Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmico / Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH

O hipercortisolismo ACTH-dependente (HAD), também chamado de doença de Cushing, é uma das endocrinopatias mais comumente diagnosticadas na espécie canina. A sintomatologia clínica ocorre, secundariamente, aos efeitos gliconeogênicos, catabólicos, antiinflamatórios e imunossupressores dos glicocorticóides sobre vários sistemas orgânicos. Há uma marcante predisposição da doença na raça poodle e casos familiais têm sido diagnosticados sugerindo uma causa genética. As alterações moleculares que levam ao desenvolvimento do HAD em cães permanecem indefinidas. Dentre os genes implicados no desenvolvimento dos corticotrofos e na regulação do eixo corticotrófico, destacam-se o Tbx19 e o Crhr1, respectivamente. O Tbx19 é um fator de transcrição obrigatório para a transcrição do gene da proopiomelanocortina (POMC) e para a diferenciação terminal dos corticotrofos. Como está presente, exclusivamente, em corticotrofos normais e adenomatosos, foi proposto seu envolvimento na secreção excessiva de ACTH na doença de Cushing. A presença de CRHR1 nos corticotrofinomas na espécie humana e canina levantou a hipótese da sua participação na tumorigênese hipofisária, promovendo uma estimulação celular prolongada, mesmo na ausência de hormônios hipotalâmicos. Um aumento da expressão do CRHR1 foi demonstrado nos tumores corticotróficos, apesar da secreção autônoma de ACTH e dos níveis portais suprimidos de CRH em pacientes humanos e caninos com doença de Cushing. Os objetivos do presente trabalho foram pesquisar a presença de mutações germinativas nas regiões codificadoras dos genes Tbx19 e Crhr1 em cães com HAD. Para tanto, estudamos 50 cães da raça poodle com hipercortisolismo ACTH-dependente (33 fêmeas e 17 machos), com idade média de 8,71 anos e 50 cães controle da mesma raça (32 fêmeas e 18 machos) com idade superior a 6 anos (média de 9,38 anos) e sem endocrinopatias. O DNA genômico foi extraído e amplificado através da reação de polimerização em cadeia (PCR), utilizando-se oligonucleotídeos (primers) específicos para os genes Tbx19 e Crhr1. Foi identificada uma nova variante alélica tanto no Tbx19 como no Crhr1, ambas não descritas na literatura. No gene Tbx19, a variante p. S343G foi encontrada em dois cães não aparentados, mas também em dois controles normais, sugerindo tratar-se de um novo polimorfismo. Já a variante p. V97M do Crhr1 foi encontrada, em heterozigose em um animal com HAD, porém não foi observada em cem alelos normais. O códon 97 está localizado no domínio extracelular aminoterminal do gene Crhr1, de extrema importância para a ligação com alta afinidade ao ligante. O estudo molecular da estrutura quartenária da proteína mutada, seguido da avaliação da energia de ligação da superfície de contato entre o hormônio e o receptor revelou um rearranjo estrutural com alteração da superfície de contato entre o CRH e o seu receptor CRHR1, resultando em uma energia de ligação 17% superior à do receptor selvagem. Em conclusão, esse estudo não identificou alterações no gene Tbx19 associadas ao hipercortisolismo ACTH-dependente canino, mas por outro lado, identificou pela primeira vez, uma mutação ativadora no Crhr1, provavelmente responsável pelo hipercortisolismo ACTH-dependente em um cão da raça poodle. / The ACTH-dependent hypercortisolism (ADH), also called Cushing\'s disease, is one of the most commonly diagnosed endocrine diseases in dogs. The symptoms occur due to glucocorticoids excess leading to gluconeogenic, catabolic, anti-inflammatory and immunosuppressive effects in multiple organs and systems. There is a high incidence of Cushing\'s disease in Poodles and familial disease has been identified suggesting a genetic involvement. The molecular changes that lead to the development of ACTH-dependent hypercortisolism in dogs remain undefined. Among genes implicated in corticotroph development and in corticotropic axis regulation, we would like to point out Tbx19 and Crhr1, respectively. Tbx19 gene is a transcription factor required for transcription of the proopiomelanocortin gene and for terminal differentiation of the corticotroph. Inactivating mutations in that gene are associated with human isolated ACTH deficiency. Since Tbx19 is present exclusively in normal and adenomatous corticotroph cells, its involvement in the secretion of ACTH in Cushing\'s disease was proposed. The presence of CRHR1 in corticotrophinomas in humans and dogs raised the possibility of its involvement in pituitary tumorigenesis, promoting prolonged cell stimulation, even in the absence of hypothalamic hormones. An increased expression of the CRHR1 mRNA was demonstrated in human and canine ACTH-secreting pituitary adenomas, despite the autonomous ACTH secretion and the low portal levels of CRH. The aim of this study was to investigate Tbx19 and Crhr1 coding region mutations in Poodle dogs with ACTH-dependent hypercortisolism. We studied 50 Poodle dogs with ADH (33 females and 17 males) with a mean age of 8.71 years and 50 control dogs of the same breed (32 females and 18 males) older than 6 years (mean 9.38 years) and without endocrinopathies. Genomic DNA was extracted from peripheral blood, amplified by the polymerase chain reaction (PCR) using specific intronic primers and submitted to automatic sequence. We identified a new allelic variant in the Tbx19 and Crhr1 coding regions. The allelic variant p. S343G in the Tbx19 gene was found in two unrelated dogs, but also in two normal controls, suggesting that this is a new polymorphism. The Crhr1 allelic variant p. V97M was found in heterozygosity in one animal with ACTH-dependent hypercortisolism, but was not observed in one hundred normal alleles. The codon 97 is located in the extracellular amino terminal domain of the Crhr1 and is extremely important for high affinity ligand binding. The molecular analysis of the quaternary structure of normal and mutated proteins, followed by evaluation of the binding energy of the contact surface between the hormone and the receptor showed a structural rearrangement of the mutated protein by changing the contact surface between the CRH and its receptor CRHR1, resulting in a binding energy 17% higher than the wild type. In conclusion, this study did not identify Tbx19 mutations associated with canine ACTH-dependent hypercortisolism, but on the other hand, we first identified a Crhr1 gain-of-function mutation probably responsible for ACTH-dependent hypercortisolism in a Poodle dog of our cohort.

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral. / BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

Na busca por novos materiais doadores de óxido nítrico (NO), o presente trabalho descreve o desenvolvimento de um filme à base de amido de mandioca, no qual foi incorporado um nitrosilo complexo de rutênio, e o estudo da liberação de NO nesse material. O nitrosilo complexo trans-[Ru(NH3)4(isn)NO](BF4)3 (RuNOisn; isn = isonicotinamida) apresenta a propriedade de liberar NO de forma controlada, por meio de fotólise (λirr = 310-370 nm) e de redução química. A incorporação desse complexo em filmes de amido foi realizada em condições brandas, resultando em um novo material para o armazenamento e liberação de NO, designado como CSx-RuNOisn. Os ensaios espectroscópicos indicaram que a esfera de coordenação do complexo RuNOisn permaneceu inalterada durante a produção dos filmes. A exposição de CSx-RuNOisn à luz (λirr = 355 nm) levou à liberação de NO e provavelmente à formação do fotoproduto trans [RuIII(NH3)4isn(H2O)]3+ no filme. A reação desse aquocomplexo de rutênio(III) com solução aquosa contendo nitrito de sódio regenerou o complexo de partida, RuNOisn. A identificação e quantificação do NO liberado durante a fotólise foi efetuada por meio da reação com oximioglobina. Durante o tempo de irradiação de 17 minutos, foram liberados 5,02 ± 0,12 μM de NO (10, 04 ± 0,24 nmol NO em 2 mL). Os ensaios de liberação de NO desencadeada por redução foram realizados utilizando-se L-cisteína como redutor. O fluxo de NO liberado a partir da reação com cisteína perdurou por mais de 7 horas, alcançando-se concentrações fisiologicamente relevantes, com fluxo médio de 1,9 pmol NO s-1 cm-2 de filme. Esse valor é comparável àquele produzido por células endoteliais, em que o fluxo de NO é de 1,67 pmol s-1 cm-2. Os resultados preliminares de degradação dos filmes in vivo sugerem que o material foi degradado pelo organismo em 30 dias. Todos os resultados alcançados sugerem que o filme CSx-RuNOisn é um candidato promissor para aplicações em meio biológico. Um novo complexo de rutênio contendo o ligante nitrosilsulfito (N(O)SO3 -) foi isolado, trans [Ru(NH3)4(isn)(N(O)SO3)](X) (isn = isonicotinamida, X = PF6- ou SiPF6 2-), e a sua estrutura cristalina determinada por difração de raio-X. A síntese desse complexo foi realizada por meio da reação entre trans-[Ru(NH3)4(isn)(NO)]3+ e íons sulfito (SO32-). O ataque nucleofílico do SO32- ocorreu no nitrogênio do ligante nitrosônio (NO) coordenado ao centro metálico de rutênio ([Ru-NO+]), originando o ligante O=N-SO3-: [RuNO+]3+ + SO32- →[Ru(N(O)SO3)]+. Observou-se que em meio aquoso, no intervalo de pH de 7,4 a 5,2 o complexo trans [Ru(NH3)4(isn)(N(O)SO3)]+ é estável, e a velocidade de decomposição (labilização do ligante isn) variou de k = 0,86 a 3,07 × 10-5 s-1. Em soluções mais ácidas (tampão ácido acético/acetato pH 4,2, 3,9, ou 1,0 M ácido trifluoroacético) o complexo trans-[Ru(NH3)4(isn)(N(O)SO3)]+ decompõe-se formando o respectivo nitrosilo complexo trans- [RuII(NH3)4(isn)NO+]3+. A reação do íon trans-[Ru(NH3)4(isn)(N(O)SO3)]+ com íons hidróxido (OH-) dá origem ao respectivo nitro complexo trans-[Ru(NH3)4(isn)(NO2)]+, que foi caracterizado por RMN de 15N e por espectroscopia eletrônica. As constantes de velocidade para essa reação são k = 6,16 ± 0,22 M-1 s-1 à T = 25oC, e k = 2,15 ± 0,07 M-1 s-1 à T = 15oC. A reação entre o nitrosilo complexo trans [RuII(NH3)4(isn)NO+]3+ e íons OH- também resulta na formação do nitro complexo trans-[Ru(NH3)4(isn)(NO2)]+. Neste caso, a constante de velocidade foi estimada entre k = 47-58 M-1 s-1 à T = 25oC, e o valor obtido experimentalmente à T = 15oC foi de k = 10,53 ± 0,29 M-1 s-1. O espectro eletrônico do íon complexo trans [Ru(NH3)4(isn)(N(O)SO3)]+ em meio aquoso apresentou uma banda larga com λ max = 362 nm (ε ∼6000 M-1 cm-1), atribuída por cálculos teóricos às seguintes transições: transferência de carga do metal para o ligante (TCML) Ru → N(O)SO3 e Ru → isn, e também d → d. Os ensaios preliminares de fotólise (λ irrad = 355 nm) do complexo trans[Ru(NH3)4(isn)(N(O)SO3)](PF6) em solução de tampão fosfato (pH 7,4) sugerem a formação das seguintes espécies nos intervalos iniciais de fotólise: i) NO, ii) SO3 •-, e iii) isn (labilizado do complexo). O mecanismo para a formação desses produtos ainda está sob investigação. / Aiming the production of new nitric oxide releasing materials (NORM), this work reports the development of a cassava starch based film, in which a ruthenium nitrosyl complex was impregnated, and evaluate the NO release from this film. Ruthenium nitrosyl complex trans-[Ru(NH3)4(isn)NO](BF4)3 (RuNOisn; isn = isonicotinamide) is able to release NO in a controlled manner through both photolysis (λirr = 310-370 nm) and chemical reduction. The incorporation of such complex into the starch-based films was performed under mild conditions, yielding a new material able to store and release NO, abbreviated as CSx-RuNOisn. Spectroscopic analysis of CSx-RuNOisn indicated that the coordination sphere of RuNOisn remained intact during film production. Exposure of CSx-RuNOisn to long wave UV-light (λirr = 355 nm) leads to NO release and likely to the formation of the paramagnetic photoproduct trans-[RuIII(NH3)4isn(H2O)]3+ in the film. Reaction of this aquoruthenium(III) complex with aqueous nitrite regenerates RuNOisn in the film. Delivery of NO upon photolysis of CSx-RuNO isn was verified and quantified by trapping with oxymyoglobin. The calculated concentration of NO released from the film was 5.02 ± 0.12 μM (10.04 ± 0.24 nmol NO in a 2 mL) after approximately 17 min of irradiation (500 laser pulses at 2 s intervals). Moreover, NO release upon chemical reduction was carried out using L-cysteine as a reductant. Cysteine-mediated NO delivery from CSx-RuNOisn persisted for more than 7 h, during which physiologically relevant NO concentrations were liberated (average flux of 1.9 pmol NO s-1 cm-2 of film). This value is comparable to that produced by endothelial cells (1.67 pmol s-1 cm-2). Preliminary results about the biodegradation of the films in vivo suggest that the films were completely absorbed by the organism in a period of 30 days. These results suggest that CSx-RuNOisn is a promising candidate for use in biological applications. A new nitrosylsulphito complex bearing the ligand (N(O)SO3-) was isolated, trans-[Ru(NH3)4(isn)(N(O)SO3)](X) (isn = isonicotinamide, X = PF6- or SiPF6-), and its structure was determined by X-Ray crystallography. This complex was obtained by the reaction between trans-[Ru(NH3)4(isn)(NO)]3+ and sulfite ions (SO32-). X-Ray results confirmed that the nucleophilic attack of the sulphite anion (SO32-) was on the nitrogen atom of the nitrosyl ligand (NO) coordinated to the ruthenium center ([Ru-NO+]), yielding the ligand O=N-SO3-: [RuNO+]3+ + SO32- → [Ru(N(O)SO3)]+. Complex trans- [Ru(NH3)4(isn)(N(O)SO3)]+ is stable in aqueous solution from pH 7.4 to 5.2, and the decomposition rates (k) (due to the isn labilization) are in the range of k = 0.86-3.07 × 10-5 s-1. In more acidic conditions, (acetate buffer pH 4.2, 3.9, and trifluoroacetic acid solution 1.0 M) complex trans-[Ru(NH3)4(isn)(N(O)SO3)]+ is converted into the respective nitrosyl trans-[RuII(NH3)4(isn)NO+]3+. Reaction of trans-[Ru(NH3)4(isn)(N(O)SO3)]+ and hydroxide ions (OH-) yielded the nitro complex trans-[Ru(NH3)4(isn)(NO2)]+, which was characterized by 15N NMR and electronic spectroscopy. Rate constants for such reaction are k = 6.16 ± 0.22 M-1 s-1 at 25oC, and k = 2.15 ± 0.07 M-1 s-1 at 15oC. In the case of complex trans-[RuII(NH3)4(isn)NO+]3+, its reaction with OH- also yield the nitro complex trans-[Ru(NH3)4(isn)(NO2)]+. The estimated rate constant for such reaction was k = 46.9-57.6 M-1 s-1 at 25oC, and the experimental value obtained at 15oC was k = 10.53 ± 0.29 M-1 s-1. The ion complex trans-[Ru(NH3)4(isn)(N(O)SO3)]+ showed an intense and broad band at 362 nm (ε∼6000 M-1 cm-1) in aqueous solutions, which was assigned by DFT calculations to the following transitions: metal to ligand charge transfer (MLCT) Ru→N(O)SO3 and Ru→isn, and d→d as well. Preliminary photolysis assays (λirrad = 355 nm) performed with complex trans-[Ru(NH3)4(isn)(N(O)SO3)](PF6) in phosphate buffer solution (pH 7,4) suggests that the following species have been formed (in the initial photolysis period): i) NO, ii) SO3•-, and iii) isn (labilized). The whole mechanism to yield such products is still under investigation.

Abstract not available

生物科技為目前世界各國極力投入之新興產業，此產業無需大量天然資源及低污染的特性，同時又能解決人類的醫療問題並提昇生活水準的未來發展空間，更使得此一新興產業吸引了眾人的眼光。 　　本論文提出一個評估的模式，讓此產業之技術、產品或專利之價值能夠經系統化的分析後完整呈現，並可基於此分析結果作出策略上之判斷，並以GnRH vaccine技術為例。因為GnRH分子之序列早已發表，經本研究分析得兩種常用的專利保護策略，第一種是將GnRH與不同的載體進行結合，而新生成一個嵌合化合物，並申請此新化合物之專利；第二種是進行GnRH分子結構上之改變以取得新序列的專利保護，取得效果優於自然設計之分子，或藉以增加與免疫載體之結合率。經分析後發現，GnRH疫苗應用理論已經相當成熟，未來幾年應該會有成熟產品通過上市之審核過程；再隨著新科技的進步，可預期將會有更多的新產品會上市，而進一步地有效提昇產品功效並降低製造成本。 　　本研究最後針對GnRH未來在人用藥品及動物用藥之未來市場加以分析，並對於以重複免疫原在重組型疫苗製備法之開發提出行銷、財務、通路、價格、推廣、臨床實驗之建議，以期能經由策略規劃的角度將生物技術的價值作最大化的管理。 / Biotechnology attracts high attentions among many countries recently. New biotechnology will provide a key solution to cure diseases and potentially will be able to increase human life quality without damaging the environment and consuming massy natural resources. 　　In this thesis, we presented a possible process, which involved in academic, marketing and patent information analysis, to effectively evaluate the feasibility and potential of any novel biological technologies, so as to provide the managerial and strategic suggestions based on the analysis. GnRH repeat immunogens vaccine development was taken as an example to demonstrate if the proposed process can be executed adequately. After discretly cross examining the collected information, we concluded that many independent GnRH vaccine patents are available but will not be effective enough to stop to new competitors to get in this sector because GnRH sequence was disclosed in very early research and nobody held the patent for it and most of GnRH patents were granted to either different GnRH-Carrier chimeras or the GnRH analog sequences. Analysis results show that there is only one company closely approaching the final product approval at this moment. However, any improved technology can easily employed to provide a better product with higher effectiveness and lower cost following the first product's upcoming marketing clearance and then make the future market very complicated. 　　Based on those findings, we further analyze the Taiwanese current environment and provide a few managerial suggestions to maximize the value of a novel GnRH vaccine preparation technology.

<p>The human endometrium undergoes dramatic changes in morphology and function during the menstrual cycle. Recurrent angiogenesis (the formation of new blood vessels) is of utmost importance for oxygen supply and nourishment of the rapidly growing endometrial tissue. </p><p>The importance of some growth factors known to stimulate new blood vessel formation both in vivo and in vitro in non-uterine tissues, for endometrial angiogenesis, was studied. Further, the possible relationship between the patterns of expression of some angiogenic growth factors and bleeding disturbances during the use of a progestin-only intrauterine contraceptive device was analyzed. Different ways of determining changes in the endometrial vascular density during the menstrual cycle were also evaluated. </p><p>The expression of the angiogenic growth factors vascular endothelial growth factors (VEGF) A, B, C, and D, fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF) and their receptors was analyzed using immunohistochemistry.</p><p>VEGF-A, -B and -C, FGF-2 and EGF and their receptors were all found to be expressed in normal human endometrium, especially in and/or around blood vessels, supporting the hypothesis that these peptides most probably contribute to the regulation of angiogenesis and blood vessel function in normal human endometrium.</p><p>There were differences in expression of some of the studied ligands and receptors in endometrium from users of an LNG-IUS with and without bleeding disturbances. We conclude that changes in the expression of these growth factors and receptors might be involved in the formation of fragile and dysfunctional blood vessels that subsequently give rise to bleeding disturbances.</p><p>The three different methods that were applied for calculating endometrial blood vessel density showed similar results and none of them indicated any significant changes during the menstrual cycle. Angiogenesis thus seems to occur mainly by blood vessel elongation and the angiogenic activity is probably related to changes in endometrial thickness and coiling of the spiral arteries.</p>

The human endometrium undergoes dramatic changes in morphology and function during the menstrual cycle. Recurrent angiogenesis (the formation of new blood vessels) is of utmost importance for oxygen supply and nourishment of the rapidly growing endometrial tissue. The importance of some growth factors known to stimulate new blood vessel formation both in vivo and in vitro in non-uterine tissues, for endometrial angiogenesis, was studied. Further, the possible relationship between the patterns of expression of some angiogenic growth factors and bleeding disturbances during the use of a progestin-only intrauterine contraceptive device was analyzed. Different ways of determining changes in the endometrial vascular density during the menstrual cycle were also evaluated. The expression of the angiogenic growth factors vascular endothelial growth factors (VEGF) A, B, C, and D, fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF) and their receptors was analyzed using immunohistochemistry. VEGF-A, -B and -C, FGF-2 and EGF and their receptors were all found to be expressed in normal human endometrium, especially in and/or around blood vessels, supporting the hypothesis that these peptides most probably contribute to the regulation of angiogenesis and blood vessel function in normal human endometrium. There were differences in expression of some of the studied ligands and receptors in endometrium from users of an LNG-IUS with and without bleeding disturbances. We conclude that changes in the expression of these growth factors and receptors might be involved in the formation of fragile and dysfunctional blood vessels that subsequently give rise to bleeding disturbances. The three different methods that were applied for calculating endometrial blood vessel density showed similar results and none of them indicated any significant changes during the menstrual cycle. Angiogenesis thus seems to occur mainly by blood vessel elongation and the angiogenic activity is probably related to changes in endometrial thickness and coiling of the spiral arteries.

Crohns sjukdom är en kronisk inflammatorisk tarmsjukdom av okänd orsak. Det tidigaste tecknet på Crohns sjukdom är mikroskopiska sår i det s.k. follikelassocierade epitelet (FAE) som täcker ansamlingar av immunceller i tarmen. FAE är specialiserat för att fånga innehåll från tarmen och transportera det till underliggande immunvävnad. Denna funktion är viktig för att inducera skyddande immunsvar, men den utgör också en ingångsväg för sjukdomsalstrande bakterier. Crohns sjukdom är associerat med ett kraftigt ökat immunsvar mot bakterier, och sjukdomsförloppet kan ändras av stress. Det övergripande syftet med avhandlingen var att studera effekterna av stress på FAE samt att undersöka rollen av FAE vid utvecklingen av tarminflammation, särskilt vid Crohns sjukdom. Inledningsvis studerades effekterna av psykologisk stress på FAE. Stressade råttor uppvisade ökad genomsläpplighet av bakterier efter stress, och passagen var högre i FAE än i vanligt epitel. Efterföljande experiment visade att stressförändringarna i slemhinnan regleras via kortikotropinfrisättande hormon och mastceller. Vidare visade det sig att vasoaktiv intestinal peptid kunde efterlikna stressens effekter på genomsläppligheten, och att detta kunde förhindras genom att blockera mastcellerna. Studier av tunntarmsslemhinna från patienter med icke-inflammatorisk tarmsjukdom och friska kontroller visade en högre passage av bakterier i FAE än i vanligt epitel. Hos patienter med Crohns sjukdom var bakteriepassagen genom FAE betydligt ökad jämfört med kontroller. Resultaten från detta avhandlingsarbete visar att stress kan förändra upptaget av bakterier från tarmen via FAE, med mekanismer som innefattar kortikotropinfrisättande hormon och mastceller. Detta har gett nya kunskaper kring regleringen av slemhinnebarriären. Vidare presenterar denna avhandling nya insikter i sjukdomsuppkomsten vid Crohns sjukdom genom att påvisa en tidigare okänd defekt i barriärfunktionen i FAE. / The earliest observable signs of Crohn’s disease are microscopic erosions in the follicle-associated epithelium (FAE) covering the Peyer’s patches. The FAE, which contains M cells, is specialised in sampling of luminal content and delivery to underlying immune cells. This sampling is crucial for induction of protective immune responses, but it also provides a route of entry for microorganisms into the mucosa. Crohn’s disease is associated with an increased immune response to bacteria, and the disease course can be altered by stress. The overall aim of this thesis was to study the effects of stress on the FAE and elucidate the role of FAE in the development of intestinal inflammation, specifically Crohn’s disease. Initially, rats were submitted to acute and chronic water avoidance stress to study the effects of psychological stress on the FAE. Stressed rats showed enhanced antigen and bacterial passage, and the passage was higher in FAE than in regular villus epithelium (VE). Further, stress gave rise to ultrastructural changes. Subsequent experiments revealed the stress-induced increase in permeability to be regulated by corticotropin-releasing hormone and mast cells. Furthermore, vasoactive intestinal peptide (VIP) mimicked the stress effects on permeability, and the VIP effects were inhibited by a mast cell stabiliser. Human studies of ileal mucosa from patients with non-inflammatory disease and healthy controls showed a higher antigen and bacterial passage in FAE than in VE. In patients with Crohn’s disease, the bacterial passage across the FAE was significantly increased compared to non-inflammatory and inflammatory controls (ulcerative colitis). Furthermore, there was an enhanced uptake of bacteria into dendritic cells, and augmented TNF-α release in Crohn’s disease mucosa. Taken together this thesis shows that stress can modulate the uptake of luminal antigens and bacteria via the FAE, through mechanisms involving CRH and mast cells. It further shows that human ileal FAE is functionally distinct from VE, and that Crohn’s disease patients exhibit enhanced FAE permeability compared to inflammatory and non-inflammatory controls. This thesis presents novel insights into regulation of the FAE barrier, as well as into the pathophysiology of Crohn’s disease by demonstrating a previously unrecognised defect of the FAE barrier function in ileal Crohn’s disease.

A fundamental question in the neurobiological study of drug addiction concerns the mechanisms mediating the motivational effects of chronic drug withdrawal. According to one theory, drugs of abuse activate opposing motivational processes after both acute and chronic drug use. The negative experience of withdrawal is the opponent process of chronic drug use that drives relapse to drug-seeking and -taking, making the identification of the neurobiological substrates mediating withdrawal an issue of central importance in addiction research. In this thesis, I identify the involvement of the neurotransmitters dopamine (DA) and corticotropin-releasing factor (CRF) in the opponent motivational a- and b-processes occurring after acute and chronic nicotine administration. I report that acute nicotine stimulates an initial aversive a-process followed by a rewarding opponent b-process, and chronic nicotine stimulates a rewarding a-process followed by an aversive opponent b-process (withdrawal). These responses can be modeled using a place conditioning paradigm. I demonstrate that the acute nicotine a-process is mediated by phasic dopaminergic activity and the DA receptor subtype-1 (D1R) but not by tonic dopaminergic activity and the DA receptor subtype-2 (D2R) or CRF activity, and the opponent b-process is neither DA- nor CRF-mediated. I also demonstrate that the chronic nicotine a-process is DA- but not CRF-mediated, and that withdrawal from chronic nicotine (the b-process) decreases tonic but not phasic DA activity in the ventral tegmental area (VTA), an effect that is D2R- but not D1R-mediated. I show that a specific pattern of signaling at D1Rs and D2Rs mediates the motivational responses to acute nicotine and chronic nicotine withdrawal, respectively, by demonstrating that both increasing or decreasing signaling at these receptors prevents the expression of the conditioned motivational response. Furthermore, I report that the induction of nicotine dependence increases CRF mRNA in VTA DA neurons, and that blocking either the upregulation of CRF mRNA or the activation of VTA CRF receptors prevents the anxiogenic and aversive motivational responses to withdrawal from chronic nicotine. The results described in this thesis provide novel evidence of a VTA DA/CRF system, and demonstrate that both CRF and a specific pattern of tonic DA activity in the VTA are necessary for the aversive motivational experience of nicotine withdrawal.