Η ριβονουκλεάση P (RNase P) είναι το ένζυμο που ευθύνεται για την ωρίμανση του 5΄ άκρου των πρόδρομων μορίων tRNA συμμετέχοντας έτσι στην πρωτεΐνοσύνθεση. H RNase P καταλύει την ενδονουκλεολυτική θραύση ενός φωσφοδιεστερικού δεσμού παρουσία ιόντων Mg2+ παράγοντας προϊόντα με 3΄ υδροξυλικά και 5΄ φωσφορικά άκρα. Η πλειοψηφία των RNase P ενζύμων που έχουν μελετηθεί μέχρι σήμερα είναι ριβονουκλεοπρωτεΐνες αποτελούμενες από μια RNA και πρωτεϊνικές υπομονάδες. Η RNA υπομονάδα της βακτηριακής RNase P είναι ένα από τα πρώτα καταλυτικά RNA που μελετήθηκαν. Η RNase P και το ριβόσωμα είναι τα μόνα γνωστά ριβοένζυμα που είναι συντηρημένα και στις τρείς φυλογεννετικές περιοχές. Δεδομένης της ζωτικής σημασίας των λεμφοκυττάρων στην ακεραιότητα του ανοσολογικού συστήματος και στην παθογένεια ευρέος φάσματος δερματολογικών και μη νοσημάτων, σκοπός της παρούσας εργασίας ήταν η ανάπτυξη μεθοδολογίας για την απομόνωση και τον καθαρισμό της RNase P από περιφερικά λεμφοκύτταρα υγιών μαρτύρων, ο προσδιορισμός της ενζυμικής της δραστικότητας, καθώς και η μελέτη της in vitro επιδράσεως δυο συνθετικών ρετινοειδών, του 13-cis ρετινοϊκού οξέος και της ασιτρετίνης, της αμινογλυκοσίδης νεομυκίνης Β, όπως και της διφωσφορικής χλωροκίνης στην δραστικότητα της λεμφοκυτταρικής RNase P. Το ένζυμο καθαρίστηκε με τη χρήση κατιοντοανταλλακτικής χρωματογραφίας φωσφοκυτταρίνης και προσδιορίστηκαν οι βέλτιστες συνθήκες δραστικότητάς του.Μετά από μελέτη της επίδρασης των συνθετικών ρετινοϊδών και της νεομυκίνης Β στην δραστικότητα της RNase P, προέκυψε ότι και τα τρία προκαλούν μια δοσοεξαρτώμενη αναστολή της δραστικότητας της RNase Ρ των ανθρωπίνων λεμφοκυττάρων. Η διφωσφορική χλωροκίνη δεν επηρεάζει τη δραστικότητα της λεμφοκυτταρικής RNase P. Λεπτομερής κινητική ανάλυση έδειξε πως η αναστολή που προκαλείται από την ασιτρετίνη είναι συναγωνιστικού τύπου, ενώ αυτή από την νεομυκίνη Β είναι μη συναγωνιστικού τύπου.Η κινητική σταθερά Km της λεμφοκυτταρικής RNase P βρέθηκε ότι ισούται με 245 nM και η Vmax με 0.42 pmol/min. Συμπερασματικά, η απομόνωση της RNase P από ανθρώπινα περιφερικά λεμφοκύτταρα καθιστά δυνατή τη μελέτη της πιθανής ανάμιξης αυτού του ριβοενζύμου στους παθογενετικούς μηχανισμούς διαφόρων αυτοάνοσων, φλεγμονωδών και νεοπλασματικών δερματοπαθειών και μπορεί να διευκολύνει τη περαιτέρω ανάπτυξη της βασιζομένης στην RNase P τεχνολογίας για τη γονιδιακή θεραπεία δερματικών και μη παθήσεων. / Ribonuclease P (RNase P) is the enzyme responsible for the 5΄ maturation of the precursor tRNA molecules, participating in tRNA biogenesis and therefore in protein synthesis. It catalyses the endonucleolytic cleavage of a phosphodiester bond in the presence of Mg2+ and results in the production of molecules that bear 3΄ hydroxyl and 5΄ phosphoric ends. Most forms of RNase P are ribonucleoproteins consisting of an essential RNA and protein subunits. The RNA component of the bacterial RNase P was one of the first identified catalytic RNAs. So far, RNase P and the ribosome are the only ribozymes known to be conserved in all kingdoms of life (bacteria, archaea and eucarya). In view of the vital importance of lymphocytes for an effective immune system, we proceeded to the RNase P isolation from human peripheral lymphocytes. The enzyme was purified with cation exchange phosphocellulose chromatography and the optimal conditions were determined. Herein, it was investigated the effect of the synthetic retinoids (cis-retinoic acid and acitretin), neomycin B, as well as chloroquine diphosphate, on the RNase P activity. Cis-retinoic acid, acitretin and neomycin B exerted a dose-dependent inhibitory effect on RNase P activity from human lymphocytes, wlile the activity was not affected in the presence of chloroquine diphosphate. A detailed kinetic analysis showed that the inhibition caused by acitretin was of competitive type, whereas that caused by neomycin B was of noncompetitive type. The kinetic constant Km of RNase P activity isolated from lymphocytes for the tRNA maturation reaction has been estimated equal to 245 nM and the Vmax value has been estimated equal to 0.42 pmol/min. Finally, the isolation of RNase P from human peripheral lymphocytes will enable the study of the possible involvement of this ribozyme in the pathogenetic mechanisms of diverse autoimmune, inflammatory and neoplastic cutaneous disorders and may facilitate the further development of RNase P-based technology for gene therapy of infectious and neoplastic dermatoses.

No description available.

A esclerose múltipla é a principal doença desmielinizante do sistema nervoso central (SNC). É considerada a principal causa de incapacidade neurológica em adultos jovens. O comprometimento cognitivo é muito comum nessa doença, envolvendo o aprendizado, a memória e a organização cortical do movimento, funções vitais que são reguladas pelo sistema colinérgico. O modelo de desmielinização tóxica induzida pelo brometo de etídio (BE) foi utilizado neste estudo, para avaliar a atividade da enzima acetilcolinesterase (AChE) no estriado (ST), hipocampo (HP), córtex cerebral (CC), hipotálamo (HY) e ponte (PN) associado ao tratamento com interferon beta (IFN-b), ciclosporina A (CsA), vitamina E (vit E) e ebselen (Ebs). Além disso, também foi investigado o efeito in vitro do BE na atividade da AChE, juntamente com os parâmetros cinéticos dessa enzima no ST, HP, CC e CB de ratos adultos. Os resultados demonstraram que o BE inibiu significativamente a atividade da AChE no ST, HP, CC e CB nas concentrações de 0,00625, 0,0125, 0,025, 0,05 e 0,1mM e a análise dos dados cinéticos mostraram uma inibição do tipo incompetitiva no ST, HP e CC, enquanto no CB a inibição foi do tipo mista. No estudo in vivo, foi observado uma inibição na atividade da AChE no CC, ST, HP, HY, PN e CB nos diferentes períodos avaliados pós-injeção do BE (3, 7, 15, 21 e 30 dias). Quando ratos desmielinizados com BE foram tratados com IFN-b e CsA, não houve alteração na atividade dessa enzima. Por outro lado, o tratamento com Vit E e Ebs foram capazes de aumentar a atividade da AChE no ST, CC e HP. Estudos imuno-histoquímicos demonstraram que nos ratos tratados com Vit E e Ebs as lesões induzidas pelo BE foram menores, sugerindo que estes compostos interferem no desenvolvimento de lesões desmielinizantes. Foi também avaliado o efeito per se do IFN-b, da CsA, da Vit E e do Ebs na atividade da AChE, os quais demonstraram um efeito inibitório sobre a atividade dessa enzima. Em plaquetas de ratos desmielinizados, foi observado uma diminuição na atividade da NTPDase e o tratamento com a Vit E e o Ebs modularam a hidrólise dos nucleotídeos de adenina. O presente trabalho demonstrou que o BE é um potente inibidor da atividade da AChE in vitro e os resultados in vivo demonstraram que a atividade dessa enzima está alterada após um evento de desmielinização tóxica no SNC. Os resultados deste estudo ajudaram a confirmar que drogas utilizadas no tratamento de pacientes com esclerose múltipla tais como o IFN-b e a CsA causam efeitos na atividade da AChE. A Vit E e o Ebs, além de demonstrarem uma interação com a neurotransmissão colinérgica, também modularam a hidrólise dos nucleotídeos de adenina em plaquetas de ratos, contribuindo no controle da coagulação plaquetária em processos desmielinizantes. Neste contexto, sugere-se que o IFN-b, a CsA, a vitamina E e o ebselen podem ser investigados em estudos futuros com a intenção de encontrar uma melhor terapia para beneficiar pacientes com patologias desmielinizantes. / Multiple sclerosis is the main demyelinating disease of the central nervous system (CNS). It is the most common cause of neurological disability among young adults. The cognitive impairment is very commum in this illness, involving learning, memory and cortical organization of the movement, vital functions regulated by the cholinergic system. The model of toxic demyelination induced by ethidium bromide (EB) was used to evaluate brain acetylcholinesterase (AChE) activity in the striatum (ST), hippocampus (HP), cerebral cortex (CC), cerebellum (CB), hypothalamus (HY) and pons (PN), associated with treatment with interferon beta (IFN-b), ciclosporine A (CsA), vitamin E (Vit E) and ebselen (Ebs). In addition, the per se effect of EB on AChE activity was studied in vitro together with the kinetic parameters of this enzyme in the ST, HP, CC and CB of adult rats. The results showed that EB in vitro significantly inhibited AChE activity in the ST, HP, CC and CB at concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1mM. The kinetic analysis demonstrated an uncompetitive inhibition in the ST, HP and CC, whereas in the CB the inhibition type was mixed. In relation to the in vivo results, AChE activity was inhibited after demyelination by EB in the CC, ST, HP, HY, PN and CB at the post-injection points of time evaluated (3-7-15-21 and 30 days). When demyelinated rats were submitted to the treatment with IFN-b and CsA, the results demonstrated that these compounds did not alter AChE activity. However, Vit E and Ebs were able to increase AChE activity in the ST, CC and HP. In addition, immunohistochemistry studies demonstrated that in Vit E and Ebs treated rats, the lesions induced by EB were smaller suggesting that these compounds somehow interfered in the development of the lesions. The per se effect of IFN-b, CsA, Vit E and Ebs were also evaluated, demonstrating that these compounds have an inhibitory effect on AChE activity. Platelets of the demyelinated rats demonstrated a reduction in NTPDase activity and the treatments with Ebs and Vit E modulated adenine nucleotide hydrolysis. The present investigation demonstrated that EB is a strong inhibitor of AChE activity in vitro and the results in vivo showed that the activity of this enzyme is altered after an event of toxic demyelination in the CNS. The results this study help the confirm that drugs used in the treatment of the patients with multiple sclerosis such as IFN-b and CsA cause effects in the AChE activity. The Vit E and Ebs besides of interaction with the cholinergic neurotransmission also modulated adenine nucleotide hydrolysis in platelets of the rats, contributing to the control of the platelet coagulant status in the demyelinating process. In this context, we can suggest that IFN-b, CsA, Vit E and Ebs may be investigated in future studies with the intention of finding a better therapy for to improve patients with demyelinating pathologies.

A esclerose múltipla é a principal doença desmielinizante do sistema nervoso central (SNC). É considerada a principal causa de incapacidade neurológica em adultos jovens. O comprometimento cognitivo é muito comum nessa doença, envolvendo o aprendizado, a memória e a organização cortical do movimento, funções vitais que são reguladas pelo sistema colinérgico. O modelo de desmielinização tóxica induzida pelo brometo de etídio (BE) foi utilizado neste estudo, para avaliar a atividade da enzima acetilcolinesterase (AChE) no estriado (ST), hipocampo (HP), córtex cerebral (CC), hipotálamo (HY) e ponte (PN) associado ao tratamento com interferon beta (IFN-b), ciclosporina A (CsA), vitamina E (vit E) e ebselen (Ebs). Além disso, também foi investigado o efeito in vitro do BE na atividade da AChE, juntamente com os parâmetros cinéticos dessa enzima no ST, HP, CC e CB de ratos adultos. Os resultados demonstraram que o BE inibiu significativamente a atividade da AChE no ST, HP, CC e CB nas concentrações de 0,00625, 0,0125, 0,025, 0,05 e 0,1mM e a análise dos dados cinéticos mostraram uma inibição do tipo incompetitiva no ST, HP e CC, enquanto no CB a inibição foi do tipo mista. No estudo in vivo, foi observado uma inibição na atividade da AChE no CC, ST, HP, HY, PN e CB nos diferentes períodos avaliados pós-injeção do BE (3, 7, 15, 21 e 30 dias). Quando ratos desmielinizados com BE foram tratados com IFN-b e CsA, não houve alteração na atividade dessa enzima. Por outro lado, o tratamento com Vit E e Ebs foram capazes de aumentar a atividade da AChE no ST, CC e HP. Estudos imuno-histoquímicos demonstraram que nos ratos tratados com Vit E e Ebs as lesões induzidas pelo BE foram menores, sugerindo que estes compostos interferem no desenvolvimento de lesões desmielinizantes. Foi também avaliado o efeito per se do IFN-b, da CsA, da Vit E e do Ebs na atividade da AChE, os quais demonstraram um efeito inibitório sobre a atividade dessa enzima. Em plaquetas de ratos desmielinizados, foi observado uma diminuição na atividade da NTPDase e o tratamento com a Vit E e o Ebs modularam a hidrólise dos nucleotídeos de adenina. O presente trabalho demonstrou que o BE é um potente inibidor da atividade da AChE in vitro e os resultados in vivo demonstraram que a atividade dessa enzima está alterada após um evento de desmielinização tóxica no SNC. Os resultados deste estudo ajudaram a confirmar que drogas utilizadas no tratamento de pacientes com esclerose múltipla tais como o IFN-b e a CsA causam efeitos na atividade da AChE. A Vit E e o Ebs, além de demonstrarem uma interação com a neurotransmissão colinérgica, também modularam a hidrólise dos nucleotídeos de adenina em plaquetas de ratos, contribuindo no controle da coagulação plaquetária em processos desmielinizantes. Neste contexto, sugere-se que o IFN-b, a CsA, a vitamina E e o ebselen podem ser investigados em estudos futuros com a intenção de encontrar uma melhor terapia para beneficiar pacientes com patologias desmielinizantes. / Multiple sclerosis is the main demyelinating disease of the central nervous system (CNS). It is the most common cause of neurological disability among young adults. The cognitive impairment is very commum in this illness, involving learning, memory and cortical organization of the movement, vital functions regulated by the cholinergic system. The model of toxic demyelination induced by ethidium bromide (EB) was used to evaluate brain acetylcholinesterase (AChE) activity in the striatum (ST), hippocampus (HP), cerebral cortex (CC), cerebellum (CB), hypothalamus (HY) and pons (PN), associated with treatment with interferon beta (IFN-b), ciclosporine A (CsA), vitamin E (Vit E) and ebselen (Ebs). In addition, the per se effect of EB on AChE activity was studied in vitro together with the kinetic parameters of this enzyme in the ST, HP, CC and CB of adult rats. The results showed that EB in vitro significantly inhibited AChE activity in the ST, HP, CC and CB at concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1mM. The kinetic analysis demonstrated an uncompetitive inhibition in the ST, HP and CC, whereas in the CB the inhibition type was mixed. In relation to the in vivo results, AChE activity was inhibited after demyelination by EB in the CC, ST, HP, HY, PN and CB at the post-injection points of time evaluated (3-7-15-21 and 30 days). When demyelinated rats were submitted to the treatment with IFN-b and CsA, the results demonstrated that these compounds did not alter AChE activity. However, Vit E and Ebs were able to increase AChE activity in the ST, CC and HP. In addition, immunohistochemistry studies demonstrated that in Vit E and Ebs treated rats, the lesions induced by EB were smaller suggesting that these compounds somehow interfered in the development of the lesions. The per se effect of IFN-b, CsA, Vit E and Ebs were also evaluated, demonstrating that these compounds have an inhibitory effect on AChE activity. Platelets of the demyelinated rats demonstrated a reduction in NTPDase activity and the treatments with Ebs and Vit E modulated adenine nucleotide hydrolysis. The present investigation demonstrated that EB is a strong inhibitor of AChE activity in vitro and the results in vivo showed that the activity of this enzyme is altered after an event of toxic demyelination in the CNS. The results this study help the confirm that drugs used in the treatment of the patients with multiple sclerosis such as IFN-b and CsA cause effects in the AChE activity. The Vit E and Ebs besides of interaction with the cholinergic neurotransmission also modulated adenine nucleotide hydrolysis in platelets of the rats, contributing to the control of the platelet coagulant status in the demyelinating process. In this context, we can suggest that IFN-b, CsA, Vit E and Ebs may be investigated in future studies with the intention of finding a better therapy for to improve patients with demyelinating pathologies.

A hérnia perineal é uma das afecções cirúrgicas frequentemente observadas na rotina da clínica cirúrgica de pequenos animais e acometem na sua maioria, cães machos, não castrados e acima de cinco anos. Apresenta elevado índice de recidivas e complicações pós-cirúrgicas, e devido a este fato, muitas técnicas foram preconizadas e utilizadas para a sua correção. Os objetivos do presente estudo foram avaliar a locomoção cinética dos cães com hérnia perineal bilateral, submetidos à técnica de transposição do músculo semitendinoso antes e após a sua transposição; verificar a capacidade de adaptação do membro pélvico operado, após a transposição do semitendinoso, até 90 dias de pós-operatório e analisar a viabilidade dessa técnica de reparação em casos de hérnia perineal bilateral. Foram operados onze cães com diagnostico de hérnia perineal bilateral, no Serviço de Cirurgia de Pequenos Animais junto ao Hospital Veterinário da Faculdade de Medicina Veterinária da Universidade de São Paulo (HOVET-FMVZ / USP). Todos foram submetidos à técnica de transposição do músculo e avaliados com exames baropodométricos antes e após a cirurgia, nos períodos de 30, 60 e 90 dias pós-operatório. As variáveis pico de força vertical (PFV) e impulso vertical (IV) foram utilizadas como parâmetros para a análise de mudanças na locomoção. A PFV e IV não mostraram diferença estatística (p> 0,05) entre os membros operados e não operados, respectivamente (18,62 0 &plusmn 4,93 e 18,51 &plusmn 3,75), indicando que não houve alteração na locomoção dos cães após a transposição do músculo semitendinoso. A diferença no comportamento dessas variáveis no período pré-operatório nos permitiu aferir que o desconforto provocado pela hérnia perineal possa interferir na locomoção do animal gerando assimetria, considerando que após o procedimento operatório a simetria da locomoção apresentou valores mais compatíveis dentro do normal. As diferenças em PFV e IV, embora não significativas, nos dá uma idéia da recuperação desses cães e sugere que a transposição não afeta a função locomotora do membro operado. / A perineal hernia is a surgical lesion frequently observed in surgical clinic for small animals, affecting mainly dogs neutered over five years. Has a high recurrence rate and postoperative complications, and due to this fact, many techniques have been proposed and used for its correction. The objectives of this study were to evaluate the kinetic movement of dogs with bilateral perineal hernia, submitted to the technique of transposition of the semitendinosus muscle before and after its implementation and verify the adaptability of the pelvic limb surgery, after transposition of the semitendinosus 90 days of postoperatively and analyze the feasibility of this technique of repair in cases of bilateral perineal hernia. Eleven dogs were operated with diagnosis of bilateral perineal hernia, on the Surgery Service of the animal small of the Veterinary Hospital of the Faculty of Veterinary Medicine, University of São Paulo (HOVET-FMVZ / USP). All patients underwent the technique of muscle transposition and evaluated baropodometric examinations before and after surgery, in periods of 30, 60 and 90 days postoperatively. The variables peak vertical force (PFV) and vertical impulse (VI) were used as parameters for the analysis of changes in locomotion. The PFV and IV showed no statistical difference (p> 0.05) between the operated and non operated limbs, respectively (18.62 &plusmn 4.93 and 18.51 &plusmn 3.75), indicating that there was no change in locomotion dogs after the transposition of the semitendinosus muscle. The difference in behavior of these variables in the preoperative period allowed us to infer that the discomfort caused by perineal hernia can interfere with the locomotion of the asymmetry animal generating, whereas after the operative procedure, the symmetry of locomotion showed more next values of the normal. The difference in PFV and IV, although not significant, gives us an idea of the recovery of these dogs and suggests that the transposition does not affect the motor function of the limb.

A displasia coxofemoral (DCF) é uma doença poligênica, hereditária que acomete várias espécies e é influenciada por diversos fatores durante o desenvolvimento causando incongruência articular. É uma das afecções ortopédicas mais comuns em cães e possui alta incidência em animais da raça Pastor Alemão. O objetivo desse estudo foi correlacionar o grau de severidade da DCF com a locomoção dos cães através da análise cinética pelo sistema de baropodometria (Tekscan®) que propicia de modo quantitativo os valores das forças verticais de reação ao solo verificando assim alterações no apoio de cães displásicos durante a locomoção. Foram formados 5 grupos de acordo com a classificação de DCF segundo as categorias A, B, C, D e E com 8 pastores alemães em cada. Os animais foram examinados para controle e descrição dos sinais apresentados, bem como realizaram o teste de análise cinética do qual foi retirado 5 passagens válidas sobre a plataforma múltipla para comparação entre as forças de reação ao solo (FRS). Na análise cinética foram avaliados: - pico de força vertical (PFV), impulso vertical (IV) e tempo de apoio (TA) com a velocidade constante entre 1,3 a 1,6 m/s. Além da metodologia convencional foi realizada a análise de cada região de apoio, a fim de verificar a distribuição das FRS nos coxins e dígitos. Dos resultados obtidos nota-se uma tendência a diminuição progressiva do PFV nos membros pélvicos (MPs) a partir do grupo de cães com displasia leve (C) e o impulso se apresentou diminuído nos cães displásicos sem qualquer correlação com a severidade da doença. Nenhuma compensação relevante foi encontrada nos membros torácicos (MTs) e não houve diferença entre o TA em cães displásicos. A simetria entre o lado direito e esquerdo foi constatada em todos os grupos pela análise convencional. A nova metodologia permitiu a descrição detalhada do padrão da curva de força em formato de M realizada pelo coxim no inicio do apoio e pelos dígitos em um segundo momento. Entretanto dada a variabilidade e assimetria do apoio nessas regiões pode-se inferir que o coxim dos MTs têm maior porcentagem de apoio em relação aos dos MPs e essa distribuição não é afetada de modo regular na DCF. Os sinais clínicos apresentados ao exame físico como a dor, claudicação, atrofia muscular e diminuição de amplitude articular, corroboram com a diminuição do apoio em cães displásicos. Conclui-se que de acordo com o grau de severidade da DCF os animais têm uma tendência a apoiarem menos nos MPs, entretanto deve-se levar em conta a condição individual de cada animal, pois há cães displásicos que possuem o mesmo padrão de apoio de cães livres de DCF. / Hip dysplasia (HD) is a polygenic disease, inherited that affects many species and is influenced by several factors during development causing joint incongruity. It is one of the most common orthopedic disorders in dogs and has a high incidence in German shepherd dogs. The objective of this study was to correlate the degree of severity of the HD with the locomotion of dogs by kinetic analysis trough the pressure walkway system (Tekscan®) that provides quantitative values of vertical ground reaction forces (GRF) to the changes in the support of dysplastic dogs during locomotion. Five groups were performed according to HD´s classification in the categories A, B, C, D and E in German shepherd dogs total of 8 animals in each group. Dogs were examined and kinetic analysis was performed with 5 valid passages on the multiple platforms were used for comparison each groups. In kinetic analysis was assessed the peak vertical force (PVF), vertical impulse (VI), and stance phase (SF) with a constant speed between 1.3 to 1.6m/s. Besides conventional methodology the analysis of support checking the distribution of GRF in the pad and digits in each region were innovative. Results had a tendency to decrease progressively the PVF in the hindlimbs (HL) from group C (mild dysplasia) and VI is decreased in dysplastic dogs without any correlation with the severity of disease. Relevant compensation was not found in the forelimbs (FL) and even in dysplastic dogs there were no difference between the SF. Symmetry between the right and left side was found in all groups by conventional analyze. The new methodology has allowed a detailed description of the standard curve of force in M-shaped held by the support pad at the beginning and the digits in a second time. However given the variability and asymmetry of support in these areas we can only assert that the FL pads have a higher percentage of support from HL and this distribution is not affected on a regular basis in the HD. The clinical signs presented by physical examination like pain, lameness, muscle atrophy and articulate amplitude decreased, corroborated with the support decrease in dysplastic dogs. We conclude that according to the severity HD degree animals have tendencies to support less at the HL. However it should be consider the individual response because there are dysplastic dogs that have the same pattern of support that dogs free of HD.

The purpose of this study was to evaluate the manual and automatic analysis methods for force-time curve analysis of the isometric mid-thigh pull (IMTP) test. The visual analysis, first derivative analysis and threshold analysis methods were used to analyse onset time and time-specific forces at 50 ms, 90 ms, 200 ms and 250 ms on the force-time curve. Ninety-three collegiate sports athletes’ trials were selected and analysed by each method. The visual analysis method was set as the reference method for paired comparisons with the first derivative analysis method and threshold analysis method. Onset time comparisons revealed that the first derivative analysis method was comparable with the visual analysis method with average difference at about 30 ms. Results from the weighted least products regression analysis and the Bland-Altman analysis showed that large fixed bias confounded by proportional bias existed in the threshold analysis method, and time-specific force variables obtained from the first derivative analysis method were closer to those from the visual analysis method when compared with the threshold analysis method. These findings suggest that the first derivative analysis method could be an effective tool for force-time curve analysis of the IMTP test.

There has been a desire to accurately interpret the inhaled pharmacokinetic (PK) profiles of drugs in humans to aid successful inhaled drug and product developments. However, challenges are layered, as 1) the drug dose delivered to the lung (DTL) from inhalers is a portion of the formulated dose but rarely determined; 2) lung delivery and regional deposition differ, depending on drug, formulation and inhaler; 3) drugs are not only absorbed from the lung but may also be from the gastrointestinal (GI) tract; and 4) in addition to absorption into the systemic circulation, multiple non-absorptive processes also eliminate drugs from the lung, such as mucociliary clearance, metabolism, phagocytosis and tissue binding. Hence, this thesis project aims to develop new lung disposition model-based analyses to derive the meaningful kinetic descriptors for lung disposition from inhaled PK profiles in humans. Two approaches, curve fitting- and moment-based approaches, were developed. Both approaches modeled the kinetics of lung disposition rate-controlled by absorption (ka) and non-absorptive loss (knal), assuming no contribution of GI absorption. An exhaustive literature review found necessary data sets for three drugs, tobramycin, calcitonin and ciprofloxacin. In the curve fitting-based approach, each inhaled PK profile was fitted to the lung disposition model, while the DTL was obtained from corresponding -scintigraphic lung deposition and the kinetic parameters of systemic disposition were fixed by separate intravenous PK profile model analysis. In the moment analysis-based approach, the mean lung residence times (MLRT) and the DTL-based bioavailability (FL) were estimated and used to determine the ka and knal values in the lung disposition model, given FL = MLRTka = ka/(ka+knal). The ka and knal values were successfully derived for all the three drugs delivered by dry powder inhalers (DPIs) and/or nebulizers (NEB) through both approaches. Their “goodness-of-fit” was reasonably satisfactory. The ka values appeared to be primarily described by partition-based diffusion affected by the three hydrophilic drug’s molecular weight. In contrast, the knal values differed, yet appeared to become plausible, with a notion of additional non-absorptive confoundedness due to lung tissue binding (tobramycin) and metabolism (calcitonin), in addition to mucociliary clearance. The ka and knal values derived by the two approaches were comparable in majority of the cases. The success of these PK modeling analyses enabled further attempts to identify most influential attributes by simulation. The systemic PK and lung exposure profiles were predicted by simulation upon ±20 % changes in each of the DTL, ka and knal values to examine changes in the systemic PK metrics (Cmax, AUC and Tmax) and local lung exposure metrics (AUClung and LRT0.5). For all three drugs, the Cmax and AUC changes were identical to changes in the DTL without changing the Tmax. In contrast, impacts of the ka and knal changes differed between drugs, depending on the relative contribution of the rate constant to their sum (ka+knal). It appeared that the major contributor of the sum (ka+knal) was that rate-controlling the kinetics of lung disposition. In conclusion, this thesis project has successfully proposed two new approaches of curve fitting and moment-based analysis by accurately deriving the kinetic descriptors of lung disposition (ka and knal) for three drugs from the inhaled PK profiles in humans. Their applications were extended to predict likely changes in the systemic PK and local lung exposure metrics by simulation. While attempts should continue with more drugs, these approaches are believed to be useful in identifying critical attributes to determine the lung disposition kinetics and thus predicting the lung kinetic behavior and systemic PK profiles of new drug entities in humans.

Problématique : Les paramètres fonctionnels extraits en Tomographie par Émission de Positons (TEP) à la 18F-Choline (FCH) et en Imagerie par Résonance Magnétique (IRM) apportent-ils une information supplémentaire par rapports aux informations déjà connues pour caractériser l’agressivité du tissu tumoral ? Objectifs : Notre travail avait pour objectifs tout d’abord (i) de mettre en évidence un éventuel lien entre les paramètres quantitatifs extraits par TEP à la FCH et et les paramètres clinicopathologiques dans le cancer de la prostate. Après cette étude préliminaire, pour quantifier au mieux l’influx de FCH par analyse dynamique de l’acquisition précoce, la deuxième étape avait pour but (ii) d’optimiser cette acquisition. Enfin (iii), le dernier objectif était de mettre en évidence un éventuel lien entre les paramètres quantitatifs extraits par TEP à la FCH et les paramètres quantitatifs extraits par IRM multiparamétrique. Résultats : Pour la première étape (i), nous avons comparé les paramètres quantitatifs extraits par TEP à la FCH et les paramètres clinico-pathologiques de 61 patients venant en TEP à la FCH pour le bilan d’extension initial. L’influx de FCH mesuré par analyse dynamique était plus élevé pour les patients avec un score de Gleason ≥ 4+3 que pour les patients avec un score de Gleason < 4 + 3. Pour la deuxième étape (ii), nous avons tout d’abord voulu optimiser la durée de l’acquisition précoce de TEP à la FCH en comparant le contraste sur bruit de 77 lésions tumorales à 5 minutes et 10 minutes après l’injection de FCH. Le contraste sur bruit à 5 minutes n’était statistiquement pas différent de celui à 10 minutes. La deuxième phase de l’optimisation de l’acquisition précoce de la TEP à la FCH consistait à déterminer quel était le meilleur échantillonnage temporel de ces 5 minutes par la comparaison de 7 échantillonnages temporels différents avec un objectif d’influx de FCH extrait de 37 lésions. L’échantillonnage temporel 12x5”-8x30” a été retenu. Pour la dernière étape (iii), nous avons comparé les paramètres TEP et IRM extraits de 14 lésions tumorales prostatiques. L’influx de FCH et la constante de transfert du gadolinium étaient corrélés, toutefois de manière modérée (r = 0,55). Conclusion : L’influx de FCH mesuré par analyse dynamique de l’acquisition précoce en TEP semble lié à la différenciation des cancers prostatiques. Cet influx semble également lié à la constante de transfert du gadolinium. Ces 2 paramètres d’imagerie semblent toutefois quantifier des processus physiopathologiques différents. Les différents résultats obtenus justifient la poursuite des travaux pour évaluer le rôle de marqueur d’agressivité des cellules cancéreuses prostatiques des différents paramètres quantitatifs obtenus par imagerie fonctionnelle par TEP à la FCH et par IRM multiparamétrique. / Research question: Do the functional parameters derived by 18F-Choline (FCH) Positon Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) add informations to characterize the aggressiveness of prostate cancer? Objectives: The first objective of this work was (i) to enhance a potential link between quantitative parameters extracted by FCH PET and clinicopathological parameters in prostate cancer. Then, after this preliminary study, in order to optimize the quantification of the FCH influx with a kinetic analysis, the second objective was (ii) to optimize the exam protocol of the FCH PET early acquisition. Finally, the last objective was (iii) to enhance a potential link between quantitative parameters extracted by FCH PET and quantitative parameters extracted by multiparametric MRI in prostate cancer. Results: For the first step (i), we compared FCH PET quantitative parameters and clinicopathological parameters extracted from 61 patients referred to the nuclear medicine department to perform an FCH PET/ CT with newly histologically proven prostate cancer and before any treatment The FCH influx measured using kinetic analysis was higher for patients with a Gleason score ≥ 4+3 than for patients with a Gleason score < 4 + 3. Concerning the second step (ii), firstly, we compared the contrast to noise ratio of 77 prostatic cancer lesions at 5 minutes and 10 minutes post-injection in order to optimize the length of the early FCH PET acquisition. No significant difference was observed. Secondly, we sought to define an optimal time sampling of the early FCH PET acquisition comparing 7 different time samplings with a FCH influx as objective extracted from 37 prostatic cancer lesions. The 12x5”-8x30” time sampling was selected. For the last step of this work (iii), we compared FCH PET and multiparametric MRI quantitative parameters extracted from 14 prostatic cancer lesions. The FCH influx was moderately correlated to the vessel permeability measured by the volume transfert constant of gadolinium (r = 0.55). Conclusion: The FCH influx extracted from the early FCH PET acquisition using kinetic analysis seems to be linked to the tumoral differentiation of prostatic cancers. This FCH influx seems also linked to the vessel permeability. However, due to the moderate degree of correlation, these two imaging parameters reflect two different processes. To confirm the results obtained in this work, other studies are needed to enhance the role of the functional parameters derived by FCH PET and multiparametric MRI as biomarkers for prostate cancer.

O objetivo da pesquisa foi avaliar os efeitos da terapia de ondas de choque extracorpórea em pacientes com osteoartrose coxofemoral secundária a displasia. Foram analisados 30 cães (Grupo I) com auxílio da plataforma de baropodometria, portadores de osteoartrose bilateral da articulação coxofemoral, que receberam como tratamento ondas de choque radiais somente em uma das articulações acometidas de modo aleatório que foi comparado ao membro contralateral com o acompanhamento de 3 meses, 18 animais (Grupo II) com displasia coxofemoral e osteoartrose bilateral medicados com condroprotetor e também comparado ao banco de 20 animais hígidos (Grupo III). O protocolo de tratamento envolveu três sessões de 2000 pulsos cada com frequência de 10 Hz de 2-3,4 bars, com intervalo de 7 dias, e a aplicação realizada sob sedação e curta anestesia. A avaliação mensal quantitativa do apoio dos membros e da eficácia do tratamento foi realizada com análise cinética utilizando sistema de baropodometria, mensurando-se o pico de força vertical (PFV) e o impulso vertical (IV).O grupo I e II tiveram em média valores do PFV e IV inferiores ao grupo III. No grupo I, observou-se um aumento em média de 10,6% do PFV e de 10,4% do IV do membro tratado, assim como uma melhora na simetria. O grupo II apresentou um aumento em média do membro pélvico esquerdo de 9% do IV, porém houve piora de 11% na simetria deste mesmo parâmetro. Os dados da análise cinética no membro tratado do grupo I atingiram em média valores próximos, mas ainda inferiores, ao grupo III de animais hígidos. Estas avaliações verificaram a eficácia das ondas de choque como tratamento conservativo de cães portadores de osteoartrose secundária a displasia, sugerindo efeitos benéficos de forma quantitativa para uma melhor locomoção. / The objective of this research was evaluate the extracorporeal shockwave therapy (ESWT) effects in patients with hip osteoarthritis secondary to hip dysplasia. Thirty dogs (Group I) with bilateral hip osteoarthritis were evaluated trough pressure walkway system. They had received unilateral radial shockwaves for treatment in random selection. The treated limb was compared with contra lateral limb during a follow up of 3 months. A comparison with the treated group with eighteen dogs under conservative treatment (Group II) and twenty healthy dogs (Group III) was also performed. Protocol treatment included three sessions of 2000 pulses each in a frequency of 10Hz with a pressure between 2 and 3.4 bar performed weekly using chemical restraint and short general anesthesia. Monthly assessment with kinetic analysis had done it with pressure walkway system. The peak of vertical force (PVF) and vertical impulse (VI) were measured. Group III kinetic analysis mean values were higher than groups I and II. Our results of group I suggest 10.6% of improvement in the treated limb in the PVF and 10.4% in the VI. In this group was also detected an improvement in the symmetry. The group II showed an improvement of 9% of VI only in the left pelvic limb but the symmetry decreased in 11% in this parameter. The kinetic analysis data of group I reached mean values of the treated limb close to healthy dogs of group III but remains slightly lower. These evaluations verified the efficacy of ESWT suggesting benefic quantitative effects in dog\'s locomotion.