Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 137-167).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 142 pages. Includes Vita. Includes bibliographical references.

The mechanisms that regulate the differentiation of stem cells (SCs) into cardiomyocytes are still unclear and the role of endogenous molecules on this process remains unexplored. One such molecule is the bioactive phospholipid lysophosphatidic acid (LPA) which accumulates in the myocardium following acute infarction and exerts multiple biological functions, including the regulation of cell growth and differentiation as well as cell survival (Tigyi et al., 2003; Sengupta, et al., 2004). Experiments were therefore carried out in this thesis to reveal whether LPA can induce the differentiation of stem cells into cardiomyocytes and to identify the signalling mechanisms that mediate this effect. All experiments were carried out in the mouse P19 carcinoma stem cell line. Treatments with LPA in the absence and presence of various pharmacological compounds were conducted in embryoid bodies (EBs) formed from the P19 cells in sterile Petri dishes over 4 days. The EBs were subsequently transferred into 6-well cell culture plates and cultured for specific time points. Lysates were generated and subjected to western blotting for expression of cardiac- specific myosin light chain -1v (MLC-1v). To look at the expression of LPA receptors (LPAR1-LPAR5) experiments were carried out by RT-PCR using specific primers for each LPA receptor and the role of the latter in mediated responses to LPA were examined in the presence of the LPAR 1/3 antagonist, Ki16425, or the LPAR 4 receptor blocker suramin. In addition, experiments were carried out investigating the role of Gαi and specific signalling pathways that may be involved in the differentiation of P19 cells. These were carried out using potent inhibitors/antagonists of Gαi inhibitor (Pertussis toxin), PI3K inhibitor (LY294002), Akt inhibitor (Akt inhibitor XIII), PKC inhibitor (Bisindolylmaleimide I BIM-I), ROCK inhibitor (Y-27632), p38-MAPK inhibitor (SB203580) and ERK1/2 inhibitor (PD98059). Further experiments were carried out to establish whether the presence of LPA results in the phosphorylation of the targeted kinases. These studies were however limited to Akt, p38 MAPK and ERK1/2. Incubation of cells with LPA resulted in the differentiation of P19 cells into cardiomyocytes as reflected by the induction of MLC-1v. The latter increased significantly above basal in a time-dependent manner, reaching a maximum 10 days after plating EBs in 6-well plates. The induction of MLC-1v was more pronounced in cells incubated with 5 μM LPA at 6 days but showed little concentration differences at day 12. RT-PCR analysis confirmed the expression of LPA receptors 1 to 4 but not 5. Pre-incubating cells with suramin and Ki16425 concentration-dependently inhibited MLC-1v expression with 0.05 mg/ml and 10 μM respectively, virtually abolishing the expression of MLC-1v. Additionally, inhibitors of LPAR1/3 and LPAR4 receptors and all the signalling inhibitors except SB203580 abolished the phosphorylation of ERK1/2. Similarly, p38 MAPK activation was completely abolished by LPAR1/3 and LPAR4 receptor antagonists, Interestingly, only LY294002 (5 μM) and Y27632 (10 μM) abolished the LPA induced activation of p38 MAPK while SB203580, BIM-I, Akt inhibitor XIII and PD95080 caused no significant changes to the phosphorylation of p38 MAPK. In conclusion, the studies carried out in this thesis have shown that LPA can induce P19 stem cells to differentiate into cardiomyocytes and they are linked to the well characterised LPA receptors (LPAR1/3 and 4). These receptors are coupled to downstream signalling pathways of which those involving the ROCK, PI3K, PKC and/or Akt may be critical, and may converge on ERK1/2. Inhibition of any of these pathways has the potential to suppress differentiation. In contrast, signalling leading to p38 activation may potentially suppress differentiation but this needs further clarification.

No description available.

Les Récepteurs Couplés à un Canal Ionique (ICCRs) sont des canaux ioniques artificielscréés par fusion d'un Récepteur Couplé aux Protéines G (RCPG) au canal ionique Kir6.2. Dansce concept, le canal agit comme un rapporteur direct des changements conformationnels desRCPGs permettant de détecter par simple mesure de courant, la fixation d'agonistes etd'antagonistes proportionnellement à leur concentration.Le signal induit étant directement corrélé à l'activité du récepteur, indépendamment desvoies de signalisation des protéines G, nous avons exploité cet avantage pour étendre le champd'applications des ICCRs au cours de cette thèse. Nous avons développé quatre applications quisont: 1) la caractérisation fonctionnelle des RCPG optimisés pour la cristallisation par insertionde domaine du lysozyme du phage T4 dans la boucle ICL3; 2) la détection de la dépendance desRCPGs au cholestérol; 3) la détection de ligands dits "biaisés" pour faciliter leur criblage; et 4) lacartographie fonctionnelle des portes du canal Kir6.2 régulées par des protéines membranairesinteragissant par le domaine N-terminal. / Ion Channel-Coupled Receptors (ICCRs) are artificial ion channels created by the fusion of a Gprotein-coupled receptor to a Kir6.2 channel. In this concept, the channel acts a direct reporter ofthe conformational changes of the GPCRs, allowing the detection by simple current recordingsof agonists and antagonists binding in concentration-dependent manner.The signal being directly correlated to the receptor activity, independently of G protein signallingpathways, we exploited this advantage to extend the field of applications of ICCRs during thisthesis. We developed 4 applications: 1) the functional characterization of the optimized GPCRsfor crystallization by insertion of the T4 phage lysozyme domain in the ICL3 loop; 2) thedetection of a cholesterol-dependence of the GPCRs; 3) the detection of the so-called "biasedligands" to simplify their screening; and 4) the functional mapping of the Kir6.2 channel gatesunder control of membrane proteins interaction with the N-terminus domain.

Os receptores medeiam a maioria das respostas fisiológicas em resposta a diversidade de estímulos. A ativação da sinalização mediada pelo receptor de angiotensina II tipo 1 é o principal responsável pelos efeitos do hormônio angiotensina II (Ang II) nos tecidos alvo. No rim concentrações fisiológicas de Ang II aumentam a atividade no túbulo proximal da isoforma 3 do trocador de Na+/H+ (NHE3). Este efeito é crucial para a manutenção do volume extracelular e pressão arterial. Evidências recentes mostraram que a ativação seletiva da sinalização enviesada da beta-arrestina/ receptor AT1 induz diurese e natriurese independentemente da sinalização via proteína G. Neste estudo testamos a hipótese de que a sinalização enviesada do receptor AT1/ beta-arrestina inibe a atividade do NHE3 no túbulo proximal, bem como investigar os possíveis mecanismos moleculares que medeio este efeito. Para tal, nós determinamos os efeitos do composto TRV120023, que se liga ao receptor AT1, bloqueando o acoplamento da proteína G e estimulando a sinalização da beta-arrestina, na função do NHE3 in vivo e in vitro. A atividade do NHE3 foi medida quer em túbulo proximal nativo, por meio de microperfusão estacionária, bem como em uma linha celular de túbulo proximal de gamba (OKP), por meio de recuperação de pH intracelular dependente de Na+. Os nossos resultados mostram que o TRV120023 na concentração de 10-7 M inibe marcadamente a atividade do NHE3 em túbulo proximal quer in vivo quer in vitro, sendo que este efeito é completamente abolido nas células silenciadas para a beta-arrestina 1 e 2 através de RNA de interferência. Adicionalmente, a estimulação do NHE3 pela Ang II é completamente suprimida pelo TRV120023 quer in vivo quer in vitro. A inibição do NHE3 pelo TRV120023 foi associada com a diminuição do NHE3 expresso na superfície da membrana plasmática em células OKP e com a redistribuição entre o corpo e a base das microvilosidades em túbulo proximal de rato. A diminuição do NHE3 na superfície da membrana plasmática em células OKP estava associado com um aumento na internalização do NHE via endocitose mediada por clatrina. A inibição do NHE3 mediada pela beta-arrestina não envolve a sinalização do receptor AT2, cAMP/ PKA, Akt e ERK1/2. Estes achados indicam que a sinalização enviesada do receptor AT1/beta-arretina inibe a atividade do NHE3 em túbulo proximal, pelo menos em parte, devido a alterações na localização subcelular do NHE3 / Cell surface receptors mediate most of our physiological responses to an array of stimulus. The triggering of the angiotensin II type I (AT1) receptor signaling is the major control point in the regulation of the ultimate effects of the peptide hormone angiotensin II (Ang II) on its target tissue. In the kidney physiological concentrations of Ang II upregulate the activity of proximal tubule Na+/H+ exchanger isoform 3 (NHE3). This effect is crucial for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the betaarrestin-biased AT1 receptor signalingpathway induces diuresis and natriuresis independent of G-protein mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule as well as investigate the underlying molecular mechanisms mediating this effect. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G protein coupling, and stimulates beta-arrestin signaling, on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. Our results showed that 10-7 MTRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro, and the effect was completely abolished in OKP cells silenced for beta-arrestin 1 and 2 by small interference RNA. Additionally, stimulation of NHE3 by Ang II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. The decreased surface NHE3 in OKP cells was associated with an increase in NHE3 internalization via clathrin mediated endocytic. Beta-arrestin mediated NHE3 inhibition did not involve AT2 receptor, cAMP/ PKA, Akt and ERK1/2 signaling. These findings indicate that biased signaling of the AT1 receptor/beta-arrestin pathway inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization

Este estudo visou observar os efeitos plásticos induzidos pelo exercício a curto prazo em regiões motoras do encéfalo de ratos. Observou-se a expressão das subunidades GluR1 e GluR2/3. Os animais foram divididos em grupos de: 3 dias(COR3), 7 dias(COR7) e 15 dias(COR15); e um grupo controle(CONT). Empregaram-se as técnicas de imuno-histoquímica e immunoblotting. A expressão de GluR1 no cerebelo, demonstrou um decréscimo em COR3. No hipocampo houve uma queda na expressão em COR3(40%), retornando aos níveis basais em COR7. No córtex cerebral observou-se uma queda da expressão com máxima queda em COR7(52%), retornando à expressão basal em COR15. O estriado não sofreu alterações na expressão de GluR1 ao longo dos primeiros 7 dias, tendo um aumento em COR15(90%). A expressão de GluR2/3 não foi alterada, exceto no cerebelo, onde houve um decréscimo em dois momentos distintos, COR3(55%) e COR15(25%), retornando à expressão basal em COR7. Os nossos dados revelam que o exercício físico a curto prazo foi capaz de promover alterações plásticas ao longo do treinamento. / This study aimed at analyzing the plastic effects of the short-term exercise upon the rat motor area. We check the expression of GluR1 and GluR2/3. We divided into 3 experimental groups based on duration of exercise: 3 days(COR3), 7 days(COR7), and 15 days(COR15); and a control group(CONT). The experimental animals were subjected to a treadmill exercise protocol. The brains were subjected to the techniques of immunohistochemistry and immunoblotting. In the cerebellum, there was a decrease for COR3(17%). In the hippocampus, there was a decrease of the GluR1 expression for COR3 (40%). In the cerebral cortex there was a drop of GluR1 expression for COR3 and COR7(52%). In the striatum, there was no change of GluR1 expression during the first seven days, with a increase for COR15(90%). The GluR2/3 expression did not change in any brain structure analyzed, except in the cerebellum, where there was a significant decrease for two distinct groups, COR3(55%) and COR15(25%). Our data show that short-term physical exercise was able to promote plastic changes during training.

L’agression de l'organisme par un agent physique, psychique ou émotionnel déclenche une réponse physiologique et comportementale qui permet à un individu de se prévenir du danger et de maintenir sa survie. Le système nerveux central a depuis longtemps été identifié comme un majeur acteur de cette réponse adaptative. Cependant, une exposition prolongée au stress conduit à des adaptations cellulaires et des réadaptations de circuits neuronaux qui contribuent à l'émergence de troubles neuropsychiatriques. L’interaction entre le système dopaminergique (DA) et sérotoninergique (5HT) a été impliquées dans ces réarrangements physiologiques et pathologiques qui influencent les comportements motivationnels de l'individu face à une menace. Fait intéressant, l'habenula latérale (Hbl), une région du cerveau très conservée entre les espèces, contrôle directement et indirectement les systèmes DA et 5HT, et son activité est modulée par des stimuli aversifs chez les humains et les animaux. De plus, l'activité de la Hbl est augmentée chez des modèles animaux de la dépression ou lors de l'induction d'un épisode dépressif chez des patients humains. Inversement, l’emploie de stratégies ayant pour cible la Hbl permettent d’améliorer certains symptômes dépressifs à la fois chez les modèles animaux de dépression et chez l’homme. Ainsi, la dérégulation de l’Hbl pourrait jouer un rôle dans l'apparition de symptômes dépressifs. Cependant, les changements moléculaires et cellulaires précoces qui occurrent au niveau de Hbl suite à l'exposition continue à un environnent aversifs restent peu connus. De plus, la plupart des modèles animaux utilisés pour interroger le rôle de Hbl dans l'état dépressif implique une exposition répétée de l’animal à des stimuli douloureux. Si la fonction de l’Hb est aberrante lors d’une exposition chronique à d’autre type de stress reste méconnu. Dans mon travail de thèse, je me suis intéressée aux adaptations cellulaires et moléculaires au niveau des neurones Hbl suite à l’exposition de différents types d'expériences aversives et leurs relatives importances pour l'expression de symptômes dépressifs. Plus précisément, je présente dans ce manuscrit, les résultats d'un premier travail qui vise à identifier les adaptations cellulaires et moléculaires de Hbl suite à l’exposition de souris à des chocs électriques et leurs rôles dans l'émergence de symptômes dépressifs. Cette étude montre que l’exposition à de brefs aléatoires chocs électriques entraine une diminution de l’expression de surface de récepteur métabotropiques gamma-aminobutyrate B (GABABRs), et par conséquent une diminution de leur fonction au niveau des neurones de l’Hbl. GABABR est un récepteur métabotropique couplé à la protéine Gi, il hyperpolarise les neurones de l’Hbl 4.par l'activation du canal potassique GIRK. La diminution de la signalisation GABABR-GIRK est accompagnée par une augmentation de l'activité de la protéine phosphatase 2 (PP2A), reconnue pour induire l’endocytose du complexe GABABR-GIRK. GABABR-GIRK contrôle étroitement l'activité Hbl, et par conséquent une diminution de leurs fonctions conduit à l’hyperexcitabilité des neurones de l’Hbl. En adoptant des stratégies visant à restaurer spécifiquement la signalisation GABABR-GIRK dans l’Hbl, telle que la surexpression GIRK, ou l'inhibition pharmacologique locale de l'activité PP2A, nous avons observé une amélioration de certains symptomes « dépressifs », établissant ainsi un lien causal entre l’aberrante diminution du signal GABABR et certain aspect de l’état dépressifs. / Prolonged exposure to aversive stimuli leads to cellular and circuit adaptations that contribute to the emergence of neuropsychiatric disorders such as depression. Interactions between the dopaminergic (DA) and the serotoninergic (5HT) systems have been implicated in these pathological adaptations ultimately influencing motivated behaviors. Interestingly, the lateral habenula (LHb), an ethologically well-conserved epithalamic region, directly and indirectly controls DA and 5HT systems, and its activity is modulated by aversive events in both humans and animals. Moreover, the activity of the LHb increases in animal models of depression and depressed human patients. Conversely, strategies that locally target the LHb have been shown to reverse depressive-like symptoms both in animal models and in humans. Altogether, this led to the hypothesis that LHb dysregulation could play a role in the emergence of depressive like symptoms. However, little is known about the early cellular and molecular adaptations that occur within the LHb after exposure to aversive events. Moreover, most of the animal models employed to interrogate the LHb role in depressive states used acute painful stimuli; whether LHb function becomes aberrant after chronic exposure to painless stressors remain elusive. In my thesis work, I explored the precise cellular and molecular adaptations of LHb neurons following exposure to different kind of unpredictable aversive experiences, and their importance for the expression of depressive like symptoms.More precisely, I will present the results of an initial work aiming to identify early cellular and molecular adaptations within the LHb following unpredictable stimuli and their importance for the emergence of depressive symptoms. This study shows that unpredictable foot-shocks lead to decreased surface expression and function of the gamma-aminobutyrate receptor (GABABR), a metabotropic receptor that hyperpolarizes LHb neurons through the activation of the G protein-coupled inwardly-rectifying potassium channels (GIRKs). This decrease of GABABR-GIRK signaling went along with an upregulation of the activity of the protein phosphatase 2 (PP2A), which is a well-known down-regulator of GABAB-GIRK complex surface expression. GABABR-GIRK signaling tightly controls LHb activity, and its downregulation consequently leads to aberrant hyperexcitability of LHb neurons. Using specific strategies to restore the GABABR-GIRK signaling within the LHb, such as GIRK overexpression, or local pharmacological inhibition of PP2A activity, we were able to ameliorate depressive like states following unpredictable foot-shocks. The second study allowed instead to establish the cellular adaptations within the LHb following a chronic non-painful aversive experience and during a critical developmental period. I showed that exposure to maternal separation in childhood (MS mice) also leads to depressive like symptoms together with a hyperexcitability of LHb neurons. This stress-driven increase in LHb activity was causally linked to a decrease of the GABABR-GIRK signaling. Moreover, using diverse reversal strategies such as chemogenetics or a therapeutically-relevant intervention such as Deep Brain Stimulation (DBS), we could selectively decrease LHb neuronal activity and consequently ameliorate the depressive like symptoms, suggesting a causal link between these two phenotypes.Altogether, the work presented in this thesis suggests that LHb neuronal hyperexcitability could represent a common substrate necessary for the expression of certain aspects of the depressive like state and further supports its relevance as a potential target in the treatment of this disorder.

O vírus respiratório sincicial humano (HRSV) é um dos agentes patogênicos respiratórios de grande importância clínica, tendo em vista que acomete 64 milhões de crianças por ano em todo o mundo. A resposta imune do hospedeiro e a variabilidade genética do HRSV podem interferir na produção de uma vacina eficaz, tal como a presença de quasispecies na população viral. O objetivo deste trabalho foi detectar quasispecies em amostras de HRSV e verificar se soros obtidos da criança na fase convalescente da doença e de sua respectiva mãe selecionam estes mutantes. Uma alteração não sinonímia foi detectada no gene F em um dos clones seqüenciados, enquanto duas alterações sinonímias e duas não sinonímias foram encontradas no gene G do HRSV, sendo as últimas no mesmo nucleotídeo. Um dos clones pré-selecionados com soro humano apresentou a mesma alteração não-sinonímia, encontrada na ausência de anticorpos no gene G. Os resultados sugerem que diferentes sequencias virais presentes em menor quantidade na população podem ser selecionadas pelo sistema imunológico do hospedeiro. / Human respiratory syncytial virus (HRSV) is one of the most important clinical respiratory pathogens, since 64 millions children in the world are infected by this agent every year. Host immunity and viral genetic variability are important factors to a vaccine development, besides quasispecies presence in the viral population. In this work, HRSV quasispecies were detected in clinical samples in absence and presence of human polyclonal serum collected by children in the convalescent phase and mother serum. A non-synonymy variation was found in the F gene in antibodies absence. Four mutations were found at HRSV G2 in polyclonal serum absence. Two were synonymy and two were non-synonymy variation, the last in the same nucleotide. A non-synonymy mutation was found in the G2 region in presence of polyclonal serum collected from child convalescent phase. This alteration was the same of the observed in absence of polyclonal serum so it is possible that host antibodies can selected different viral minority sequences present in the population.