Gout is one form of inflammatory arthritis, which is caused by the precipitation of crystals of monosodium urate (MSU) in the joints. Acute gout is associated with sudden and painful inflammatory episodes characterized by high neutrophil infiltration. In spite of years of study gout treatment remains a challenge due to its relative ineficcacy. Thus, search for new and efficient therapies is necessary. The objective of this study was to investigate the involvement of glibenclamide in a model of acute gout in rats induced by MSU. MSU crystals produced nociception and edema when injected into the ankle joint of rats. Treatment with glibenclamide (3 mg/kg, s.c.) or dexamethasone (8 mg/kg, s.c., used as a positive control) decreased spontaneous nociception (67% ± 11 and 70 ± 7% inhibition, respectively) and edema (28 ± 7% and 77 ± 7% inhibition, respectively) induced 6 hours after MSU injection. The number of leukocyte infiltrates in the synovial fluid as well as the release of interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) significantly increased at 6 hours after injection of MSU joint, but these effects were not reversed by treatment with glibenclamide (3 mg/kg, s.c.). In contrast, dexamethasone reduced the leukocyte infiltration and release of IL-1β and PGE2. To confirm if the dose of glibenclamide was able to block the KATP channels, we determined the levels of glucose in the blood of animals. Glibenclamide decreased (23 ± 2%) and dexamethasone increased the blood glucose of the rats compared to vehicle-treated animals / MSU. Therefoe, the effects of glibenclamide on nociception and edema induced MSU, suggests that this sulfonylurea may be an interesting option as an adjunct therapy in pain observed in acute attacks of gout. / A gota é uma forma de artrite inflamatória, causada pela precipitação de cristais de urato monossódico (MSU) nas articulações. A forma aguda de gota está associada a episódios inflamatórios súbitos e dolorosos caracterizados por uma grande infiltração de neutrófilos. Apesar dos anos de estudo sobre a gota, o seu tratamento ainda é um desafio pela relativa ineficácia dos fármacos disponíveis no mercado. Assim, a busca por novos agentes terapêuticos mais efetivos e seguros se faz necessário. Desta forma, o objetivo deste estudo foi investigar o possível potencial farmacológico da glibenclamida em um modelo de gota aguda induzida por MSU em ratos. Os cristais de MSU produziram nocicepção e edema quando injetados na articulação do tornozelo de ratos. O tratamento com glibenclamida (3 mg/kg, s.c.) ou dexametasona (8 mg/kg, s.c., usada como controle positivo) reduziu a nocicepção espontânea (67 ± 11% e 70 ± 7% de inibição, respectivamente) e o edema (28 ± 7% e 77 ± 7% de inibição, respectivamente) induzidos pelo MSU, 6 horas após a injeção do cristal. O número de leucócitos infiltrados no líquido sinovial, assim como a liberação de interleucina 1β (IL-1β) e de prostaglandina E2 (PGE2) foram consideravelmente aumentados, 6 horas após a injeção de MSU na articulação, porém esses efeitos não foram revertidos pelo tratamento com glibenclamida (3 mg/kg, s.c.). Em contrapartida, dexametasona reduziu a infiltração de leucócitos e a liberação de IL-1β e de PGE2. Para confirmar se a dose utilizada de glibenclamida foi capaz de bloquear os canais de KATP, foi avaliado os níveis de glicose no sangue dos animais. A glibenclamida reduziu (23 ± 2%) e a dexametasona aumentou a glicemia dos ratos quando comparado aos animais tratados com veículo /MSU. Assim, frente aos efeitos desempenhados pela glibenclamida sobre a nocicepção e edema induzidos pelo MSU, sugere-se que esta sulfonilureia possa ser uma opção interessante como um tratamento adjuvante na dor observada em ataques agudos de gota.

The fever response, besides being part of host defense response to infection or inflammation, is associated with discomfort and anxiety and may constitute a risk for febrile seizures in children. Therefore, antipyretic therapy is routinely prescribed for febrile patients. The animal models of fever using the systemic injection of lipopolysaccharide (LPS) and Baker yeast, described in the literature, are suitable for screening of novel antipyretics, but they do not provide information regarding the mechanism of action of these compounds. Therefore, the present study aimed to describe and validate a model of fever induction by prostaglandin (PG) E2, the final mediator of febrile response in the central nervous system, in young male Wistar rats (25-30 days of age). In this protocol, PGE2 was injected intrathecally without implantation of cannula. Rectal temperature (TR) was recorded every thirty minutes for three hours after PGE2 injection (08:00 11:00 h). The intrathecal (i.t.) injection of PGE2 10 ηg in 100 μL/animal induced fever in the animals, which was prevented by administration of EP1 and EP3 receptors antagonists, but did not by antagonist of EP4 receptor. In addition, the classic antipyretics dipyrone and acetaminophen, at doses that had no effect per se on TR of animals, did not revert the fever induced by i.t. injection of PGE2. This model seems suitable to investigate whether the action of antipyretics occurs upstream or downstream the prostaglandin coupling in EP receptors. In addition, this protocol is advantageous from the technical, ethical and economical point of view compared to others PGE2-induced fever protocols described in the literature, because trepanation for cannula implantation is not required, reducing the inflammatory response, animals suffering and experimental costs. / A febre, apesar de fazer parte da resposta de defesa do hospedeiro à infecção ou inflamação, está associada com desconforto e ansiedade, além de representar um risco iminente de convulsões febris em crianças. Por isso, terapia antipirética é rotineiramente prescrita a pacientes febris. Os modelos animais de febre empregando a injeção sistêmica de lipopolissacarídeo (LPS) e fermento de padeiro, descritos na literatura, são úteis para a triagem de novos antipiréticos, mas não fornecem informações a respeito do mecanismo de ação desses compostos. Diante disso, o presente estudo objetivou padronizar e validar um modelo de indução de febre por prostaglandina (PG) E2, o mediador final da resposta febril no sistema nervoso central, em ratos machos jovens da raça Wistar (25-30 dias). Neste protocolo, a PGE2 foi injetada pela via intratecal (i.t.), não necessitando a implantação de cânula. A temperatura retal (TR) foi registrada a cada trinta minutos durante três horas após a injeção da PGE2 (08:00-11:00 h). A injeção i.t. de PGE2 10 ηg em 100 μL/animal induziu febre nos animais, a qual foi prevenida pela administração de antagonistas dos receptores EP1 e EP3, mas não por antagonista do receptor EP4. Além disso, os antipiréticos clássicos dipirona e paracetamol, em doses que não tiveram efeito per se na TR dos animais, não reverteram a febre induzida por PGE2 i.t. Este modelo parece útil para investigar se a ação dos antipiréticos ocorre antes ou depois da ligação da PGE2 em seus receptores EP. Além disso, este protocolo é vantajoso do ponto de vista técnico, ético e econômico em relação aos outros protocolos de indução de febre por PGE2 descritos na literatura, porque a trepanação para implantação de cânula não é necessária, reduzindo a resposta inflamatória, o sofrimento dos animais e os custos experimentais.

Corpus luteum is a transient endocrine structure formed from the ruptured ovarian follicle. Its main function is to secrete P4, a pro-gestational hormone, essential for establishment and maintenance of pregnancy in mammals. The modulators of CL structure and function are classified as trophic and lytic factors. The luteotrophic factors include pituitary hormones, growth factors, intra luteal factors and cytokines, while luteolytic factors include PGF2α and oxytocin. The interplay between luteotrophic and luteolytic factors regulates luteal steroidogenesis. The precise timing of expression of various enzymes/proteins required for synthesis and metabolism of P4 constitutes an important process in the overall regulation of CL function. The three hormones LH/CG, E2 and PRL are regarded as luteotrophic factors crucial for control of CL function in mammals. Depending on species, either individually or all three hormones in the form of luteotrophic complex have been shown to participate in the regulation of CL function. In addition to the well-established endocrine role of E2, its secretion is the hallmark of the ovulating follicle, has an important role in the intraovarian growth, differentiation and survival of cells. Chapter I provides a comprehensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in bovines and rodents. In Chapter II, studies have been carried out to examine 20α-HSD expression and its activity in the CL of buffalo cow. During induced and spontaneous luteolysis, rapid fall in circulating P4 is one of the early signs of initiation of luteolytic process in several species. In rodents, it is well recognized that during luteolysis, P4 is catabolized into inactive metabolite, 20α-OHP by the reaction of 20α-HSD enzyme during luteolysis. Experiments were carried out to determine 20α-HSD expression and activity throughout the luteal phase and during induced luteolysis in the buffalo cow. Circulating P4 concentration declined rapidly in response to PGF2α treatment, but HPLC analysis of serum samples did not reveal changes in circulating 20α-OHP levels in buffalo cows. In contrast, pseudo pregnant rats receiving PGF2α treatment showed higher 20α-OHP levels at 24 h post treatment. qPCR expression of 20α-HSD in CL during different stages of luteal phase and PGF2α-treated buffalo cows was carried out and higher expression of 20α-HSD was observed at 3 and 18 h post treatment, but its activity was not altered post PGF2α treatment at other time points examined. The expression of the transcription factor Nurr77 which is involved in increased expression of 20α-HSD increased several fold 3 h post PGF2α treatment similar to the observation in PGF2α-treated pseudo pregnant rats. The results suggested that the synthesis rather than catabolism of P4 appears to be primarily affected by PGF2α treatment in buffalo cows in contrast to increased metabolism of P4 as seen in rodents. In bovines, to date no luteotropic actions for E2 has been demonstrated and whether E2 has direct effect on CL function has also not been reported. Expression of CYP19A1 gene that encodes aromatase enzyme although gets down regulated post ovulation but its expression recovers in the CL and also E2 biosynthesis has been reported in the bovine CL. Recently it was observed that CYP19A1 expression was consistently down regulated following administration of luteolytic dose of PGF2α. Experiments were conducted to examine the expression of ERα and ERβ in the CL throughout the buffalo estrous cycle as well as examined the luteal E2 levels post PGF2α treatment. The results indicated that ER expression was detectable during different stages of CL and that circulating and luteal E2 levels declined post PGF2α treatment. It was hypothesized that decrease in luteal E2 levels leads to down regulation of ER signaling and changes in expression of E2 responsive genes in the CL. To test the hypothesis, 89 genes which were regarded as E2 responsive genes were selected and the previously published global gene expression data of the buffalo CL was mined for E2 responsive genes. It was observed that 57 of 89 genes regarded as E2 responsive genes were found to be differentially expressed. Since non pregnant buffalo CL is not regarded as major site of E2 production, to validate the authenticity of differentially E2 expressed genes post PGF2α, CL of another species, the macaque, which is known to secrete abundant E2 was included for the analysis. Incidentally, the global gene expression data for the PGF2 α treated macaques (in which CYP19A1 gene expression also gets down regulated) has previously been reported from the laboratory. Here again, it was observed that nearly 79 of 89 genes were identified to be differentially expressed. To further determine the consequences of decreased ER signaling, molecules associated with survival and apoptosis were examined. The results indicated decreased expression (both mRNA and protein levels) of Akt, Bax and Bcl-2 genes. The results suggested an important role for E2 on CL function in the buffalo cow. In Chapter III, several experiments were conducted in another model system, pregnant rat, in which aromatase expression and therefore E2 production is high in the CL. Experiments were conducted to examine the effects of E2 inhibition and E2 replacement on the expression of genes. For this purpose, pregnant rats were treated with a specific aromatase inhibitor on day 12-15 of pregnancy. Together with AI, exogenous E2 was administered to another group of pregnant rats. The CL collected from different groups of rats on day 16 of pregnancy was subjected to microarray analysis. The analysis post validation of microarray data has shown that clusters of genes could be segregated into various pathways involving luteal steroidogenesis, immune system, various growth factors and apoptotic processes, all directed towards the regulation of CL function. The involvement of E2 in luteal cell proliferation and lipid deposition well corroborated with protein levels for cyclin D1 and ki67 and the results of oil red O staining, respectively. There have been reports implicating PI3K/Akt signaling in cyclin D1 accumulation, but mechanism of action does not appear to involve transcriptional activation of cyclin D1. The results of the present study indicate a decrease in cyclin D1 protein levels due to inhibition of PI3K/Akt signaling by AI treatment which is prevented upon administration of E2 during AI treatment. The findings provide a comprehensive overview for the mechanisms associated with the cell survival, progression, etc. The bioinformatics approach provided complete landscape of functional changes affected by the upstream regulators of genes associated with survival and apoptosis. Also, the findings further strengthen the hypothesis of involvement of E2 in the regulation of CL function by way of activation of Akt, the primary mediator of PI3K signaling in the regulation of cellular component that affect cell survival. In the present study, IGFBP5 which was up regulated during luteal inhibition of E2 with AI treatment was selected for further studies. Although IGFBP5 is known to be associated with follicular atresia in the rat ovary, there is limited data for the involvement of IGFBP5 in either a growth stimulatory or inhibitory action on ovarian cells. Based on present findings, a causal link between reduced ERα transcriptional activities resulting in inhibition of Akt/PKB in the presence of IGFBP5 expression could be proposed. Further, the cellular hypertrophy mediated by E2 has been speculated due to increased proliferation of vascular endothelial cells, blood supply and thus nutrients. E2, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat CL of pregnancy. In CL, the prominent IGFBP5 mRNA expression in different types of luteal cells has not been reported. The mRNA expression for IGFBP5 across the two types of luteal cells showed higher expression in SLC. Hence, in the present study, it has been speculated that prevention of conversion of SLC to LLC due to lack of E2 biosynthesis in presence of AI might be acting as a source for the increased IGFBP5 levels during mid pregnancy in rat CL and brings about changes associated with lack of E2. Various receptor studies on rat CL have demonstrated the lack of progesterone receptor (PR) mRNA expression in the rat CL negating its involvement as an autocrine/paracrine regulator of CL function through an intracellular receptor, but the involvement of non-PR involvement in mediating such mechanism further strengthens the role of ERs. The luteotrophic complex formation in pregnant rat principally by PRL and E2 has been discussed at length in Chapter III. PRL appears to maintain luteal ER content in the CL during rat pregnancy which further determines the luteotrophic and luteolytic actions of E2. Further, study on expression of E2 responsive genes would help in identifying E2 regulating molecules to get a clear picture on the role of E2 in understanding regulation of the CL function. The interaction of E2 with growth factor signaling including the IGF pathway has been well established in different species and this interaction is tightly linked to ERα expression, an observation interpreted as physiological coupling of growth factor and stress signaling pathways. Attempts were made towards understanding cross talk between the E2 signaling and the IGF1 signaling in few experiments carried out in Chapter IV. Based on the results, it can be proposed that a causal link exists between reduced ERα transcriptional activity and inhibition of Akt/PKB in the presence of IGFBP5. The present study has shown the activity of IGF on ERα activity mediated partly via PI3K/Akt pathway. Hence, the finding further speculates that inhibitory effect of IGFBP5 on E2 induced ERα function was due to sequestration of IGF1, possibly present in serum or produced within the cells. Another striking observation was the down regulation of glucocorticoid receptor (GR) gene, NR3C1, in the data of earlier studies [Priyanka, 2009, GEO accession number GSE8371 and Kunal, 2014, GEO accession number GSE27961] and the present study has been compared and discussed in this thesis. Glucocorticoids provide key signals for differentiation of fetal and placental tissues. Therefore, regulation of glucocorticoid access to the placenta and fetus is recognized as an important determinant of prognosis outcome and subsequent development of the postnatal phenotype. Differential regulation of these genes in CL post E2 deprivation and replacement further emphasize the regulation of CL via various biological, cellular and molecular functions. Interestingly, besides transcriptional regulation of IGF axis components, E2 activated ERα also rapidly influence the activity of IGF axis related to signaling proteins in a non-genomic manner, especially by the PI3K/Akt pathway. PI3K/Akt pathway analysis has been carried out in E2 inhibition and replacement experiments. To further confirm the observations of E2 and growth factor interaction, experiments have been set up with exogenous GH for increasing circulating levels of IGF in the system. The findings suggest that the non-genomic signaling pathway activated by the phosphorylation of ERα induced by E2 gets inhibited in the presence of AI result in increased expression of IGFBP5. The reduction in circulating IGF1 in pregnant rats may be associated with the effect on IGFBP, important for determining biological action of IGF1. The changes observed in the present study emphasize the exclusive effects of the IGFBP5 on the CL function brought about perturbations in luteal E2 content. The experiments described in the present thesis aim at understanding the mechanism responsible for decreased serum and luteal P4 post PGF2α treatment in buffalo cows, i.e. whether PGF2α acts on biosynthetic or catabolic process of P4. In the present study, experiments were designed to elucidate the role of E2 in regulation of CL function, since down regulation of CYP19A1 gene mRNA was one of the early events observed in buffalo cows post PGF2α treatment. This line of research work was extended to rodents, a species that secretes high levels of E2 during pregnancy. Genome wide transcriptional changes data revealed differential expression of several E2 responsive genes following E2 inhibition and replacement treatments. The results revealed importance of ER-mediated PI3K/Akt signaling essential for regulation of many transcriptional regulatory molecules in the CL and an interesting involvement of IGFBP5 as a link between E2 and IGF signaling. These findings further provide an insight into the role of IGFBP5 in E2-mediated actions in rat CL during pregnancy. In conclusion, the present findings suggest inhibitory effect of IGFBP5 on E2-induced ERα function and hence, its selection as a target molecule for regulation of CL function and for many beneficial processes involved in anti-carcinogenic properties can be thought of.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

No description available.

Les accouchements prématurés constituent un problème médical majeur en constante augmentation et ce, malgré tous les efforts mis en œuvre afin de contrer le déclenchement des contractions avant terme. Cette thèse relate du ''design'' rationnel d'un nouvel agent thérapeutique (i.e., tocolytique) qui serait capable de 1) arrêter les contractions, et 2) prolonger la gestation. Pour ce faire, une nouvelle cible, la prostaglandine F2α et son récepteur ont été sélectionnés et le peptidomimétisme a été choisi afin de résoudre cette problématique. L'introduction contient un historique rapide de la conception à la synthèse (''drug design'') du peptide parent, le PDC113, premier peptide a avoir démontré des aptitudes tocolytiques suffisantes pour faire du peptidomimétisme. La deuxième partie de l'introduction présente les concepts du peptidomimétisme appliqués au PDC113 qui ont permis d'accéder au PDC113.824, inhibiteur allostérique du récepteur de la prostaglandine F2α, et explique comment ce mime nous a permis d'élucider les mécanismes de signalisation intracellulaire impliqués dans la contraction musculaire lisse. Cette thèse présente la conception, la synthèse et l'étude structure-activité de mimes de repliement de tour β au sein du mime peptidique original (PDC113.824) dans lequel nous avons remplacé l'azabicycloalkane central (l'indolizidin-2-one) par une série d'autres azabicycloalcanes connus et des acides aza-aminés dont nous avons élaboré la synthèse. Dans un premier temps, une nouvelle stratégie de synthèse en solution de l'aza-glycyl-proline à partir de la diphényle hydrazone et du chloroformate de p-nitrophényle a été réalisée. Cette stratégie a permis d'éliminer les réactions secondaires de cyclisation intramoléculaires communément obtenues lors de l'introduction d'acides aza-aminés avec les protections traditionnelles de type carbamate en présence de phosgène, mais aussi de faciliter l'accès en une étape à des dérivés peptidiques du type aza-glycyle. L'élongation de l'aza-glycyl-proline en solution nous a permis d'accéder à un nouveau mime tetrapeptidique du Smac, un activateur potentiel de l'apoptose au sein de cellules cancéreuses. Par la suite, nous avons développé une stratégie de diversification sélective de l'azote α du résidu azaglycine en utilisant différents types d'halogénures d'alkyle en présence de tert-butoxyde de potassium. Afin de valider le protocole d'alkylation de l'aza-dipeptide, différents halogénures d'alkyle ont été testés. Nous avons également démontré l'utilité des aza-dipeptides résultants en tant que ''building block'' afin d'accéder à une variété d'azapeptides. En effet, l'aza-dipeptide a été déprotégée sélectivement soit en N-terminal soit en C-terminal, respectivement. D'autre part, la libération de l'amine de l'ester méthylique de l'aza-alkylglycyl-proline a conduit à une catégorie de composés à potentiel thérapeutique, les azadicétopipérazines (aza-DKP) par cyclisation intramoléculaire. Enfin, notre intérêt quant au développement d'un nouvel agent tocolytique nous a amené à développer une nouvelle voie de synthèse en solution du PDC113.824 permettant ainsi d'élucider les voies de signalisation intracellulaires du récepteur de la prostaglandine F2α. Afin de valider l'importance de la stéréochimie et d'étudier la relation structure/ activité du mime, nous avons remplacé l'indolizidin-2-one (I2aa) centrale du PDC113.824 par une série d'autres azabicycloalcanes et azadipeptides. Les azabicycloalcanes D-I2aa, quinolizidinone, et indolizidin-9-one ont été synthétisés et incorporés au sein du dit peptide ne donnant aucune activité ni in vitro ni ex vivo, validant ainsi l'importance du tour β de type II' pour le maintien de l'activité biologique du PDC113.824. Finalement, l'insertion d'une série de dérivés aza(alkyl)glycyl-prolyles a mené à de nouveaux inhibiteurs allostériques du récepteur de la PGF2α, l'un contenant l'azaglycine et l'autre, l'azaphénylalanine. Cette thèse a ainsi contribué, grâce à la conception et l'application de nouvelles méthodes de synthèse d'aza-peptides, au développement de nouveaux composés à potentiel thérapeutique afin d'inhiber le travail prématuré. / Premature birth is a steadily increasing major medical problem, in spite of efforts made to counter the onset of preterm contractions. This thesis describes the rational design of a new therapeutic agent (i.e., tocolytic) capable of 1) stopping uterine contractions, and 2) prolonging gestation. The prostaglandin F2α receptor was explored for tocolytic development and a peptidomimetic approach was developed to produce modulators of this novel target. A brief discussion introduces the impact of preterm birth and history on the design and synthesis of a peptide lead, PDC113, to address this unmet medical need. Subsequently, the peptidomimetic approach is described for converting PDC113 to the small molecule PDC113.824, which was shown to be an allosteric modulator of prostaglandin F2α receptor, which mediates intracellular signalling pathways involved in uterine contraction. This thesis presents the design, synthesis and structure-activity relationship study of the peptide mimic PDC113.824, in which we have replaced the central β-turn mimic moiety, the indolizidin-2-one amino acid, by a series of other previously reported azabicycloalcane turn mimics and novel aza-amino acids. To accomplish the latter, new solution-phase methods for synthesizing aza-glycyl-proline analogs were developed starting from diphenyl hydrazone and p-nitrophenyl chloroformate. This strategy has surmounted side reactions such as the intramolecular cyclization commonly obtained with traditional coupling reactions employing alkyl-carbazates and phosgene, facilitating access to aza-glycyl-proline derivatives by a one step reaction. The modification of the aza-glycyl-proline using conventional strategies led to a new Smac mimic (a proapoptotic molecule), with potential as a tetrapeptide activator of apoptosis in cancer cells. Subsequently, we focused on the diversification of the aza-glycine residue by selective alkylation using different alkyl halides in the presence of potassium tert-butoxide. To validate the alkylation protocol, various alkyl groups were employed, and the usefulness of the resulting aza-dipeptides as ''building blocks'' was examined. Conditions were developed for selectively unmasking the protecting groups at the N- and C-terminal of the aza-dipeptide. In addition, removal of the amine protection of aza-alkylglycyl-proline methyl ester gave access to a class of compounds with therapeutic potential, the aza-diketopiperazines (aza-DKP), by intramolecular cyclization. Finally, our interest in developing a new tocolytic agent led us to develop a new solution-phase synthetic route to PDC113.824 for elucidating the intracellular signalling pathways of prostaglandin F2α receptor. To explore the importance of stereochemistry and to study the relationship between structure and activity, we replaced the central indolizidin-2-one (I2aa) of PDC113.824 by a series of others azabicycloalcanes and aza-dipeptides. The D-I2aa, quinazolidinone and indolizidin-9-one analogs were synthesized and incorporated into the mimic; however, none exhibited activity neither in vitro nor ex vivo, thus indicating the importance of a type II' β-turn for maintaining biological activity of PDC113.824. Finally, the synthesis of a series of aza(alkyl)glycyl-prolyl analogs led to new allosteric modulators of the PGF2α receptor, one containing aza-glycine and another aza-phenylalanine. This results reported in this thesis have contributed to the development of novel agents with promise for inhibiting preterm labor by the conception and application of new methods for making aza-peptides.

No description available.

ウコン利用の一形態であるウコン熱水エキスの抗炎症作用について検証することを目的として本研究を行った。内皮細胞もしくはマクロファージを使用した細胞試験を実施し、ウコン熱水エキスの抗炎症作用を実証した。また、その作用機序を一部明らかにし、その作用に寄与する成分を一部明らかにした。さらに、臨床試験を実施し、ウコン熱水エキスが健常者の気分状態に対する作用を評価し、その摂取が疲労を改善する可能性があることが示唆された。 / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

Indiana University-Purdue University Indianapolis (IUPUI) / Adult hematopoietic stem cells (HSC) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment or self-renewal. We have demonstrated that mouse and human HSC express prostaglandin E2 (PGE2) receptors, and that short-term ex vivo exposure of HSC to PGE2 enhances their homing, survival and proliferation, resulting in increased long-term repopulating cell and competitive repopulating unit (CRU) frequency. HSC pulsed with PGE2 are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon multiple serial transplantations, with full multi-lineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression and enhances their migration to SDF-1α in vitro and homing to bone marrow in vivo and stimulates HSC entry into and progression through cell cycle. In addition, PGE2 enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. While PGE2 pulse of HSC promotes HSC self-renewal, blockade of PGE2 biosynthesis with non-steroidal anti-inflammatory drugs (NSAIDs) results in expansion of bone marrow hematopoietic progenitor cells (HPC). We co-administered NSAIDs along with the mobilizing agent granulocyte-colony stimulating factor (G-CSF) and evaluations of limiting dilution transplants, assays monitoring neutrophil and platelet recoveries, and secondary transplantations, clearly indicate that NSAIDs facilitate mobilization of a hematopoietic graft with superior functional activity compared to the graft mobilized by G-CSF alone. Enhanced mobilization has also been confirmed in baboons mobilized with G-CSF and a NSAID. Increases in mobilization are the result of a reduction of signaling through the PGE2 receptor EP4, which results in marrow expansion and reduction in the osteoblastic HSC niche. We also identify a new role for cannabinoids, an eicosanoid with opposing functions to PGE2, in hematopoietic mobilization. Additionally, we demonstrate increased survival in lethally irradiated mice treated with PGE2, NSAIDs, or the hypoxia mimetic cobalt chloride. Our results define novel mechanisms of action whereby eicosanoids regulate HSC and HPC function, and characterize novel translational strategies for hematopoietic therapies.

Chronic kidney disease (CKD) is defined as glomerular filtration rate (GFR) less than 60 mL/min/1.73 m2 for 3 months and is characterized by progressive loss of renal function. The second leading cause of CKD is hypertension. More than half of CKD patients also suffer from hypertension. Arteries and arterioles adjust to the fluctuations in the systematic blood pressure through a mechanism called autoregulation. In the kidneys, autoregulation protects the delicate glomeruli capillaries from high blood pressure and occurs through myogenic constriction (MC). MC refers to contraction of arterioles in response to an increase in the blood pressure. Chronically hypertensive individuals and animal models have an enhanced MC, leading to minimal renal injury despite their elevated blood pressure. Experimental and clinical evidence point to a role for the MC in the pathogenesis of the CKD, however, the mechanism through which preglomerular arterial MC contributes to CKD has not been fully elucidated. This thesis showed that augmented MC in chronically hypertensive animal models was due to increased thromboxane A2 prostaglandin that was not released from the endothelium (Chapter 2). Nevertheless, inhibiting MC while also reducing the blood pressure prevented salt-induced renal injury even though the blood pressure was still not normalized compared to the normotensive controls (Chapter 3). The resulting improvement in renal structure and function could be attributed to the reduction in the blood pressure, albumin, and uromodulin (UMOD) excretion (Chapter 3). UMOD is a kidney-specific glycoprotein that, based on a genome-wide association study have the strongest association to CKD (Chapter 3). Comparing two CKD hypertensive animal models further revealed that CKD progression was independent of the blood pressure and strongly associated with UMOD excretion levels (Chapter 4). Collectively, the data discussed in this thesis demonstrates potential therapeutic targets in CKD hypertensive animal models. / Dissertation / Doctor of Philosophy (PhD)