No description available.

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada / Background: The identification of genetic variants that predispose increased susceptibility to nicotine dependence becomes increasingly important for the prevention and smoking treatment. In the context of personalized medicine, the main aims of this study were to evaluate whether the CHRNA2, CHRNA3, CHRNA5 and CHRNB3 polymorphisms are associated with the level of dependence in smokers and the result of smoking treatment. Methods: This cohort study enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. PAF comfort scale was used to evaluate the comfort of the patient during treatment. The CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968 and rs2036527 and CHRNB3 rs6474413 polymorphisms were genotyped by high resolution melting analysis. Results: Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively; compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P=0.03, n=389; P=0.01, n=391). The GA or AA genotypes to the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR=1.63; 95%CI=1.04 to 2.54; P=0.03 and OR=1.59, 95%CI=1.02 to 2.48; P=0.04; respectively), in a multivariate model. We found no association of these polymorphisms with FTND score for nicotine dependence. For the CHRNA2 rs2472553, CHRNA3 rs1051730 and CHRNB3 rs6474413 polymorphisms no significant associations were found with phenotypes studied. Conclusion: The CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These results can contribute to major advances in personalized medicine based therapy

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada / Background: The identification of genetic variants that predispose increased susceptibility to nicotine dependence becomes increasingly important for the prevention and smoking treatment. In the context of personalized medicine, the main aims of this study were to evaluate whether the CHRNA2, CHRNA3, CHRNA5 and CHRNB3 polymorphisms are associated with the level of dependence in smokers and the result of smoking treatment. Methods: This cohort study enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. PAF comfort scale was used to evaluate the comfort of the patient during treatment. The CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968 and rs2036527 and CHRNB3 rs6474413 polymorphisms were genotyped by high resolution melting analysis. Results: Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively; compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P=0.03, n=389; P=0.01, n=391). The GA or AA genotypes to the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR=1.63; 95%CI=1.04 to 2.54; P=0.03 and OR=1.59, 95%CI=1.02 to 2.48; P=0.04; respectively), in a multivariate model. We found no association of these polymorphisms with FTND score for nicotine dependence. For the CHRNA2 rs2472553, CHRNA3 rs1051730 and CHRNB3 rs6474413 polymorphisms no significant associations were found with phenotypes studied. Conclusion: The CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These results can contribute to major advances in personalized medicine based therapy

Inspiriert von den natürlich vorkommenden Antibiotika der Gramicidin Familie und ihrer d-(alt)-l Aminosäuresequenz, die es diesen Oligopeptiden ermöglicht, eine beta–helikale Sekundärstruktur einzunehmen, war das Hauptziel dieser Arbeit die Synthese und Charakterisierung von Peptiden und diversen Pseudopeptiden mit regulärer all-l und d-(alt)-l Sequenz und die Untersuchung des Einflusses dieser stereochemischen Variation auf die Strukturen und Eigenschaften dieser Verbindungen. Zusätzlich ergab der Austausch von Amid-Bindungen im Peptid-Rückgrat durch verschiedene Isostere diverse, teils einzigartige Pseudopeptid-Strukturen, wohingegen Verzweigung des linearen Peptid-Rückgrates zu sphärischen Molekülen führte. Alle Projekte zielten auf die Entwicklung und Synthese diskreter Oligomere für Strukturuntersuchungen, sowie auf die Einbindung der jeweiligen Strukturelemente in Polymere. Die Polymerization geeigneter Monomere zu Polymeren soll zu makro- und supramolekularen Nano-Objekten führen. Die divergent/konvergente Synthese einer Serie von Oligo-d-(alt)-l-lysinen zielte auf die Generierung hydrophiler, pH-sensitiver nanotubularer Strukturen. Schrittweiser Austausch von Amid-Bindungen des Peptid-Rückgrates durch Ester-(alt)-Urea-Einheiten führte zu all-l und d-(alt)-l Oligopseudoleucinen mit 50% und 0% Amid-Bindungs-Anteil. Design, Synthese und Polymerisation von AB-“Click”-Monomeren, basierend auf all-l and l-(alt)-d lysin Dipeptiden, ergaben hochmolekulare, Triazol-enthaltende Polypseudopeptide, deren Seitenketten mit Pyrenbuttersäure quantitativ postfunktionalisiert werden konnten. Die Einführung von Verzweigung in Glutamat-Peptide ergab chirale Dendrimere mit adressierbaren fokalen und periphären Funktionalitäten, sowie variabler Ladungsdichte. Design, Synthese und Polymerisation eines Glutamat basierenden AB2-“Click”-Monomers lieferte verwandte chirale hyperverzweigte Polypseudopeptide. / Inspired by the naturally occurring antibiotics of the Gramicidin family and their d-(alt)-l amino acid sequence, enabling these oligopeptides to adopt a beta–helical secondary structure, the work presented in this thesis targeted the syn-thesis and characterization of peptides and diverse pseudopeptides with regular all-l and d-(alt)-l sequences and the influence of this stereochemical variation on the compounds’ structures and properties. Further diversification of the struc-tures as obtained by replacing amide bonds in the peptide backbone with differ-ent isosteres, affording unique pseudopeptide structures. In addition spherical molecules were generated by introducing branching into the linear peptide scaf-fold. Throughout all projects, the aim was the design and synthesis of discrete oligomers for structural investigations and the incorporation of the respective structural elements into polymers via the polymerization of suitable monomers, in order to generate nanoscale macromolecular and supramolecular objects. The divergent/convergent synthesis of a series of oligo-d-(alt)-l-lysines targeted the generation of hydrophilic, pH-sensitive nanotubular structures. The stepwise replacement of peptide backbone amide bonds with ester-(alt)-urea moieties afforded all-l and d-(alt)-l oligopseudoleucines with 50% and 0% amide content. The design, synthesis, and polymerization of an AB-“Click”-monomer, based on all-l and l-(alt)-d lysine dipeptides afforded high molecular weight, triazole con-taining polypseudopeptides. Quantitative coupling to pyrene butyric acid afforded the respective side chain labeled polymers. The introduction of branching into glutamate peptides afforded fully chiral den-drimers with addressable focal and peripheral functionalities and variable charge density. The design, synthesis, and polymerization of a glutamate based AB2-“Click”-monomer led to related chiral hyperbranched polypseudopeptides.

Introdução: Os receptores do peptídeo insulinotrópico dependente de glicose (GIPR) e do hormônio luteinizante (LHCGR) são receptores acoplados à proteína G com amplo padrão de expressão tecidual. A expressão anômala destes receptores tem sido descrita em casos de hiperplasia adrenal macronodular independente de ACTH (AIMAH) e em alguns adenomas, resultando em aumento da secreção hormonal (cortisol, andrógenos e aldosterona) pelo cortex adrenal. O papel destes receptores em outras formas de hiperplasia, como a doença adrenocortical nodular pigmentosa primária (PPNAD), aumento da adrenal associado à neoplasia endócrina múltipla tipo 1 (MEN1), e em carcinoma do córtex adrenal tem sido pouco investigado; sendo assim, considera-se relevante estudar a expressão destes receptores nos pacientes com tumores adrenocorticais esporádicos, nos pacientes com AIMAH, PPNAD e aumento adrenal associado à MEN1. Objetivos: 1) Caracterização molecular dos casos de neoplasia endócrina múltipla tipo 1 e PPNAD: pesquisa de mutações dos genes MEN1 e PRKAR1A e análise da perda de heterozigose (LOH) destes genes no tecido adrenal destes pacientes. 2) Quantificar a expressão do GIPR e do LHCGR em tecido adrenocortical normal, tumoral, hiperplásico e correlacionar a expressão destes com a classificação histológica dos tumores adrenocorticais. Pacientes: 55 pacientes (30 adultos) com tumores adrenocorticais (37 adenomas e 18 carcinomas); 7 pacientes com AIMAH, 4 com MEN1, 1 com PPNAD e tecidos controles (adrenal; testículo e pâncreas). Métodos: extração de DNA genômico, RNA e síntese de DNA complementar (cDNA); amplificação por PCR das regiões codificadoras dos genes MEN1 e PRKAR1A seguida por seqüenciamento automático. Pesquisa de LOH pela amplificação de microssatélites por PCR e análise pelo programa GeneScan. Quantificação da expressão do GIPR e do LHCGR por PCR em tempo real pelo método TaqMan e estudo de imunohistoquímica para GIPR nos tumores adrenocorticais. Resultados: identificação de 3 mutações (893+ 1G>A, W183X e A68fsX118) e dois polimorfirmos (S145S e D418D) no gene MEN1 e uma mutação (Y21X) no PRKAR1A. Ausência de LOH nos tecidos adrenais estudados. A expressão do GIPR e do LHCGR foi identificada em tecidos adrenais normais, tumorais e hiperplásicos. O nível de expressão do GIPR foi mais elevado nos tumores adrenocorticais malignos que nos benignos tanto no grupo pediátrico (mediana= 18,1 e 4,6, respectivamente; p <0,05), quanto no grupo adulto (mediana = 4,8 e 1,3 respectivamente; p <0,001). O nível de expressão do LHCGR, no grupo pediátrico, foi elevado tanto nos tumores benignos quanto nos malignos (mediana= 6,4 e 4,3, respectivamente). No grupo adulto os níveis de expressão deste receptor foram extremamente baixos nos tumores malignos em relação aos benignos (mediana= 0,06 e 2,3, respectivamente; p <0,001). A imunohistoquímica para o GIPR foi variável e não correlacionada à expressão do gene GIPR. Não houve diferença nos níveis de expressão do GIPR e do LHCGR nas hiperplasias do córtex adrenal. Conclusões: a presença de LOH e mutação em heterozigose composta do gene MEN1 e do PRKAR1A foram afastadas como mecanismos responsáveis pelo aumento adrenal tanto nos pacientes com MEN1 como no paciente com PPNAD. A hiperexpressão de GIPR está associada a malignidade nos tumores adrenocorticais nos grupos adulto e pediátrico e a baixa expressão de LHCGR está associada a malignidade nos tumores adrenocorticais somente no grupo adulto. / Introduction: The glucose- dependent insulinotropic peptide receptor (GIPR) and luteinizing hormone receptor (LHCGR) are G-protein coupled receptors with a wide tissue expression pattern. The aberrant expression of these receptors has been described in cases of ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in some adenomas, resulting in the increase of adrenal cortex hormonal secretion (cortisol, androgens and aldosterone). The role of these receptors in other forms of adrenocortical hyperplasia, such as primary pigmented nodular adrenocortical disease (PPNAD), adrenal enlargement associated with multiple endocrine neoplasia type 1 (MEN1), and adrenocortical carcinoma has been scarcely investigated. Thus, the study of the expression of these receptors in patients with sporadical adrenocortical tumors, AIMAH, PPNAD and adrenal enlargement associated to MEN1 was considered important. Objectives: 1) Molecular study in patients with multiple endocrine neoplasia type 1 and PPNAD: mutation screening of MEN1 and PRKAR1A genes and analysis of the loss of heterozygosis (LOH) of these genes in the adrenal lesions of these patients. 2) To quantify the GIPR and LHCGR expression, in normal, tumor and hyperplasic tissue and to correlate the expression of these receptors with the adrenocortical tumor histology. Patients: 55 patients (30 adults) with adrenocortical tumors (37 adenomas and 18 carcinomas); 7 patients with AIMAH, 4 with MEN1, 1 with PPNAD and control tissue (adrenal, testis and pancreas). Methods: Extraction of genomic DNA, RNA and synthesis of complementary DNA (cDNA); PCR-amplification of the coding regions of MEN1 and PRKAR1A, followed by direct sequencing. LOH study using polymorphic marker amplification by PCR and GeneScan software analysis. Quantification of GIPR and LHCGR expression using realtime PCR -TaqMan method and GIPR immunohistochemistry study in adrenocortical tumors. Results: Identification of 3 mutations (893+ 1G>A, W183X and A68fsX118) and two polymorphic alterations (S145S and D418D) in MEN1 and a mutation (Y21X) in the PRKAR1A gene; LOH was not identified in adrenal tissue. The GIPR and LHCGR expression was identified in normal, tumor and hyperplasic adrenal tissues; the GIPR expression level was more elevated in malignant tumors compared to benign tumors in pediatric (median = 18.1 and 4.6, respectively; p <0.05) and adult patients (median = 4.8 and 1.3 respectively; p <0.001). The LHCGR expression in pediatric patients was elevated in benign as well as in malignant tumors (median = 6.4 and 4.3, respectively). In the adult group, the expression level of these receptors was extremely low in malignant tumors in relation to benign ones (median = 0.06 and 2.3, respectively; p <0.001). The GIPR immunohistochemistry was variable and did not correlate with GIPR gene expression. No difference between GIPR and LHCGR expression levels was observed in the different forms of hyperplasia. Conclusions: The presence of LOH and mutations in compound heterozygosis of MEN1 and PRKAR1A genes were ruled out as the mechanisms responsible for the adrenal enlargement in patients with multiple endocrine neoplasia type 1. GIPR overexpression is associated with malignant adrenocortical tumors in the adult and pediatric patients and low LHCGR expression is associated with malignant adrenocortical tumors only in the adult patients.

No description available.

Introdução: Os receptores do peptídeo insulinotrópico dependente de glicose (GIPR) e do hormônio luteinizante (LHCGR) são receptores acoplados à proteína G com amplo padrão de expressão tecidual. A expressão anômala destes receptores tem sido descrita em casos de hiperplasia adrenal macronodular independente de ACTH (AIMAH) e em alguns adenomas, resultando em aumento da secreção hormonal (cortisol, andrógenos e aldosterona) pelo cortex adrenal. O papel destes receptores em outras formas de hiperplasia, como a doença adrenocortical nodular pigmentosa primária (PPNAD), aumento da adrenal associado à neoplasia endócrina múltipla tipo 1 (MEN1), e em carcinoma do córtex adrenal tem sido pouco investigado; sendo assim, considera-se relevante estudar a expressão destes receptores nos pacientes com tumores adrenocorticais esporádicos, nos pacientes com AIMAH, PPNAD e aumento adrenal associado à MEN1. Objetivos: 1) Caracterização molecular dos casos de neoplasia endócrina múltipla tipo 1 e PPNAD: pesquisa de mutações dos genes MEN1 e PRKAR1A e análise da perda de heterozigose (LOH) destes genes no tecido adrenal destes pacientes. 2) Quantificar a expressão do GIPR e do LHCGR em tecido adrenocortical normal, tumoral, hiperplásico e correlacionar a expressão destes com a classificação histológica dos tumores adrenocorticais. Pacientes: 55 pacientes (30 adultos) com tumores adrenocorticais (37 adenomas e 18 carcinomas); 7 pacientes com AIMAH, 4 com MEN1, 1 com PPNAD e tecidos controles (adrenal; testículo e pâncreas). Métodos: extração de DNA genômico, RNA e síntese de DNA complementar (cDNA); amplificação por PCR das regiões codificadoras dos genes MEN1 e PRKAR1A seguida por seqüenciamento automático. Pesquisa de LOH pela amplificação de microssatélites por PCR e análise pelo programa GeneScan. Quantificação da expressão do GIPR e do LHCGR por PCR em tempo real pelo método TaqMan e estudo de imunohistoquímica para GIPR nos tumores adrenocorticais. Resultados: identificação de 3 mutações (893+ 1G>A, W183X e A68fsX118) e dois polimorfirmos (S145S e D418D) no gene MEN1 e uma mutação (Y21X) no PRKAR1A. Ausência de LOH nos tecidos adrenais estudados. A expressão do GIPR e do LHCGR foi identificada em tecidos adrenais normais, tumorais e hiperplásicos. O nível de expressão do GIPR foi mais elevado nos tumores adrenocorticais malignos que nos benignos tanto no grupo pediátrico (mediana= 18,1 e 4,6, respectivamente; p <0,05), quanto no grupo adulto (mediana = 4,8 e 1,3 respectivamente; p <0,001). O nível de expressão do LHCGR, no grupo pediátrico, foi elevado tanto nos tumores benignos quanto nos malignos (mediana= 6,4 e 4,3, respectivamente). No grupo adulto os níveis de expressão deste receptor foram extremamente baixos nos tumores malignos em relação aos benignos (mediana= 0,06 e 2,3, respectivamente; p <0,001). A imunohistoquímica para o GIPR foi variável e não correlacionada à expressão do gene GIPR. Não houve diferença nos níveis de expressão do GIPR e do LHCGR nas hiperplasias do córtex adrenal. Conclusões: a presença de LOH e mutação em heterozigose composta do gene MEN1 e do PRKAR1A foram afastadas como mecanismos responsáveis pelo aumento adrenal tanto nos pacientes com MEN1 como no paciente com PPNAD. A hiperexpressão de GIPR está associada a malignidade nos tumores adrenocorticais nos grupos adulto e pediátrico e a baixa expressão de LHCGR está associada a malignidade nos tumores adrenocorticais somente no grupo adulto. / Introduction: The glucose- dependent insulinotropic peptide receptor (GIPR) and luteinizing hormone receptor (LHCGR) are G-protein coupled receptors with a wide tissue expression pattern. The aberrant expression of these receptors has been described in cases of ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in some adenomas, resulting in the increase of adrenal cortex hormonal secretion (cortisol, androgens and aldosterone). The role of these receptors in other forms of adrenocortical hyperplasia, such as primary pigmented nodular adrenocortical disease (PPNAD), adrenal enlargement associated with multiple endocrine neoplasia type 1 (MEN1), and adrenocortical carcinoma has been scarcely investigated. Thus, the study of the expression of these receptors in patients with sporadical adrenocortical tumors, AIMAH, PPNAD and adrenal enlargement associated to MEN1 was considered important. Objectives: 1) Molecular study in patients with multiple endocrine neoplasia type 1 and PPNAD: mutation screening of MEN1 and PRKAR1A genes and analysis of the loss of heterozygosis (LOH) of these genes in the adrenal lesions of these patients. 2) To quantify the GIPR and LHCGR expression, in normal, tumor and hyperplasic tissue and to correlate the expression of these receptors with the adrenocortical tumor histology. Patients: 55 patients (30 adults) with adrenocortical tumors (37 adenomas and 18 carcinomas); 7 patients with AIMAH, 4 with MEN1, 1 with PPNAD and control tissue (adrenal, testis and pancreas). Methods: Extraction of genomic DNA, RNA and synthesis of complementary DNA (cDNA); PCR-amplification of the coding regions of MEN1 and PRKAR1A, followed by direct sequencing. LOH study using polymorphic marker amplification by PCR and GeneScan software analysis. Quantification of GIPR and LHCGR expression using realtime PCR -TaqMan method and GIPR immunohistochemistry study in adrenocortical tumors. Results: Identification of 3 mutations (893+ 1G>A, W183X and A68fsX118) and two polymorphic alterations (S145S and D418D) in MEN1 and a mutation (Y21X) in the PRKAR1A gene; LOH was not identified in adrenal tissue. The GIPR and LHCGR expression was identified in normal, tumor and hyperplasic adrenal tissues; the GIPR expression level was more elevated in malignant tumors compared to benign tumors in pediatric (median = 18.1 and 4.6, respectively; p <0.05) and adult patients (median = 4.8 and 1.3 respectively; p <0.001). The LHCGR expression in pediatric patients was elevated in benign as well as in malignant tumors (median = 6.4 and 4.3, respectively). In the adult group, the expression level of these receptors was extremely low in malignant tumors in relation to benign ones (median = 0.06 and 2.3, respectively; p <0.001). The GIPR immunohistochemistry was variable and did not correlate with GIPR gene expression. No difference between GIPR and LHCGR expression levels was observed in the different forms of hyperplasia. Conclusions: The presence of LOH and mutations in compound heterozygosis of MEN1 and PRKAR1A genes were ruled out as the mechanisms responsible for the adrenal enlargement in patients with multiple endocrine neoplasia type 1. GIPR overexpression is associated with malignant adrenocortical tumors in the adult and pediatric patients and low LHCGR expression is associated with malignant adrenocortical tumors only in the adult patients.

[ES] Los tumores malignos del cerebro y del sistema nervioso central representan el 21% de los tumores en niños y son la segunda causa principal de muerte por cáncer pediátrico después de la leucemia. Los gliomas de alto grado siguen siendo incurables y poseen altas tasas de mortalidad: una supervivencia a cinco años del 4,7 % para pacientes con glioblastoma multiforme (GBM) y menos de un año en pacientes con glioma pontino intrínseco difuso (DIPG). Los bajos niveles de penetración del fármaco a través de la barrera hematoencefálica y la baja tasa de supervivencia asociada resaltan en la necesidad de nuevas propuestas de tratamiento para esta necesidad clínica no cubierta. La administración intranasal ofrece un enfoque no invasivo prometedor que elude el metabolismo hepato-gastrointestinal y la barrera hematoencefálica, constituyendo una ruta directa de la nariz al cerebro. Los poliglutamatos (PGA) representan excelentes candidatos para la administración de agentes terapéuticos en el cerebro debido a su biodegradabilidad y multivalencia, lo que permite la unión covalente de fármacos y grupos directores que puedan contribuir a cruzar las diferentes barreras biológicas existentes desde la nariz hasta el cerebro. La conjugación covalente del agente terapéutico a la cadena polimérica ofrece una estabilidad prolongada en la circulación sanguínea y un mayor control sobre la liberación del fármaco en el microambiente tumoral. Esta tesis se centra en el diseño racional y desarrollo de nuevos conjugados PGA-fármaco y una plataforma intranasal segura y eficiente para la administración dirigida y la liberación del fármaco en el cerebro como tratamiento del glioma pediátrico. Se desarrolló una familia de conjugados de poliglutamatos que incorporan palbociclib, un inhibidor de CDK, utilizando diferentes espaciadores sensibles a estímulos, así como diferente carga de fármacos. Estudiamos el efecto de la carga y conjugación de un fármaco sobre la conformación en solución de PGAs lineales y en forma de estrella. Con dispersión de rayos X de ángulo pequeño y dicroísmo circular, demostramos la evolución del conjugado polipeptídico al aumentar la carga de fármaco. Además, establecimos el vínculo entre la conformación de los conjugados y su actividad biológica en células GBM y DIPG derivadas de pacientes. Nuestros hallazgos ilustran la necesidad de una comprensión profunda de las propiedades fisicoquímicas de los nanosistemas estudiados que pueden ayudar a predecir su resultado biológico. Para el cribado rápido y la validación de la formulación intranasal, establecimos con éxito una plataforma de detección ex vivo basada en células de difusión verticales de Franz con mucosa nasal de oveja. Varios sistemas basados en PGA lineal y en forma de estrella modificadas con diferentes ligandos (incluyendo el ácido docosahexaenoico, ácido hialurónico (HA), odorranalectina), así como sistemas entrecruzados del PGA en forma de estrella y la mezcla física con un HA entrecruzado (HA-CP®). A continuación, se realizó estudios de biodistribución mediante administración intranasal de los candidatos seleccionados, seguidos de la cuantificación ex vivo con la técnica IVIS y el ensayo de fluorescencia estándar después de la homogeneización de órganos y los estudios histológicos del cerebro. Los datos obtenidos mostraron claramente la presencia de las diferentes plataformas en el cerebro e, su internalización en células del bulbo olfativo y una buena difusión en el cerebro a través de diferentes áreas, llegando incluso a detectarse en el hipocampo. Los candidatos seleccionados se han escalado y en la actualidad su evaluación biológica en un modelo in vivo está en curso. En conclusión, se ha diseñado, desarrollado y validado diferentes plataformas basadas en PGA capaces de llegar al cerebro a través de la administración intranasal, constituyendo una base prometedora para el tratamiento de múltiples trastornos relacionados con el cerebro. / [CA] Els tumors malignes del cervell i del sistema nerviós central representen el 21% dels tumors en nens i són la segona causa principal de mort per càncer pediàtric després de la leucèmia. Els gliomes d'alt grau que continuen sent incurables amb taxes de mortalitat altes: una supervivència a cinc anys del 4,7 % per a pacients amb glioblastoma multiforme (GBM) i menys d'un any en pacients amb glioma pontí intrínsec difús (DIPG). Els nivells baixos de penetració del fàrmac a través de la barrera hematoencefàlica i la baixa taxa de supervivència associada ressalten en la necessitat de noves propostes de tractament per a una necessitat clínica no satisfeta. L'administració intranasal ofereix un enfocament no invasiu prometedor que eludeix el metabolisme hepatogastrointestinal i la barrera hematoencefàlica, cosa que permet l'administració directa del nas al cervell. Els poliglutamats (PGA) representen excel·lents candidats per a l'administració al cervell a causa de la seva biodegradabilitat i multivalència, la qual cosa dóna suport a la introducció covalent de fàrmacs i grups objectiu i pot facilitar la transició del nas al cervell. La conjugació covalent de l'agent terapèutic a la cadena polimèrica ofereix una estabilitat prolongada en la circulació sanguínia i un major control sobre l'alliberament del fàrmac al microambient tumoral. Aquesta tesi se centra en el desenvolupament de nous conjugats PGA-fàrmac dissenyats racionalment i una plataforma intranasal segura i eficient per a l'administració dirigida i l'alliberament del fàrmac al cervell com a tractament del glioma pediàtric. Es va desenvolupar una família de conjugats de poliglutamat que incorporen l'inhibidor de CDK palbociclib, utilitzant diferents enllaçadors sensibles a estímuls i nivells variables de càrrega de fàrmacs. Estudiem l'efecte del fàrmac hidrofòbic conjugat sobre la conformació en solució de PGA lineals i en forma d'estrella. Amb dispersió de raigs X d'angle petit i dicroisme circular, demostrem l'evolució del sistema conjugat en augmentar la càrrega de fàrmac. A més, establim el vincle entre la conformació dels conjugats i la seva activitat biològica provada en cèl·lules GBM i DIPG derivades de pacients. Les nostres troballes van il·lustrar la necessitat d'una comprensió profunda de les propietats fisicoquímiques dels nanosistemes estudiats que poden ajudar a predir-ne el resultat biològic. Per a la detecció ràpida de la formulació intranasal basada en els portadors de PGA, vam establir amb èxit una plataforma de detecció ex vivo basada en cèl·lules de difusió verticals de Franz amb mucosa d'ovella. Diversos sistemes basats en PGA lineal i en forma d'estrella amb fraccions conjugades (inclòs l'àcid docosahexaenoic, àcid hialurònic (HA), odorranalectina), així com partícules reticulades (a través de cicloaddició azida-alquí o enllaços disulfur) i la barreja física amb un HA es van provar els polímers creuats (HA-CP®). Els estudis de biodistribució després de l'administració intranasal es van fer en candidats seleccionats, seguits de la quantificació ex vivo amb la tècnica IVIS i l'assaig de fluorescència estàndard després de l'homogeneïtzació d'òrgans i els estudis histològics del cervell. Les dades obtingudes van mostrar clarament el transport del nas al cervell, la internalització cel·lular dels conjugats al bulb olfactiu i, el que és més interessant, una bona difusió del cervell a través de diferents àrees, arribant fins i tot a l'hipocamp. Els sistemes seleccionats es van ampliar i la preparació per a l'avaluació biològica en un model in vivo està en curs. En general, vam ser capaços de dissenyar, desenvolupar i validar diferents plataformes basades en PGA capaces d'arribar al cervell mitjançant l'administració intranasal, la qual cosa podria ser la base per al tractament de múltiples trastorns relacionats amb el cervell. / [EN] Malignant brain and central nervous system tumors account for ~21% of tumors in children and represent the second leading cause of pediatric cancer deaths after leukemia. Standard chemotherapy allows a ~95% five-year survival rate for patients with low-grade gliomas. Unfortunately, high-grade gliomas remain generally incurable and suffer from high mortality rates - a 4.7% five-year survival for glioblastoma multiform (GBM) patients and less than one year in patients with diffuse intrinsic pontine glioma (DIPG). Low levels of drug delivery through the blood-brain barrier (BBB) and the associated poor survival rate highlight the necessity for novel treatment approaches for an unmet clinical need. Intranasal administration offers a promising non-invasive approach that circumvents hepato-gastrointestinal metabolism and the BBB, thereby enabling direct nose-to-brain delivery. Polyglutamates (PGAs), represent excellent candidates for brain delivery due to their biodegradability and multivalency, which supports the covalent introduction of drugs and targeting moieties to facilitate the nose-to-brain transition. Covalent conjugation of therapeutic agents to the polymeric main chain offers prolonged stability in the blood circulation and increased control over the drug release in the tumor microenvironment if adequately designed considering endogenous triggers, including acidic pH, an increased reductive environment, or the overexpression of specific proteases. This thesis focuses on developing novel, rationally-designed PGA-drug conjugates as a safe and efficient intranasal platform for targeted delivery and drug release in the brain as a pediatric glioma treatment. We developed a family of PGA-drug conjugates incorporating the CDK inhibitor palbociclib that employed different stimuli-responsive linkers and varying drug loading levels and studied the effect of drug loading on the solution conformation of linear and star-shaped PGAs. We demonstrated system evolution upon increased drug loading with SAXS and CD. Moreover, we established a link between conjugate conformation and their biological activity, as evaluated in patient-derived GBM and DIPG cells. Our findings illustrated the necessity of a deep understanding of the physico-chemical properties of studied nanosystems that can aid in predicting their biological outcome. For the development of an intranasal formulation, we successfully established an ex vivo screening platform based on vertical Franz diffusion cells with sheep mucosa. We evaluated several systems based on linear and star-shaped PGA with conjugated moieties (including docosahexaenoic acid, hyaluronic acid, odorranalectin), crosslinked particles (via azide-alkyne cycloaddition or disulfide bonds), and physical mixtures with a hyaluronic acid cross polymer (HA-CP®). After intranasal administration with selected candidates, we performed biodistribution studies, followed by ex vivo quantification with the IVIS spectrum in vivo imaging system and fluorescent assays after organ homogenization and brain histological studies. The obtained data demonstrated nose-to-brain transportation, internalization of conjugates in the olfactory bulb and robust diffusion through different brain areas, even reaching the hippocampus. We also scaled up selected systems and preparations for biological evaluation in an in vivo model that is currently ongoing. Overall, we were able to design, develop, and validate different PGA-based platforms capable of arriving at the brain via intranasal, which might be the base for the treatment of multiple brain-related disorders. / Melnyk, T. (2024). Drug Dellivery to the Brain Using Polymer Therapeutics as an Intranasal Platform for Pediatric Glioblastoma Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203498

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L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC. / Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.