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Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T19:24:15Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:15:45Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:15:53Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) / Made available in DSpace on 2018-05-21T20:15:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) Previous issue date: 2016-08-26 / Sem bolsa / O sistema calicreínas-cininas (SCC) está envolvido em diversos processos biológicos como modulação de dor, vasodilatação, permeabilidade vascular, edema e inflamação, tendo seus efeitos mediados por dois receptores específicos acoplados a proteína G, o receptor B1 e B2 de cininas. O SCC tem sido recentemente relacionado com a homeostase da glicose e resistência à insulina. O objetivo do presente estudo foi investigar a participação do receptor B1 (B1R) de cininas nos processos metabólicos relacionados à obesidade e ao diabetes mellitus tipo 2 em camundongos obesos. Para isso foram utilizados camundongos deficientes para a leptina e nocautes para o B1R de cininas (obB1). Quando comparados, os animais obB1 mostraram uma redução na ingestão alimentar em todo período avaliado (4° a 26° semana de vida); menor ganho de peso, tanto aos 3 meses (obWT, 63,79 ± 1,59; obB1, 51,22 ± 1,09 g, p < 0,001) quanto aos 6 meses de idade (obWT, 74,61 ± 1,04; obB1, 57,98 ± 1,58 g, p < 0,001); e menor depósito de gordura. No teste de tolerância a glicose, animais obB1 apresentaram maior captação de glicose do que seus controles quando jovens (obWT, 46045 ± 3492; obB1, 30875 ± 2311 (mg/dL).min, p< 0,01). Adicionalmente, foi observado aumento do peso do fígado nestes animais, quando mais velhos (obWT, 3,29 ± 0,24; obB1 4,09 ± 0,06 g, p<0,001). Em conclusão, o silenciamento do gene do B1R oferece uma proteção contra a obesidade com um significativo impacto na homeostase da glicose. Estes resultados sugerem que o SCC pode ser um novo alvo para desenvolvimento de novas estratégias farmacológicas para o tratamento de doenças metabólicas como obesidade e diabetes mellitus tipo 2. / The kallikrein-kinin system (KKS) is involved in many biological processes such as modulating pain, vasodilation, vascular permeability, edema and inflammation, with effects mediated by two specific receptors coupled to G protein, B1 and B2 kinin receptor. The KKS has recently been associated with glucose homeostasis and insulin resistance. The aim of this study was to investigate the participation of B1 receptor (B1R) of kinins in the metabolic processes related to obesity and type 2 diabetes in obese mice. For this we used mice deficient for leptin and knockouts for B1R kinin (obB1). When compared, the animals obB1 showed a reduction in food intake in the whole study period (4 to 26 ° week of life); less weight gain, both at 3 months (obWT, 63.79 ± 1.59; obB1, 51.22 ± 1.09 g, p <0.001) and at 6 months of age (obWT, 74.61 ± 1, 04; obB1, 57.98 ± 1.58 g, p <0.001); and less fat deposit. In the glucose tolerance test, obB1 animals had higher glucose uptake than their controls when young (obWT, 46045 ± 3492; obB1, 30875 ± 2311 (mg / dL) .min, p <0.01). Additionally, there was an increase in liver weight in these animals, while older (obWT, 3.29 ± 0.24, 4.09 ± 0.06 g obB1, p <0.001). In conclusion, the B1R gene silencing offers a protection against obesity have a significant impact on the glucose homeostasis. These results suggest that the KKS may be a new target for the development of new pharmacologic strategies for treating metabolic diseases such as obesity and type 2 diabetes mellitus.

For pain research, the orofacial region is unique in a number of ways. The region has complex local anatomy, including substantial sensory innervation from neural pathways, and muscles of facial expression that convey important information concerning pain intensity and associated psychological traits. Although chronic orofacial pain conditions appear prevalent, useful documentation on pain intensity ratings using well established instruments is sparse. In particular, two conditions, atypical facial pain and atypical odontalgia, are poorly understood in aetiology so that definitive treatment modalities are severely limited. The region's local biofluid, saliva, has been used to diagnose various local and systemic disease states, and to quantitate drug concentrations. However, recent studies indicate that saliva also contains some of the same peptides, e.g. bradykinin, that are involved in pain mechanisms. It may be that pharmacological-pharmacokinetic studies of these peptides could shed more information on thesignificance of their presence in saliva. This thesis consists of four major sections. Section 1 comprises of three clinical studies investigating orofacial pain. Section 2 deals with clinical laboratory studies of saliva. Section 3 is concerned with the development of chromatographic methods to assay bradykinin and its pharmacokinetics in saliva. Section 4 uses chromatography for the identification of novel salivary peptides. This thesis, then, presents clinical studies of orofacial pain and pharmacological investigations of saliva as the local biofluid.

Neuropathic pain is a prevalent disease with no effective, safe treatments and limited knowledge on the mechanisms involved. One target for neuropathic pain treatment may be the blockade of Dynorphin A (Dyn A). Dyn A is a unique endogenous ligand that possesses well-known neuroinhibitory effects via opioid receptors and neuroexcitatory effects that are mediated through the bradykinin 2 receptors (B2Rs). Extensive SAR was carried out to develop a ligand for the blockade of the excitatory actions of Dyn A at the B2R. A lead ligand was able to block Dyn A-induced hyperalgesia in naïve animals and was effective in a neuropathic pain model. However, the ligand was susceptible to enzymatic degradation. In an effort to increase the stability, modifications of the ligand using non-natural amino acids were performed. Analogues substituted at or near the N-terminus with a D-isomer retained binding at the receptor as well as provided a large increase in stability. These ligands were also found to be non-toxic in a cell toxicity assay. Dyn A has been found to not activate the classical signaling of the B2R, PI hydrolysis or Ca²⁺ mobilization. In an effort to determine Dyn A's signaling, a study was done examining up-regulation of phosphorylated proteins. It was found that Dyn A did not activate; pERK, 7 PKC isoforms or PKA. A well known B2R antagonist, HOE140, was found to have low affinity at rat and guinea pig brain B2Rs but high affinity in the guinea pig ileum. Further examination revealed that this discrepancy in binding may arise from a different isoform of the B2R that has not been previously examined. To date, we have discovered Dyn A analogues that have high affinity for the B2R, are very stable, and have low toxicity. The signaling pathway is still not fully understood, but further studies are underway. Also, there is evidence that the B2R in which the analogues are interacting at may be a different form than what has previously been described. Targeting this different isoform of the B2R with our current stable ligands may provide beneficial therapeutics for the treatment of neuropathic pain without the cardiovascular liabilities.

The neuropeptides dynorphin and cholecystokinin (CCK), and their associated pronociceptive effects were investigated in the RVM. Utilizing a nerve-injury model (SNL), RT-PCR analysis revealed increases (p<0.05) of prodynorphin mRNA, and bradyinkin, B1- and B2-receptor mRNA, post-SNL, 14-days, 2-days, and 14-days, respectively. Administration of dynorphin into the RVM produced both acute and long-lasting (>30-days) tactile hypersensitivity. Administration of the B1-antagonist, DALBK and the B2-antagonist, Hoe-140, into the RVM significantly attenuated dynorphin-induced tactile hypersensitivity. Nerve-injury induced tactile hypersensitivity was significantly reversed by RVM administration of dynorphin antiserum or the B2-antagonist, Hoe-140. These data suggest that dynorphin is up-regulated in the RVM in nerve-injury, and via the activation of bradykinin receptors in the RVM, produces abnormal pain. Like dynorphin, CCK is up-regulated in the RVM in nerve-injury, with studies suggesting that elevated levels of CCK in the RVM mediate pronociceptive activity through CCK2 receptor activation, resulting in enhanced spinal nociceptive transmission. At present, it is unknown what key neurotransmitters are mediating this RVM CCK-driven effect at the level of the spinal cord. Here, spinal cerebrospinal fluid (CSF) levels of serotonin (5-HT) and prostaglandin E2 (PGE2) were measured in the lumbar spinal cord in naïve rats following CCK administration into the RVM. Following RVM CCK microinjection, an approximate 5-fold increase in spinal (CSF) PGE2 levels was observed, as compared to baseline controls. PGE2 levels showed a progressive increase with peak levels observed at the 80-minute post-CCK injection timepoint, whereas 5-HT levels in the spinal CSF remained unchanged following CCK administration into the RVM. This release of PGE2 coincided with the timecourse for CCK-induced mechanical hypersensitivity. Administration of the CCK2-antagonist YM022 prior to CCK into the RVM, significantly attenuated (>50%) the release of PGE2 in the spinal cord. The non-selective COX-inhibitor naproxen and the 5-HT3 antagonist ondansetron, both administered intrathecally, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. In summary, these data suggest a bradykinin- or CCK2-receptor antagonist could be used alone or in conjunction with current therapies in the treatment of chronic pain.

国立情報学研究所で電子化したコンテンツを使用している。

For pain research, the orofacial region is unique in a number of ways. The region has complex local anatomy, including substantial sensory innervation from neural pathways, and muscles of facial expression that convey important information concerning pain intensity and associated psychological traits. Although chronic orofacial pain conditions appear prevalent, useful documentation on pain intensity ratings using well established instruments is sparse. In particular, two conditions, atypical facial pain and atypical odontalgia, are poorly understood in aetiology so that definitive treatment modalities are severely limited. The region's local biofluid, saliva, has been used to diagnose various local and systemic disease states, and to quantitate drug concentrations. However, recent studies indicate that saliva also contains some of the same peptides, e.g. bradykinin, that are involved in pain mechanisms. It may be that pharmacological-pharmacokinetic studies of these peptides could shed more information on thesignificance of their presence in saliva. This thesis consists of four major sections. Section 1 comprises of three clinical studies investigating orofacial pain. Section 2 deals with clinical laboratory studies of saliva. Section 3 is concerned with the development of chromatographic methods to assay bradykinin and its pharmacokinetics in saliva. Section 4 uses chromatography for the identification of novel salivary peptides. This thesis, then, presents clinical studies of orofacial pain and pharmacological investigations of saliva as the local biofluid.

Beatmungsbedingte Lungenschäden in der Therapie des akuten Atemnotsyndroms (ARDS) sind aufgrund der inhomogenen Vorschädigung der Lunge praktisch unvermeidbar. Die unphysiologische mechanische Belastung der Lunge führt über Volutrauma, Atelektotrauma und Biotrauma nicht selten zur Exazerbation des Syndroms und trägt zur hohen Mortalität des ARDS bei. Pharmakologische Interventionsmöglichkeiten sind Gegenstand der aktuellen Forschung. Diesbezüglich vielversprechend ist die zentrale Komponente des Kinin-Kallikrein-Systems, namentlich Bradykinin, das über seinen B2-Rezeptor anti-apoptotische Signalwege aktivieren kann und somit zellprotektive Wirkung besitzt. In der vorliegenden Arbeit wurde untersucht, in welcher Weise zyklische mechanische Dehnung die Konzentration einzelner Komponenten des Kinin-Kallikrein-Systems in isolierten alveolären Epithelzellen (Typ II) der Ratte beeinflusst. Dafür wurden die alveolären Typ-II-Zellen auf speziellen BioFlex®-Membranen kultiviert und für 24 Stunden zyklisch mit hoher Dehnungsamplitude gedehnt. Anschließend wurden mit etablierten Analysemethoden in Zellüberständen bzw. Zelllysaten die Konzentrationen von Kininogen 1, Bradykinin und vom B2-Rezeptor gemessen, sowie die Aktivität des Enzyms Kallikrein und des Bradykinin-Abbaus bestimmt - jeweils im Vergleich mit Überständen bzw. Lysaten ungedehnter AT-II-Zellkulturen. Es zeigte sich dehnungs-bedingt eine Zunahme der Bradykinin-Produktion durch Kininogen und Kallikrein und eine stark gesteigerte Bradykinin-abbauende Aktivität, sodass sich der Bradykininspiegel insgesamt verringerte. Die Konzentration des B2-Rezeptors blieb unverändert. Detailliertes Wissen über den Einfluss zyklischer mechanischer Dehnung auf die Einzelkomponenten des Kinin-Kallikrein-Systems ist eine Grundvoraussetzung, um die zellprotektive Wirkung von Bradykinin im Sinne einer pharmakologischen Interventionsmöglichkeit bei ARDS nutzbar machen zu können.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-25Bitstream added on 2014-06-13T19:14:40Z : No. of bitstreams: 1 santos_kf_me_sjrp.pdf: 789178 bytes, checksum: c9d3ae49b4891343eda0dc12453c315f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Angiotensina II, um peptídeo hipertensivo, e bradicinina, um peptídeo hipotensivo, são fatores humorais cruciais para a regulação da pressão sanguínea. A enzima chave desse sistema é a enzima conversora de angiotensina que produz angiotensina II a partir de angiotensina I e degrada bradicinina. A descoberta dos primeiros inibidores naturais dessa enzima, os peptídeos potenciadores de bradicinina (BPPs), tornou possível o desenvolvimento dos primeiros medicamentos utilizados no controle da pressão arterial humana. Caracteristicamente, os BPPs contêm de 5 a 13 resíduos de aminoácidos apresentando um resíduo de piroglutâmico no N-terminal e um resíduo de prolina no Cterminal. O precursor de BPPs encontrado na glândula de veneno de Bothrops jararaca contém 256 resíduos de aminoácidos e codifica para sete BPPs alinhados em tandem seguidos pelo peptídeo natriurético tipo-C. Até o momento, não se conhecem os mecanismos envolvidos para a liberação desses peptídeos da proteína precursora. Dessa forma, a resolução da estrutura dessa proteína pode contribuir para a elucidação do mecanismo evolvido no processamento do precursor para a liberação dos BPPs. Duas construções da proteína precursora de BPPs (domínio BPP e domínios BPP+CNP) da glândula de veneno de B. jararaca foram expressas, purificadas e suas identidades confirmadas por experimentos de western blotting. A pureza das amostras foi avaliada por SDS-PAGE e a presença de enovelamento após expressão heteróloga foi observada por experimentos de dicroísmo circular e fluorescência. Os ensaios de cristalização não foram promissores. Isso pode ser explicado pela baixa concentração da proteína usada no experimento. Assim, devido ao baixo nível de expressão de ambas as proteínas, métodos para maximização da expressão foram empregados resultando em significante... / Angiotensin II, a hypertensive peptide, and bradykinin, a hipotensive peptide, are crucial humoral factors for the regulation of blood pressure. The key enzyme for this system is the angiotensin-converting enzyme that produces angiotensin II from angiotensin I and degrades bradykinin. The discovery of the first natural inhibitors for this enzyme, the bradykinin potentiating peptides (BPPs), made it possible to develop the early drugs aimed at controlling unbalanced cardiovascular functions. Characteristically, BPPs contain 5 to 13 amino acid residues that have a pyroglutamyl residue at the N-terminus and a praline residue at the C-terminus. The BPP precursor protein contains 256 amino acid residues coding for seven BPPs aligned in tandem followed by the C-type natriuretic peptide. At present, there are no suggested mechanisms for understanding the release of BPPs from the precursor protein. Two constructs of the BPP precursor protein (BPP domain and BPP+CNP domains) from the venom gland of Bothrops jararaca were over-expressed, purified and the identity of both recombinant proteins confirmed by western blotting experiments. The purity of the samples was assessed by SDS-PAGE and the protein fold after expression was observed by circular dichroism and fluorescence experiments. Crystallization assays were not successful, probably due to the low protein concentration used for the experiment. Considering the low expression level observed for both recombinant proteins, the experimental methods were optimized to maximize the yield and resulted in high protein amounts in inclusion bodies. Methods were applied aiming at the solubilization of the proteins from the insoluble fraction and protein purification under denaturing conditions was carried out yielding high amounts of pure protein. Until this moment, none of the procedures were successful in producing refolded proteins. Work ...(Complete abstract click electronic access below)

Resumo: Angiotensina II, um peptídeo hipertensivo, e bradicinina, um peptídeo hipotensivo, são fatores humorais cruciais para a regulação da pressão sanguínea. A enzima chave desse sistema é a enzima conversora de angiotensina que produz angiotensina II a partir de angiotensina I e degrada bradicinina. A descoberta dos primeiros inibidores naturais dessa enzima, os peptídeos potenciadores de bradicinina (BPPs), tornou possível o desenvolvimento dos primeiros medicamentos utilizados no controle da pressão arterial humana. Caracteristicamente, os BPPs contêm de 5 a 13 resíduos de aminoácidos apresentando um resíduo de piroglutâmico no N-terminal e um resíduo de prolina no Cterminal. O precursor de BPPs encontrado na glândula de veneno de Bothrops jararaca contém 256 resíduos de aminoácidos e codifica para sete BPPs alinhados em tandem seguidos pelo peptídeo natriurético tipo-C. Até o momento, não se conhecem os mecanismos envolvidos para a liberação desses peptídeos da proteína precursora. Dessa forma, a resolução da estrutura dessa proteína pode contribuir para a elucidação do mecanismo evolvido no processamento do precursor para a liberação dos BPPs. Duas construções da proteína precursora de BPPs (domínio BPP e domínios BPP+CNP) da glândula de veneno de B. jararaca foram expressas, purificadas e suas identidades confirmadas por experimentos de western blotting. A pureza das amostras foi avaliada por SDS-PAGE e a presença de enovelamento após expressão heteróloga foi observada por experimentos de dicroísmo circular e fluorescência. Os ensaios de cristalização não foram promissores. Isso pode ser explicado pela baixa concentração da proteína usada no experimento. Assim, devido ao baixo nível de expressão de ambas as proteínas, métodos para maximização da expressão foram empregados resultando em significante...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Angiotensin II, a hypertensive peptide, and bradykinin, a hipotensive peptide, are crucial humoral factors for the regulation of blood pressure. The key enzyme for this system is the angiotensin-converting enzyme that produces angiotensin II from angiotensin I and degrades bradykinin. The discovery of the first natural inhibitors for this enzyme, the bradykinin potentiating peptides (BPPs), made it possible to develop the early drugs aimed at controlling unbalanced cardiovascular functions. Characteristically, BPPs contain 5 to 13 amino acid residues that have a pyroglutamyl residue at the N-terminus and a praline residue at the C-terminus. The BPP precursor protein contains 256 amino acid residues coding for seven BPPs aligned in tandem followed by the C-type natriuretic peptide. At present, there are no suggested mechanisms for understanding the release of BPPs from the precursor protein. Two constructs of the BPP precursor protein (BPP domain and BPP+CNP domains) from the venom gland of Bothrops jararaca were over-expressed, purified and the identity of both recombinant proteins confirmed by western blotting experiments. The purity of the samples was assessed by SDS-PAGE and the protein fold after expression was observed by circular dichroism and fluorescence experiments. Crystallization assays were not successful, probably due to the low protein concentration used for the experiment. Considering the low expression level observed for both recombinant proteins, the experimental methods were optimized to maximize the yield and resulted in high protein amounts in inclusion bodies. Methods were applied aiming at the solubilization of the proteins from the insoluble fraction and protein purification under denaturing conditions was carried out yielding high amounts of pure protein. Until this moment, none of the procedures were successful in producing refolded proteins. Work ...(Complete abstract click electronic access below) / Orientador: Raghuvir Krishnaswamy Arni / Coorientador: Mirian Akemi Furue Hayashi / Banca: Fátima Pereira de Souza / Banca: Patrick Jack Spencer / Mestre