The metanephric kidney consists of two types of epithelia; the Wolffian duct-derived ureteric bud and the nephrogenic components that originate from mesenchymal-toepithelial transitions in the metanephric mesenchyme. The ureteric bud forms when inductive signals from the metanephric mesenchyme stimulates the evagination of an epithelial tube from the Wolffian duct into the mesenchyme. Reciprocal signalling between the ureteric bud and the metanephric mesenchyme regulates the branching of the ureteric bud and the induction of nephron formation. Inductive and inhibitory signalling of ureteric bud growth and branching has been shown by several protein families, however, the mechanical aspects of ureteric bud branching and nephrogenesis are largely unknown. I investigated the roles of Rac1-GTPase and Rho-kinase during kidney development. These proteins are important regulators of the cytoskeleton where Rac1 is a promoter of actin filament polymerisation and Rho-kinase directly stimulates the formation and contraction of actin-myosin stress fibres. Using a cell-permeable inhibitor, Rac1 was inhibited with no effects on nephron formation or subsequent segmentation and patterning. Inhibition of active Rac1 significantly reduced the level of ureteric bud branching and also resulted in lower proliferation rates. Rho-kinase was similarly targeted using two inhibitors. Rho-kinase inhibition had important effects on nephron formation and nephron maturation. Inhibition of Rhokinase resulted in decreased levels of nephron formation and severely morphologically abnormal nephrons. The formation of apical-basal polarity was disturbed as was the development of the visceral and parietal epithelia; precursors of the renal corpuscle. Inhibition of Rho-kinase led to abnormal formation of the proximal-distal axis and abnormal segmentation of the nephron. The effects of Rho-kinase inhibition were partially mimicked by direct targeting of actin-myosin contractions using a myosin-ATPase inhibitor. This demonstrated that Rho-kinase is necessary during multiple stages of nephrogenesis and maturation, at least in part, as a result of its ability to regulate actin-myosin contraction. These results show that Rac1 and Rho-kinase play important roles during several aspects of kidney development and highlights the significance of further investigating the mechanisms involved during kidney organogenesis.

Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to vasoconstrictors, and enhanced myogenic responsiveness. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of the fundamental control mechanisms at this level of the circulation. One of the key elements within most vascular networks, including the cerebral circulation, is the presence of myogenic tone, an intrinsic process whereby resistance arteries constrict and reduce their diameter in response to elevated arterial pressure. This process is centrally involved in the ability of the brain to maintain nearly constant blood flow over a broad range of systemic blood pressures. The overall goal of this dissertation was to investigate the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. To reveal the contributions of various signaling factors in this process, measurements of diameter, intracellular Ca2+ concentration ([Ca2+]i), membrane potential and ion channel activity were performed. Initial work determined that two purinergic G protein-coupled receptors, P2Y4 and P2Y6 receptors, play a unique role in mediating pressure-induced vasoconstriction of PAs in a ligand-independent manner. Moreover, a particular transient receptor potential (TRP) channel in the melastatin subfamily, i.e. TRPM4, was also identified as a mediator of PA myogenic responses. Notably, the observations that inhibiting TRPM4 channels substantially reduces P2Y receptor-mediated depolarization and vasoconstriction, and that P2Y receptor ligands markedly activate TRPM4 currents provide definitive evidence that this ion channel functions as an important link between mechano-sensitive P2Y receptor activation and the myogenic response in PAs. Next, the signaling cascades that mediate stretch-induced TRPM4 activation in PA myocytes were explored. Interestingly, these experiments determined that the RhoA/Rho kinase signaling pathway is involved in this mechanism by facilitating pressure-induced, P2Y receptor-mediated stimulation of TRPM4 channels, leading to subsequent smooth muscle depolarization, [Ca2+]i increase and contraction. Since Rho kinase is generally accepted as a 'Ca2+-sensitization' mediator, the present, contrasting observations point to an underappreciated role of RhoA/Rho kinase signaling in the excitation-contraction mechanisms within the cerebral microcirculation. Overall, this dissertation provides evidence that myogenic regulation of cerebral PAs is mediated by mechano-sensitive P2Y receptors, which initiate the RhoA/Rho kinase signaling pathway, subsequent TRPM4 channel opening, and concomitant depolarization and contraction of arteriolar smooth muscle cells. Revealing the unique mechanochemical coupling mechanisms in the cerebral microcirculation may lead to development of innovative therapeutic strategies for prevention and treatment of microvascular pathologies in the brain.

L'os est un tissu dynamique résultant de l'équilibre entre l'activité de résorption des ostéoclastes (OCs) et l'activité de formation des ostéoblastes. Cette fonction ostéoclastique se met en place à l'intérieur de la zone de scellement qui est formée à travers une profonde réorganisation du cytosquelette médiée par la GTPase Rho, Rac1. Récemment, le laboratoire a démontré l'implication essentielle de Dock5, un facteur d'échange de Rac1, dans la formation de la zone de scellement et consécutivement, dans la dégradation du tissu osseux. En effet, les analyses des OCs matures issus de souris Dock5-/- différenciés sur support minéralisé, montrent une diminution importante du nombre de cellules présentant cette structure. Ce résultat est corrélé à la diminution drastique de la surface des lacunes de résorption formées par les OCs Dock5-/-. De plus, les études in vivo révèlent une augmentation de la masse de l'os trabéculaire des souris Dock5-/- adultes.Dans ce contexte, d'une part, nous avons analysé la voie de signalisation de Dock5 et de ces partenaires impliqués dans cette réorganisation du cytosquelette d'actine. Nos études protéomiques nous ont conduit à porter un intérêt plus particulier à la Tensine3 (Tns3), une protéine d'adhésion. Le domaine END de la Tns3 interagit avec Dock5 et nous avons localisé ces deux protéines au niveau de la ceinture de podosomes des OCs matures. De plus, nous avons montré que l'inhibition de la Tns3 perturbe l'organisation de la ceinture de podosomes des OCs et que la coexpression de la Tns3 et de Dock5 augmente le niveau de Rac1 actif dans les cellules HEK293. L'ensemble de nos résultats suggèrent, à travers son rôle de recruteur et d'activateur de Dock5, une importante implication de la Tns3 dans l'organisation de la ceinture de podosomes dépendante de Dock5.D'autre part, pour déterminer le stade développemental à partir duquel la fonction de résorption des OCs est nécessaire au développement normal de l'os, nous avons effectué des analyses histologiques sur les souris Dock5-/- durant l'ossification endochondrale. Nos données démontrent que l'activité de résorption des OCs n'est pas impliquée dans la dégradation du tissu cartilagineux minéralisé. Notre modèle d'étude, les souris Dock5-/-, nous a permis de dissocier l'implication de l'activité de résorption des OCs de la fonction de la métalloprotéase 9 (MMP9) ostéoclastique. La MMP9 ostéoclastique est un acteur crucial de la physiologie du cartilage hypertrophique durant le processus d'ossification endochondrale. Nos études sur l'os trabéculaire révèlent que l'activité de résorption des OCs devient essentielle à 7 jours de développement post-natal pour dégrader l'os minéralisé. L'ensemble de nos résultats suggèrent pour la première fois une implication spatiale et temporelle, complémentaire de l'activité de résorption des OCs et de la fonction MMP9 ostéoclastique durant le développement endochondral. / Bone is a dynamic tissue resulting from the bone resorption activity of osteoclasts (OCs) compensated by the bone formation activity of osteoblasts. This osteoclastic function takes place inside a structure called sealing zone. It is formed through a deep actin cytoskeleton remodeling involving the RhoGTPase Rac1. Recently, the laboratory demonstrated the essential implication of Dock5, a Rac1 exchange factor, in establishing the sealing zone and consequently in bone degradation. Indeed, in vitro analyses of OCs differentiated from Dock5-/- mice show an important decrease of cells having a sealing zone. This result is correlated with a drastic diminution of resorption pits surface as compared to control OCs. In addition, in vivo studies revealed an increase of Dock5 -/- adult mice trabecular bone mass. In this context, on one hand, part of my work was to investigate the signaling pathway of Dock5 and its partners implicated in this actin cytoskeleton reorganization. Proteomic analysis led us to focus our interest on Tensine3 (Tns3), an adhesion protein. We defined the END domain of Tns3 as a Dock5 interaction domain and localized these two proteins in podosomes belt of mature osteoclasts. Moreover, we shown that silencing of Tns3 disturbed podosomes belt organization of osteoclasts and that coexpression of Tns3 and Dock5 increased Rac1 activity in HEK293 cells. Taken together, our results suggest an important implication of Tns3 in Dock5-dependent podosomes belt organization, through a Dock5 localization and a Dock5 activation role.My second aim was to determine the developmental stage from which OCs resorption function is necessary for bone to develop normally. We performed histological analyses of Dock5-/- mouse bone during endochondral ossification. Our analyzes demonstrate that OCs resorption activity is not implicated in mineralized hypertrophic cartilage degradation. Our model of study, the Dock5-/- mice, allowed us to distinguish the implication of OCs resorption activity from osteoclastic metalloprotease 9 (MMP9) function. This led us to define osteoclastic MMP9 as crucial actor in physiology of hypertrophic cartilage during the endochondral ossification process whereas Ocs resorption activity becomes essential at 7 days of post-natal development to degrade mineralized bone. Taken together, our results suggest for the first time, a complementary spatial and temporal implication of resorption activity of OCs and osteoclastic MMP9 function during endochondral development.

The Hap2/3/4/5 complex is a heme-activated, CCAATT binding, global transcriptional activator of genes involved in respiration and mitochondrial biogenesis in the yeast species Saccharomyces cerevisiae. Hap4 is the regulatory subunit of the complex and its levelsdetermine the activity of the complex. Hap4 is known to play a signaling role in response toenvironmental conditions; however, little is known about the regulation of Hap4 levels or how it responses to a cell’s functional state. The activity of the Hap2-5 complex is known to be reduced in respiratory-deficient cells. In Liu Lab, it has previously been found that a link between Hap4 stability, mediated through 26S proteasome-dependent degradation, and dependence on mitochondrial functional state plays a regulatory role on downstream targets of the Hap complex. However, the mechanism behind this regulation is still largely unknown. In normally functioning yeast cells, Hap4 is a highly unstable protein with a half-life of ~10 min. We have observed that loss of mitochondrial DNA in respiratory deficient rho 0 cells has a role in the further destabilization of Hap4 to a half-life of ~4 min through the ubiquitin-proteasome pathway. Through the screening of a collection of mutants defective in E2 ubiquitin-conjugating enzymes, we show that Hap4 is greatly stabilized in ubc1Δubc4Δ double mutant cells. We also show that Hap4 stabilization in the ubc1Δubc4Δ mutant leads to increased activity of the Hap2-5 complex, indicating that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria through ubiquitin/proteasome-dependent degradation of Hap4. Furthermore, studies on Hap4 mutants involving two highly conserved cysteine residues led to a proposed mechanism behind the regulation of Ubc4 activity towards Hap4 in response to changes in the cellular redox state.

No description available.

L’efficacité de la réponse immunitaire adaptative repose tout particulièrement sur la motilité des lymphocytes T naïfs entre la circulation sanguine et les organes lymphoïdes secondaires, leur permettant ainsi de rencontrer un antigène spécifique. De nombreuses voies de signalisation sont impliquées dans ce phénomène. En particulier, les Rho GTPases y jouent un rôle central, par leur capacité à moduler le cytosquelette d’actine. Nous avons identifié la protéine Fam65b comme nouveau régulateur de la circulation des lymphocytes T. En effet, nous avons montré que la diminution de l’expression de Fam65b dans des LT primaires humains induit une augmentation de leur polarisation, leur adhésion et leur migration in vitro. Afin d’étudier son rôle dans un contexte plus physiologique, nous avons développé au laboratoire une souris Fam65b-/-, dans laquelle l’expression de Fam65b est supprimée dans le lignage T. Les lymphocytes T issus de ces souris présentent un contenu global en F-actine réduit, une plus grande quantité de L-sélectine et d’intégrines actives à leur surface, et une migration moins rapide et moins rectiligne que leurs équivalents WT. Nous n’avons pu observer, avec nos méthodes, aucune différence significative de polarisation, de migration in vitro ou d’entrée dans les organes lymphoïdes secondaires pour les LT Fam65b-/-. Nous avons identifié les Rho GTPases comme médiateurs de ces effets de Fam65b. Nous avons observé, en cytométrie de flux, que les niveaux de RhoA-GTP et de Rac-GTP sont plus élevés dans les LT murins Fam65b-/-, et que cela est aussi vrai pour RhoA-GTP dans les LT humains exprimant de faibles niveaux de Fam65b. Nous avons identifié, dans des expériences in vitro, le mécanisme par lequel Fam65b inhibe l’activité de RhoA, puisqu’il ralentit sa charge en GTP par les protéines GEF. Nous avons montré, par des techniques de biochimie, que l’activation de RhoA en aval d’une stimulation chimiokine est permise par la dissociation de RhoA et de Fam65b, probable conséquence de la phosphorylation de Fam65b. Cette dissociation a aussi été observée pour Fam65b et Rac1, mais les mécanismes mis en jeu restent à déterminer. D’autre part, l’expression de Fam65b est sous le contrôle du facteur de transcription FOXO1, connu pour son rôle dans le contrôle de l’écotaxie (homing) via la régulation de l’expression de molécules permettant l’entrée dans les ganglions lymphatiques. Fam65b, régulateur atypique de l’activité des Rho GTPases, représente donc un lien inédit entre la voie PI3K/FOXO1 et les Rho GTPases. / The motility of naive T lymphocytes between the blood and secondary lymphoid organs is essential to the efficiency of the adaptative immune response, and allows those cells to meet their cognate antigen. Numerous signaling pathways are involved in this phenomenon, such as Rho GTPases, modulators of the actin cytoskeleton. We have identified Fam65b as a new regulator of T lymphocytes recirculation. We have shown that a decrease of Fam65b expression in human primary T cells increases the morphological polarization, the adhesion and the in vitro migration of those cells. Looking for a more physiological model, we developed, in the lab, a Fam65b KO (Knock-Out) mouse, specific to the T lineage. In those animals, T cells showed decreased levels of F-actin, an increase in the display of L-selectin and integrins, and a slower and less straight migration, compared to WT (Wild-Type) T cells. On the other hand, we weren't able to see any significant differences in the morphological polarisation, the in vitro migration or the homing capacity of the Fam65b KO T cells. We have identified Rho GTPases as mediators of the effects of Fam65b. We showed, in flow cytometry, that the amount of RhoA-GTP and Rac-GTP are increased in the Fam65b KO cells. The RhoA-GTP levels are also increased in human primary T cells expressing low levels of Fam65b. We have identified, in in vitro experiments, that Fam65b slows down RhoA loading with GTP by its GEF proteins, thus inhibiting RhoA activity. Moreover, we showed that Fam65b dissociates from RhoA after chemokine stimulation of T cells, thus allowing RhoA activation. The phosphorylation of Fam65b is a probable cause to this phenomenon. Fam65b also dissociates from Rac1 in these conditions, although no mechanism is yet known. Furthermore, the transcription factor FOXO1 controls the expression of Fam65b. FOXO1 is also known to control the homing capacity of T cells, since it controls the expression of molecules involved in the entry of lymphocytes in the lymph nodes. Fam65b, an atypical regulator of Rho GTPases activity, thus represents a new connection between the PI3K/FOXO1 and the Rho GTPases pathways.

O carcinoma espinocelular de boca (CEB) é a neoplasia maligna mais comum da cavidade oral, correspondendo à aproximadamente 94% dos casos dessa região. Apesar dos diversos estudos moleculares e celulares do CEB, a taxa de sobrevida dos pacientes é de aproximadamente 50%, devido principalmente ao tamanho do tumor, metástase em linfonodos regionais, grau de diferenciação das células e sítio anatômico. O microambiente tumoral do CEB, é extremamente complexo e diversificado, tendo como característica principal um estado inflamatório crônico imunossupressivo. Este microambiente é sustentado pela liberação de diferentes citocinas inflamatórias, como IL-6, TNF- - atividades exercidas tanto pelas células tumorais quanto pelas estromais. Dentre essas atividades, tem sido relatado na literatura que as citocinas inflamatórias são capazes de aumentar a migração e a capacidade de invasão das células tumorais. Entre as células estromais, os macrófagos são as mais abundantes e participam da manutenção do microambiente tumoral. De acordo com o estímulo, podem ser polarizados M1, com papel pró-inflamatório e antitumoral, e M2, com papel anti-inflamatório e pró-tumoral. O objetivo desta tese foi compreender o papel dos macrófagos no prognóstico de CEB e das citocinas inflamatórias IL-6, TNF- - linhagens celulares de CEB. Para verificar o papel dos macrófagos no prognóstico, foi realizada uma revisão sistemática na qual foram incluídos apenas os estudos que utilizavam amostra de pacientes com CEB e avaliavam o prognóstico com marcadores para macrófagos. Foi observado que maiores concentrações de macrófagos CD68+ e CD163+ estavam relacionados com pior prognóstico de pacientes com CEB, embora não tenha sido possível concluir qual região tumoral a presença destas células seja mais importante 7 para o desfecho. Para analisar o papel das citocinas inflamatórias IL-6, TNFILensaios in vitro utilizando duas linhagens celulares, SCC25 e Cal27, em condições promotoras de migração sob a influência dessas citocinas. Foi observado que a citocina IL-6 foi capaz de aumentar a velocidade de migração e a direcionalidade tanto da SCC25 quanto da Cal 27 e que esta melhora na capacidade migratória ocorreu através de um crosstalk entre a via de sinalização relacionada a IL6 (STAT3) e a via reguladora de migração celular, Rho GTPase Rac1. Estes dados reforçam o papel do microambiente tumoral no processo de progressão tumoral e sugerem potenciais alvos terapêuticos como a modulação do perfil da população de macrófagos e o papel de interleucinas no controle de invasão tecidual e metástase. / Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, corresponding to approximately 94% of the cases in this region. Despite the diverse molecular and cellular studies of OSCC, the patient survival rate is approximately 50%, mainly due to tumor size, regional lymph node metastasis, cell differentiation and anatomic site. The OSCC tumor microenvironment is extremely complex and diverse, with the main characteristic being an immunosuppressive chronic inflammatory state. This microenvironment is supported by the release of different inflammatory cytokines, such as IL-6, TNF- - and enhance the activities of both tumor and stromal cells. Among these activities, it has been reported in the literature that inflammatory cytokines are capable of increasing migration and invasiveness of tumor cells. Among stromal cells, macrophages are the most abundant and participate in the maintenance of the tumor microenvironment. According to the stimulus, macrophages can be polarized in M1, with pro-inflammatory and anti-tumoral role, and M2, with antiinflammatory and pro-tumoral role. Thus, the aim of this thesis was to evaluate the role of macrophages in the prognosis of OSCC and the influence of inflammatory cytokines IL-6, TNF- - OSCC cell lines. To assess the role of macrophages in the prognosis, a systematic review was conducted in which only studies using a sample of OSCC patients were evaluated and the prognosis was evaluated with macrophage markers. It was observed that higher concentrations of CD68 + and CD163 + macrophages were related to worse prognosis in patients with OSCC, although it was not possible to conclude which tumor region the presence of these cells is more important for the outcome. In order to analyze the role of the inflammatory cytokines IL-6, TNF- - atory 9 behavior of OSCC cells, in vitro assays using two cell lines, SCC25 and Cal27, were performed in migration-promoting conditions under the influence of these cytokines. It was observed that IL-6 was able to increase the speed migration and directionality of both SCC25 and Cal 27 and that this improvement in migratory capacity occurred through a crosstalk between the IL6-related signaling pathway (STAT3) and cell migration-related pathway, RhoGTPase Rac1. These data reinforce the role of the tumor microenvironment in the tumor progression process and suggest potential therapeutic targets such as the modulation of the profile of the macrophages population and the role of interleukins in the control of tissue invasion and metastasis.

Nitric oxide (NO) is the principal mediator of erectile function. NO is released from the nerves and endothelium of small arteries in the penis and diffuses into surrounding smooth muscle to vasodilate through activation of soluble guanylate cyclase (sGC). Erectile dysfunction (ED) occurs in 50% of men between the ages of 40 and 70. It is likely that the pathology of ED results from impairment of NO formation or bioavailability in penile tissue. Iatrogenic nerve damage occurring during prostatectomy can attenuate neurotransmission and release of vasodilators from cavernosal nerves. Oxidative stress from chronic conditions such as diabetes and cardiovascular disease generates reactive oxygen species that can oxidize NO and decrease the molecule's bioavailability. The "gold standard" treatment for ED involves use of oral PDE-5 inhibitors that rely on an intact NO-signaling mechanism for efficacy. Although these therapies are easy to use, they are not effective in many patients suffering from ED associated with pathological conditions of decreased NO bioavailability. Rho-kinase inhibitors, sGC stimulators and sGC activators offer three new interventions that may demonstrate efficacy in treating ED associated with low NO bioavailability. Our results suggest that erectile responses to Rho-kinase inhibitors are not modulated by muscarinic receptor blockade, soluble guanylate cyclase inhibition or cavernosal nerve injury in the rat and that Rho-kinase inhibitors are additive and do not potentiate the endogenous NO-mediated erectile response. Our results with BAY 41-8543 show that this sGC stimulator has significant erectile activity and can potentiate erectile responses to low levels of exogenous and endogenously released NO. These results suggest that BAY 41-8543 would be useful in the treatment of ED occurring following nerve damage from prostatectomy. The sGC activator BAY 60-2770 has very potent erectile activity that is enhanced significantly in conditions of oxidative stress when erectile responses to endogenous NO or sGC stimulators are severely diminished. In oxidizing conditions erectile activity of sGC activators may be enhanced further with concomitant PDE-5 inhibitor therapy, providing evidence that sGC activators may be used alone and in combination with existing treatments to improve erectile function in patients who are non-responsive to standard therapeutic options for ED. / acase@tulane.edu

Receptor tyrosine kinases such as ErbB2 contribute greatly to human malignant transformation, but the role that other receptors such as ß2 adrenergic receptor (B2 AR)play in cancer is ill defined. Furthermore, while some GTPases such as Ras and RhoA promote oncogenesis, RhoB has been suggested to have tumor suppressive activity. In this thesis the tumor suppressive activity of ß2 adrenergic receptors through blockade of the Ras/Raf/Mek/Erk pathway is demonstrated. Furthermore, this thesis provides strong evidence in support of a tumor suppressive activity of RhoB, but not RhoA, in delaying EbB2 mammary oncogenesis in a transgenic mouse model. Chapter 1 describes a chemical biology approach that identifies a beta 2 adrenergic receptor agonist, ARA-211 (also known as pirbuterol) that suppresses the growth of cultured cells and of human tumors grown in nude mice by a mechanism involving stimulation of the ß2 AR, cAMP production and activation of PKA, which in turn leads to the inactivation of C-Raf, Mek1/2 and Erk1/2. Chapter 2 describes the translation of these findings by ex-vivo treatment of fresh human tumor biopsies, with the ultimate goal of validating this novel therapeutic approach. Chapter 3 describes the generation of transgenic mice that over express ErbB2 along with either RhoB or RhoA to determine the effects of these two small GTPases on ErbB2-mediated mammary tumorigenesis. The findings indicate that overexpression of RhoB, but not RhoA, results in decreased multiplicity and delay in the tumor onset mediated by ErbB2 overexpression. In summary, this thesis work resulted in the discovery of how crosstalk between the ß2 AR/cAMP/PKA circuit with the Raf/Mek/Erk1/2 cascade leads to tumor suppression; and the discovery of the suppression of ErbB2-mediated breast cancer by the GTPase RhoB.

Drug development is a long and expensive process. It is often not until potential drug candidates are administered to humans that accurate quantification of their pharmacokinetic characteristics is achieved. The goal of developing quantitative structure-pharmacokinetic relationships (QSPkRs) is to relate the molecular structure of a chemical entity with its pharmacokinetic characteristics. In this thesis artificial neural networks (ANNs) were used to construct in silico predictive QSPkRs for various pharmacokinetic parameters using different drug data sets. Drug pharmacokinetic data for all studies were taken from the literature. Information for model construction was extracted from drug molecular structure. Numerous theoretical descriptors were generated from drug structure ranging from simple constitutional and functional group counts to complex 3D quantum chemical numbers. Subsets of descriptors were selected which best modeled the target pharmacokinetic parameter(s). Using manual selective pruning, QSPkRs for physiological clearances, volumes of distribution, and fraction bound to plasma proteins were developed for a series of beta-adrenoceptor antagonists. All optimum ANN models had training and cross-validation correlations close to unity, while testing was performed with an independent set of compounds. In most cases the ANN models developed performed better than other published ANN models for the same drug data set. The ability of ANNs to develop QSPkRs with multiple target outputs was investigated for a series of cephalosporins. Multilayer perceptron ANN models were constructed for prediction of half life, volume of distribution, clearances (whole body and renal), fraction excreted in the urine, and fraction bound to plasma proteins. The optimum model was well able to differentiate compounds in a qualitative manner while quantitative predictions were mostly in agreement with observed literature values. The ability to make simultaneous predictions of important pharmacokinetic properties of a compound made this a valuable model. A radial-basis function ANN was employed to construct a quantitative structure-bioavailability relationship for a large, structurally diverse series of compounds. The optimum model contained descriptors encoding constitutional through to conformation dependent solubility characteristics. Prediction of bioavailability for the independent testing set were generally close to observed values. Furthermore, the optimum model provided a good qualitative tool for differentiating between drugs with either low or high experimental bioavailability. QSPkR models constructed with ANNs were compared with multilinear regression models. ANN models were shown to be more effective at selecting a suitable subset of descriptors to model a given pharmacokinetic parameter. They also gave more accurate predictions than multilinear regression equations. This thesis presents work which supports the use of ANNs in pharmacokinetic modeling. Successful QSPkRs were constructed using different combinations of theoretically-derived descriptors and model optimisation techniques. The results demonstrate that ANNs provide a valuable modeling tool that may be useful in drug discovery and development.