Activation of the immune system in gestating mothers has been identified as an important environmental risk factor for neuropsychiatric disorders. Maternal immune activation (MIA) animal models have been used to explore how the maternal immune system may cause expression of pathophysiology in offspring. Protein tyrosine phosphatase (PTP1B) is recruited during inflammation and its regulatory proteins are modulated in MIA. Disrupted regulation of PTP1B has been linked to mental disorders such as Rett Syndrome and anxiety. We asked if ablating neuronal PTP1B could protect from the expression of some neuropsychiatric phenotypes that appear in MIA models. In our MIA model induced with poly I:C injection at gestational day 9.5, we observed increased locomotion and sensorimotor gating and reduced anxiety in 3-month-old male offspring while females showed decreased sensorimotor gating. These effects were not replicated in PTP1B KO mice indicating a role of PTP1B in affecting locomotion and anxiety level in MIA. This model promotes a more balanced understanding of MIA and introduces PTP1B as a player in MIA-induced behaviour changes.

La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellules. / Mitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells.

Submitted by Gabriel Keine Kuga (xgabriel@gmail.com) on 2018-09-13T16:26:51Z No. of bitstreams: 1 Dissertação - Gabriel Keine Kuga - PPG Ciencias da Motricidade.pdf: 2345917 bytes, checksum: 7b943b730b7884c42ec2e4a70eea0d04 (MD5) / Approved for entry into archive by Ana Paula Santulo Custódio de Medeiros null (asantulo@rc.unesp.br) on 2018-09-13T17:08:25Z (GMT) No. of bitstreams: 1 kuga_gk_me_rcla.pdf: 2335049 bytes, checksum: d95d86caf2a2ad20a26629e663df42d5 (MD5) / Made available in DSpace on 2018-09-13T17:08:25Z (GMT). No. of bitstreams: 1 kuga_gk_me_rcla.pdf: 2335049 bytes, checksum: d95d86caf2a2ad20a26629e663df42d5 (MD5) Previous issue date: 2018-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A insulina e o fator neurotrófico derivado do cérebro (BDNF) no hipocampo promovem a plasticidade sináptica e a formação da memória no hipocampo. Por outro lado, o envelhecimento está relacionado ao declínio cognitivo e é o principal fator de risco para a doença de Alzheimer (DA). No entanto, a comunidade científica tem feito grande esforço para elucidar os mecanismos moleculares responsáveis pela patogênese da DA que são disparados pelo envelhecimento. A Proteina Tirosina Fosfatase 1B (PTP1B) está relacionada a vários processos deletérios nos neurônios e pode ser alvo promissor para novas terapias, e também regulada pelo exercício físico. Neste contexto, nosso estudo teve como objetivo investigar as alterações relacionadas com o envelhecimento sobre o conteúdo de PTP1B, sinalização da insulina e conteúdo de β-amilóide no hipocampo de ratos de meia-idade, bem como o possível efeito terapêutico do exercício físico. Ratos Wistar jovens (3 meses de idade) e de meia-idade (sedentários e exercitados) (17 meses de idade) foram submetidos ao teste do Labirinto Aquático de Morris (MWM), ao teste de tolerância à glicose e à análise molecular do tecido hipocampal através da técnica de Western Blot. Os dados foram analisados através da Análise de Variância (ANOVA) one-way com nível de significância estabelecido abaixo de 0,05. Os ratos realizaram protocolo de exercício físico de natação durante 5 dias consecutivos, com 2 horas de duração por dia. Através dos resultados, verificou-se que o envelhecimento resultou em aumento do peso corporal e intolerância à glicose, bem como diminuiu o processo de aprendizagem no MWM. Observou-se também que os ratos de meia-idade têm níveis mais altos de PTP1B, e isso está relacionado a menor fosforilação de Substrato do Receptor de Insulina-1 (IRS-1), Proteína Kinase B (Akt), Glicogênio Sintase Kinase β (GSK3β), e Receptor Tirosina Kinase Beta (TrkB). Além disso, o processo de envelhecimento aumentou o conteúdo β-amilóide no hipocampo. Por outro lado, o exercício físico foi eficiente em melhorar a tolerância à glicose e o desempenho no MWM, bem como em restaurar a fosforilação da Akt e reduzir o conteúdo de β-amilóide. Em conclusão, este estudo fornece novas evidências de que o conteúdo de PTP1B no hipocampo está aumentada com o envelhecimento e isto está relacionado com alterações cognitivas, e, por outro lado, o exercício físico atenua esse processo em ratos envelhecidos. / The insulin and Brain-Derived Neurotrophic Factor (BDNF) signaling in the hippocampus promote synaptic plasticity and memory formation. On the other hand, aging is related to the cognitive decline and is the main risk factor for Alzheimer’s Disease (DA). Nevertheless, a great effort has been made by the scientific community to elucidate the molecular mechanism of aging-related DA pathogenesis. The Protein-Tyrosine Phosphatase 1B (PTP1B) is related to several deleterious processes in neurons and emerges as a promising target for new therapies, like as physical exercise. In this context, our study aims to investigate the age-related changes in hippocampal PTP1B content, insulin signaling, β-amyloid content in the hippocampus of middle-aged rats, and the possible therapeutic effect of exercise. Young (3 months-old) and Middle-aged (17 months-old) Wistar rats were submitted to Morris-water maze (MWM) test, glucose tolerance test, and to molecular analysis in the hippocampus. The rats performed a 2-hour swim physical exercise protocol for 5 consecutive days. Aging resulted in increased body weight, and glucose intolerance and decreases learning process in MWM. Interestingly, the Middle-Aged rats have higher levels of PTPB, and this is related to lower phosphorylation of Insulin Receptor Substrate-1 (IRS-1), Protein Kinase B (Akt), Glycogen Syntase Kinase β (GSK3β), and Tyrosine Kinase Receptor Beta (TrkB). Also, the aging process increased β-amyloid content in the hippocampus. On the other hand, the physical exercise was efficient to improve glucose tolerance and MWM performance, as well as to restore Akt phosphorylation and reduce β-amyloid content. In conclusion, this study provides new evidence that PTP1B content in the hippocampus is increased with aging and this is related to cognitive alterations, and physical exercise can attenuate this process in middle-aged rats.

No description available.

NV669 est un aminosterol dérivé de la squalamine qui a montré posséder des propriétés anti-cancéreuses. L'objectif de cette étude a été de rechercher les effets bénéfiques de NV669 sur des modèles de cancers humains pancréatiques et hépatiques et de comprendre les mécanismes cellulaires et moléculaires impliqués dans la diminution de la croissance tumorale par le traitement avec NV669. Les lignées cellulaires humaines pancréatiques (BxPC3 et MiaPaca-2) et hépatiques (HepG2 et Huh7) ont été traitées avec NV669 à différentes concentrations et à différents temps. Les résultats ont montré que NV669 inhibe la prolifération des cellules cancéreuses en induisant l'arrêt du cycle cellulaire en phase G2/M via le complexe cycline B1/Cdk1 et en induisant l'apoptose via le clivage de la caspase-8 et de PARP-1, et la fragmentation de l’ADN.De plus, nos recherches in vitro ont révélé que NV669 inhibe l’activité phosphatase de PTP1B et l’expression de FAK. NV669 affecte l’expression des molécules d’adhérence CDH-1, -2 et -3 dans les lignées BxPC3 et Huh7 qui forment des monocouches cellulaires. Cela suggère qu’en inhibant PTP1B, NV669 induirait l’apoptose.Par la suite, nos résultats in vivo ont montré que NV669 inhibe la croissance des xénogreffes pancréatiques et hépatiques tumorales avec une diminution significative de la prolifération cellulaire et une augmentation de l'apoptose des cellules tumorales. Par conséquent, nos recherches suggèrent que l’analogue de la squalamine, NV669, pourrait être un agent anti cancéreux, utilisé seul ou en association avec d’autres médicaments, dans le traitement de l’adénocarcinome pancréatique et du carcinome hépatocellulaire. / NV669 is an aminosterol derived from squalamine found to possess strong antiangiogenic and anticancer effects. The aim of this study was to investigate NV669’s beneficial effects on human pancreatic and hepatic cancer models and to understand the cellular and molecular mechanisms involved in tumor growth decrease upon treatment with NV669.Pancreatic (BxPC3, MiaPaCa-2) and hepatic (HepG2, Huh7) cancer cells were treated with NV669, and the effects on proliferation, on cell cycle and death were determined. The results showed that NV669 inhibited the viability of cancer cells, induced cell cycle arrest through the regulation of G2/M phase via a decrease in the expression of cyclin B1 and phosphorylated Cdk1 and the induction of apoptosis via cleaved caspase-8 and PARP-1 and fragmented DNA. Moreover, in vitro NV669 inhibits PTP1B activity and FAK expression. NV669 impacts on the expression of adhesion molecules CDH-1, -2 and -3 in BxPC3 and Huh7 lines that form cell monolayers. This suggests that NV669 by inhibiting PTP1B would induce apoptosis. Subsequently, our in vivo results showed that NV669 inhibited the growth of pancreatic and hepatic tumor xenografts with a significant decrease in proliferation cell and an increase of tumor cell apoptosis. Therefore, NV669 may serve as an alternative anticancer agent, used alone or in association with other medications, for the treatment of pancreatic adenocarcinoma and hepatocellular carcinoma.

Le diabète de type 2 et le cancer représentent des problèmes majeurs de santé publique. Une cible thérapeutique importante de ces affections est la protéine tyrosine phosphatase PTP1B. Cette dernière est connue pour réguler la voie de l’insuline en déphosphorylant le récepteur de l’insuline, IR ou le substrat du récepteur de l’insuline, IRS. Cependant lesfonctions de PTP1B, le mécanisme par lequel cette phosphatase est régulée restent très ou pas connus. Deux études ont notamment décrites des effets opposés de l’activité de PTP1B suite à la phosphorylation sur résidu de Ser50 de PTP1B par CLK1/CLK2, des kinases LAMMER d’une part et AKT d’autre part. AKT a aussi été montré de phosphoryler la kinase LAMMER CLK1. Par ailleurs, le rôle de PTP1B dans la régulation de la voie Ras/MAPK et donc dans le cancer est un sujet très controversé. L’objectif premier de ce travail de thèse a été d’analyser, le rôle de Ptp61F (l’orthologue de Drosophile de PTP1B) dans la voie de l’insuline de Drosophile, l’interaction entre la phosphatase et la kinase LAMMER de Drosophile, DOA, le rôle de cette phosphatase dans la voie RAS/MAPK. Pour se faire, nous avons utilisé la puissance génétique de laDrosophile pour générer un mutant du gène Ptp61F qui a été caractérisé et son rôle dans les voies de signalisation a été étudié. Cette étude a montrée que, Ptp61F interagit avec IR comme PTP1B chez les mammifères. Elle montre que Ptp61F régule les acteurs clés de la voie de l’insuline Pi3K/Akt. Elle a également montrée que Ptp61F pouvait réguler les fonctions du gène de la kinase LAMMER de Drosphile, Doa. Elle montre enfin que Ptp61F interagit avec nombreuses composantes de la voie RASMAPK de Drosophile (Egfr, Ras, rl (ERK humain)) en réprimant la fonction de chacun de ces gènes et que Rl serait un substrat direct de PTP61F. Les informations selon lesquelles, Ptp61F interagit avec Akt et le gène de la kinaseLAMMER de Drosophile, Doa ont été utilisées dans la deuxième étude pour montrer le rôle que les kinases LAMMER (notamment CLK2, Cdc-like kinase 2) pouvaient jouer dans la voie de signalisation de l’insuline au niveau moléculaire en utilisant les cellules de neuroblastome humain SH-SY5Y. Il en ressort que la kinase CLK2 joue un rôle importantdans cette voie de signalisation. CLK2 est induit par l’insuline et son expression augmente avec le temps. PTP1B interagit in vitro et in vivo avec CLK2. La surexpression de CLK2 induit la baisse de la phosphorylation de AKT par un mécanisme qui pourrait passer par PTP1B, puisque in vitro, CLK2 phosphoryle PTP1B et ce dernier interagit avec AKT in vivo. C’est le résidu de Ser50 de PTP1B qui est phosphorylé et cette phoshphorylation réprime l’activité de PTP1B in vitro. On n’observe cependant pas AKT capable de phosphoryler PTP1B in vitro suggérant que la phosphorylation de PTP1B par AKT serait dépendante du contexte cellulaire. / Type 2 diabetes and cancer represent the major public health problems. One important therapeutic target for these pathologies is the protein tyrosin phosphatase PTP1B. The phosphatase is known to negatively regulates the insulin signaling pathway by dephosphorylating the insulin receptor, IR or the insulin receptor substrate, IRS. However,PTP1B functions and its regulation mechanism remain poorly known. Two studies has notably described opposite effects of PTP1B activity following phosphorylation of its Ser50 residue either by CLK1/CLK2, LAMMER kinases or by AKT. Furthermore, AKT, a main insulin signaling pathway component, has been shown to phosphorylate the LAMMER kinaseCLK1 following insulin stimulation. In addition, the role of PTP1B in the regulation of the RAS/MAPK signaling pathway and hence in cancer is a very controversial subject. The first objective of this work was to analyse, the role of Ptp61F (the Drosophila ortholog of human PTP1B) in the Drosophila insulin pathway, the interaction between the phosphatase and the Drosophila LAMMER kinase gene, Doa, the role of Ptp61F in the RAS/MAPK signaling pathway. To achieve these, we took advantage of the genetic powerful of Drosophila to generate a Ptp61F gene mutant which has been characterized and its role in signaling pathways has been studied. This study showed that Ptp61F interacts with IR like PTP1B in mammals. It shows that Ptp61F regulates key components of insulin signaling pathway Pi3K/Akt. It also shows that Ptp61F is able to regulate the Drosophila LAMMER kinase gene, Doa. Finally, we noted that Ptp61F interacts by inhibiting the activity of severalcomponent of the RAS/MAPK signaling pathway of Drosophila (Egfr, Ras, rl (human ERK)) and conclude that Rl coud be a direct substrate of PTP61F. The data showing that Ptp61F interacts with Akt and the Drosophila LAMMER kinase gene, Doa, were the basis for the second study in order to show the role that the mammal LAMMER kinase CLK2 (Cdc-like kinase 2) could play in the insulin signaling pathway at molecular level using the human neuroblastoma cell line SH-SY5Y. From this second study, we show that CLK2 play an important role in insulin signaling. CLK2 is induced by insulin and its expression increases with time. PTP1B interacts in vivo and in vitro with CLK2. Overexpression of CLK2 impairs AKT phosphorylation by a mechanism which could involved PTP1B, since in vitro, CLK2 phosphorylates PTP1B and the latter interacts withAKT in vivo. It is the Ser50 residue of PTP1B being phosphorylated by CLK2 and this phosphorylation event represses PTP1B activity in vitro. AKT cannot phosphorylates PTP1B in vitro, suggesting that the phosphorylation of PTP1B by AKT could be cellular environment dependant.

Protein-tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphorylated tyrosines by a 2-step mechanism involving nucleophilic attack by cysteine and general acid catalysis by aspartic acid. In most PTPs the aspartic acid resides on a flexible protein loop, consisting of about a dozen residues, called the WPD loop. PTP catalysis rates span several orders of magnitude, and differences in WPD loop dynamics have recently been show to correlate with the rate of enzymatic catalysis. The rate of WPD loop motion could possibly be related to a widely conserved tryptophan residue on the WPD loop. Therefore, point mutants were made in PTP1B (a human PTP) to the conserved tryptophan residue and their effects on catalytic rate and chemical reaction were studied. The results of these studies are presented in this thesis.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder amongst older adults. Features of this disease include accumulation of amyloid-β (Aβ) plaques, neurofibrillary tau tangles (NFT), neuroinflammation, and neurodegeneration. These result in a progressive decline in memory and executive function in patients. Anxiety-related behaviours are disparaging comorbidities of AD, but how they arise in patients remains elusive. Protein-tyrosine phosphatase 1B (PTP1B) has been associated with Aβ pathology and with anxiety in separate paradigms, but whether PTP1B is involved in anxiety-related behaviours in AD mouse models is unknown. The objective of this project was to compare anxiety-related behaviours between the hAPP-J20 (Aβ pathology) and PS19 (Tau pathology) mouse models of AD and determine whether PTP1B is involved in these behaviours. Another major objective of this project was to investigate the role of PTP1B in tau pathology in the PS19 mouse model in anxiety-related brain regions, since this has not been previously examined. Using key anxiety-testing paradigms such as the elevated plus maze (EPM) and the open field test (OF), an age-based dimorphism in the onset of an inappropriately lowered anxiety response in the J20 and PS19 mouse models was identified. Furthermore, it was shown that this abnormal anti-anxiety baseline phenotype could be normalized with selective PTP1B inhibition by the drug trodusquemine and by genetic neuronal ablation. Finally, in PS19 mice at 8 months of age, it was shown that PTP1B blockade has the therapeutic effect of relieving neurotoxic phospho-tau burden and neuroinflammation. Together, these findings suggest that unleashed PTP1B may serve as a potential therapeutic target, with a possible role in AD-associated anxiety-related behaviours and AD pathology.

No description available.

While the molecular defect that cause sickle cell disease has well been established, the cause of vaso-occlusive crisis remains elusive and largely debated upon. Majority of studies have linked the painful episodes to polymerization of sickle hemoglobin following its deoxygenation. The variability of the disease symptoms among patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator of Hb F induction and a widely used treatment, has ameliorated the complication of SCD but it is only effective in 50% of the patients. Expression of Hb F lowers the content of Hb S in blood and hence reduces oxidative stress caused by Hb S denaturation. Sickle cell disease severity depends on several factors. Most importantly, the ability of red cell to sickle dominates all other determinants. While deoxygenation of sickle hemoglobin may be inevitable, the duration with which the red cell remains in the deoxygenated state can be manipulated. Deoxygenation is a transient process that when compared to the time taken to develop the long filaments of deoxyhemoglobin to causes severe sickling, the red cell would have been cycled back to the lungs and re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle cells would flow as fast as healthy erythrocytes, the detrimental impacts of sickling such as vaso-occlusive crisis, would not be a concern for this disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation through auto-oxidation, which imposes oxidative stress to the cells. The oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which subsequently impair their constitutive action against the tyrosine kinases. In the end, a net tyrosine phosphorylation state in the red cell membrane proteins, most notably the transmembrane protein band 3, succeeds. Band 3 tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage to the membrane thus engendering membrane destabilization. A destabilized erythrocyte sheds membrane fragments in form of microvesicles/microparticles and discharges free hemoglobin into the extra cellular matrix. In consequence, the microparticles power initiation of coagulation cascade through activation of thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is necessary for vasodilation and induces further oxidative stress within the microvasculature, and activates expression of adhesion receptors on the endothelium. Taken together, these events culminate in entrapment of red cells (not naming leucocytes and platelets) in the microvasculature, blockade of blood vessels and further damage of erythrocytes through prolonged deoxygenated state thus terminating in tissue injury, strokes, and organ damage, amid vaso-occlusive episodes which always require hospitalization and extensive medical care for survival. Band 3 tyrosine phosphorylation and membrane weakening is not unique just to SCD, but also a druggable target for malaria. Malaria, a disease that is touted as the evolutionary cause of sickle cell disease, surprisingly thrives through the same mechanism. Briefly, malaria parasite consumes hemoglobin for its DNA synthesis, and in the process generate reactive oxygen species from denatured hemoglobin that feeds into the oxidative stress which triggers band 3 tyrosine phosphorylation. In this case however, a destabilized membrane offers perfect conditions for merozoites’ (malaria daughter parasites) egress/exit out of the cell to begin infecting other red cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s death. Treatment of diseased cells with imatinib and other Syk inhibitors effectively reversed membrane weakening. A stabilized membrane not only survives longer in circulation to alleviate SCD symptoms but also traps and starves malaria parasite leading to termination of the parasitic infection. With band 3 tyrosine phosphorylation at center stage, this dissertation explores the above events in an effort to unveil a novel therapy for sickle cell and malaria diseases. First, the therapeutic strategy regarding SCD is discussed in detail beginning with non-transfused patients and ending in additional mechanistic study on inactivation of the principal erythrocyte’s protein tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial proof of concept on efficacy of imatinib in treatment of malaria as a monotherapy and its efficacy when used in a triple combination therapy with the standard of care treatment. Finally, I outline an alternative possible mechanism of action of quinine against malaria.