluid and ammonium reabsorptions, are corrected to healthy values by a 50% knockout (impairment) of the sodium-hydrogen transporter. Next, we use the dynamic model to detect different diseased states of the proximal tubule transporters. We have accomplished this task by using Bayesian estimation to estimate transporter density parameters (a metric for kidney health) using measured signals from the proximal tubule. This approach is validated with experimental rat data, while further investigations are conducted into the performance of the estimation in the presence of varied input signals, signal resolutions, and noise levels. Estimation accuracy within 20% of true transporter density and within 4% of true fluid and solute reabsorption was achieved for all combinations of diseased transporters. We concluded that including chloride and bicarbonate concentrations improved estimation accuracy, whereas including formic acid did not. This is an important conclusion as it can help physicians determine which blood tests to order for diagnosing kidney disease; to our knowledge, this is a first. It was also found that sodium and glucose proximal tubule concentrations are most affected by changes in the sodium-hydrogen and sodium-bicarbonate transporters. This conclusion provides insight into the interplay between solute transporter density and sodium and glucose concentrations in the proximal tubule. Such knowledge paves the way for new transporter targeted therapies.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Les cellules souches adultes assurent le renouvellement des cellules différenciées, un mécanisme indispensable à la régénération des organes soumis au vieillissement et aux agressions extérieures. Des cellules souches ont été identifiées dans les tubules de Malpighi (TM) qui assurent les fonctions rénales chez la Drosophile. Ces cellules souches rénales (CSR) proviennent à l'origine de précurseurs des cellules souches intestinales (CSI) qui migrent et colonisent les TM pendant la métamorphose. Nous montrons que le facteur de transcription shavenbaby (svb) est exprimé au sein des CSR. Svb est connu pour contrôler la morphogenèse épidermique durant le développement de la Drosophile tandis que son homologue chez l'Homme, OvoL, est impliqué dans la transition épithélio-mésenchymateuse et est dérégulé dans certains cancers. Nous avons découvert que la principale fonction de svb au sein des CSR est de prévenir l'apoptose. En effet, la perte de fonction de svb, spécifiquement au sein des CSR adultes, induit leur disparition progressive et cet effet est abolit par l'expression d'inhibiteurs d'apoptose. Tout comme dans l'épiderme, nous avons pu observer que la fonction de svb est supportée par sa maturation protéolytique induite par les gènes polished-rice et ubr3. De plus, nous démontrons que Svb interagit avec Yorkie (Yki) un membre de la voie de signalisation Hippo, connue pour contrôler le nombre de CSI. Ce complexe Svb/Yki régule l'expression de l'inhibiteur apoptotique DIAP1 afin de maintenir un nombre normal de CSR. Ces travaux ont donc permis d'identifier un nouveau membre de la voie de signalisation Hippo et de découvrir un mécanisme inattendu de protection des cellules souches contre la mort cellulaire qui pourrait expliquer leur capacité de résistance à l'apoptose. / Throughout adult life, homeostasis of fundamental functions requires cell renewal to compensate for tissue damage and cell death. The renal (Malpighian) tubules in Drosophila are responsible for excretion of metabolic waste like kidney in vertebrates. Although Malpighian tubules are thought to be very stable during development, evidence of adult cell renewal have been showed and stem cells identified. Renal and nephric stem cells (RNSC) derived from intestinal stem cell precursors that colonize Malpighian tubules during metamorphosis. We showed that the gene shavenbaby (svb) which encodes a transcription factor belonging to the OVO-Like family (OVOL) is expressed in RNSCs. In vertebrates, OvoL factors act as guardian of epithelial integrity, while in Drosophila, Svb is well identified for its role in epidermal cell shape remodeling. The transcriptional activity of Svb requires its proteolytic processing mediated by the SmORF peptides Pri encoded by the polished-rice gene. Here, we show that this processing occurs also in RNSC and that the main function of svb in these cells is to protect them from apoptosis. Svb loss of function induces a progressive disappearance of RNSC that can be rescued by blocking programmed cell death. At the molecular level, we found that Svb physically interacts with Yorkie, the downstream effector of the hippo pathway to favor the expression of the inhibitor of apoptosis DIAP1. To conclude, our work identified a new member of the hippo pathway and uncovered an additional way to protect stem cell from apoptosis that may explain their capacity to resist apoptosis.

Nous avons décrit la présence de tubules inter-cellulaires [inter-cellular tubules (ICTs)], qui apparaissent entre les deux cellules filles lors de la cytokinèse d’une cellule infectée. Nos données suggèrent que ces structures sont des vestiges de SITs qui connectaient les SCVs initialement présentes dans la cellule mère et qui ont été distribuées dans les cellules filles. Les effecteurs de T3SS2 sont nécessaires à la formation de ces tubules. De plus, nous avons établit une corrélation entre la formation des ICTs et la distribution asymétrique des vacuoles bactériennes entre les cellules filles. Les protéines effectrices de T3SS2 peuvent donc modifier la distribution des bactéries pendant la cytokinèse. Il a été démontré l’existence de différents types de SITs, de composition différentes et qui interagissent avec différents compartiments de la cellule hôte. Pendant la deuxième partie de ma thèse, nous avons caractérisé des tubules dépourvus de protéines de l’hôte mais riches en protéines effectrices [LAMP1-negative tubules (LNT)]. Nous avons montré que les effecteurs de T3SS2 SseF et SseG sont nécessaires à la formation de ces structures. L’inhibition de la formation des LNTs par la suppression de sseF/G est corrélée à un recrutement réduit de LAMP1 sur les SCVs. Cela suggère que la formation des tubules favorise la capture et le transport de LAMP1 vers les SCVs. Nous avons également observé une interaction indépendante de SKIP entre Arl8b et les deux domaines de l’effecteur SifA. En absence de SifA ou de Arl8b, les tubules ont une capacité limitée à capturer les protéines membranaires lysosomales. / We describe the presence of inter-cellular tubules (ICTs) that arise between daughter cells during cytokinesis of an infected cell. Our data suggest that these structures are remnants of SITs that connect bacterial vacuoles originally present in the parent cell and that have been distributed between daughters. T3SS-2 effectors are required for the formation of these tubules. Importantly, there is a correlation between the formation of ICTs and the asymmetric distribution of bacterial vacuoles in daughters. Thus, T3SS-2 effector proteins can manipulate the distribution of bacteria during cytokinesis. This may further increase bacterial spreading and the systemic character of the infection. Different kinds of SITs with diverse host protein contents have been characterised, suggesting the capacity of these tubules to interact with different host compartments. In the second part of my thesis, we performed a biochemical and functional characterization of LAMP1-negative tubules (LNT) that are decorated with effector proteins but essentially devoid of host proteins. We show that T3SS2 effectors SseF and SseG are required for the formation of these structures. The inhibition of LNTs formation by deletion of sseF/G is correlated with a reduced recruitment of LAMP1 to the SCVs. It suggests that formation of tubules favours the capture and the transport of LAMP1 towards the SCV to keep vacuole stable. An additional observation added to this study is that there is a SKIP-independent interaction between Arl8b and both domains of SifA. In the absence of SifA or Arl8b, tubules have a limited capacity to capture host membrane proteins from the late endosomal compartments.