Os peptídeos INKKI e YPVQPFTE foram isolados a partir da hidrólise da b-caseína bovina e correspondem às seqüências 26-30 e 114-121 respectivamente. A atividade proliferativa foi avaliada em culturas primárias de linfócitos T. A atividade antitumoral in vitro foi realizada em culturas de B16F10. Foi utilizado grupo com 40 camundongos da linhagem C57BL/6J para avaliar a atividade antitumoral. Nossos resultados mostraram que o peptídeo INKKI apresentou resposta proliferativa semelhante ao mitógeno comercial PHA. O peptídeo YPVEPFTE mostrou ter ação proliferativa maior do que a apresentada pelo mitógeno comercial PHA. O peptídeo INKKI mostrou ação quimiotáxica. O tratamento in vitro mostrou que somente o pentapeptídeo INKKI induz seletiva atividade citotóxica para as células de melanoma. Os animais portadores de tumores dorsais apresentaram significativa inibição da capacidade de crescimento e a metastatização. Conclui-se que os peptídeos apresentam ação significativa tanto nos experimentos in vitro como in vivo sugerindo um possível papel fisiológico. / Peptides INKKI and YPVQPFTE were isolated from the bovine b-casein after hydrolysis corresponding to the 23-30 and 114-121 sequence, respectively. Evaluation of the proliferative activity in primary cultures of lymphocytes. The activity antitumor in vitro was accomplished culture of was studied. Groups with 40 C57BL/6J lines mice had been used to evaluate the antitumoral activity. Our results showed that peptide presented similar proliferative response to the PHA commercial mitogen. The peptide YPVEPFTE showed to have proliferative action larger than presented by the commercial mitogen. The peptide INKKI showed in the chemotactic action. The treatment in vitro had shows that the peptide INKKI induces selective citotoxicity. The bearing animals of dorsal tumors had presented significant inhibition of the capacity of growth and the spread of methastasis. Thus, the peptides casein present significant action in in vitro and in vivo experiments, suggesting a possible physiologic role.

O colágeno é a proteína fibrosa predominante da matriz extracelular e é a maior proteína constituinte do tecido conectivo. Alterações nas taxas de colágeno tipo I na derme ocorrem durante o envelhecimento. A introdução de um agente eficaz para a manutenção da pele durante o envelhecimento é importante, possibilitando a redução destes efeitos. Neste projeto foram avaliados os efeitos proliferativos e de biossíntese de colágeno, em fibroblastos humanos de derme normal tratados com colágeno bovino e derivados de diferentes perfis moleculares, colágeno super hidrolisado, colágeno hidrolisado e gelatina, em diferentes experimentos, adesão e viabilidade celular, síntese de colágeno e análises das fases do ciclo celular. Os resultados sugerem que o colágeno bovino e derivados induzem as propriedades adesivas nos fibroblastos, não desencadeiam efeitos citotóxicos e induzem a biossíntese de colágeno pelos fibroblastos. Os resultados de síntese de colágeno indicam que concentrações menores de colágeno super hidrolisado e colágeno hidrolisado podem ser utilizadas em relação à amostra de gelatina. Comparando os resultados após 48 horas de cultura na síntese de colágeno e nas modificações na fase S do ciclo celular, as células tratadas com as amostras de colágeno super hidrolisado e colágeno hidrolisado induziram aumentos significativos. / Collagen is the predominant fibrous protein of the extracellular matrix and it is a major protein constituting connective tissue. Alterations in the rate of collagen type I deposition occur during aging. The introduction of an efficient agent for the maintenance of the skin during the aging process is important as it can reduce those effects. Thus, the proliferative effects and collagen biosynthesis in normal dermis of human fibroblasts were evaluated in this project. The fibroblasts were treated with bovine collagen and its derivates from different molecular mass, as well as super hydrolysate collagen, hydrolysate collagen and gelatine. Different assays were applied as adhesion, cell viability, collagen biosynthesis, and cell cycle phases analysis. The results suggest that the bovine collagen and its derivates can induce to adhesion properties in fibroblasts, they do not produce cytotoxic effects, and they can induce the collagen biosynthesis by fibroblasts. The results also show that super hydrolysate collagen and hydrolysate collagen can be used in lower concentrations than gelatine in collagen biosynthesis analysis. Analyzing the results after 48 hours of culture, the cells treated with the samples of super hydrolysate collagen and hydrolysate collagen had significant improvement in the collagen biosynthesis and in the modifications in the S phase of the cell cycle.

Made available in DSpace on 2016-01-26T18:55:35Z (GMT). No. of bitstreams: 1 daniel.pdf: 186929 bytes, checksum: 3b546022c9b10042ce06a35c124600cd (MD5) Previous issue date: 2012-04-13 / The silver staining technique for AgNOR nucleolar organizer regions (NORs) is characterized by marking proteins linked to the ribosomal ribonucleic acid, evaluating cell proliferation. The aim was to study the validity of the AgNOR staining technique in the testicular cell proliferation in crossbred ovine. Was used treis pairs forty crossbred sheep testes between six and ten months old.Wilcoxon and Spearman tests were used with a significance level of 5%. The mean NORs count in cells of the gonads right and left were respectively: spermatogonia (8.77±1.14 and 9.04±0.96), spermatocyte (4.99±2.00 and 6.20±2.07, P<0.05), Leydig (8.05±2.82 and 7.89±2.29) and Sertoli cells (8.07±1.88 and 7.61±2.16; P<0.05). There was a correlation between the mean values for right and left sides for the number of NORs (P<0.05) between Leydig x spermatocytes (0.49); spermatocytes x Sertoli (0.49) and Sertoli x Leydig (0.96). The study demonstrates that the AgNOR staining technique is indicated to evaluate the cell proliferative potential in ovine testis with practical implementation and low cost. / A coloração pela prata das regiões organizadoras de nucléolos (NORs) é caracterizada por marcar proteínas ligadas ao ácido ribonucléico ribossômico, avaliando a proliferação em células normais ou neoplásicas. O objetivo do trabalho foi estudar em testículos de ovinos obtidos em matadouro a validade do uso da técnica de coloração AgNOR na proliferação de células saudáveis da linhagem espermatogênica. Utilizou-se 43 pares de testículos de ovinos mestiços entre seis e 10 meses de idade. Testes de Wilcoxon e Spearman foram empregados, com nível de 5%. As médias das NORs nas células das gônadas direita e esquerda foram respectivamente: espermatogônia (8,77±1,14 e 9,04±0,96), espermatócito (4,99±2,00 e 6,20±2,07; P<0,05), Leydig (8,05±2,82 e 7,89±2,29) e Sertoli (8,07±1,88 e 7,61±2,16; P<0,05). Houve correlação (P<0,05) entre os lados para o número de NORs: espermatócito x Leydig (0,49); espermatócito x Sertoli (0,49) e Leydig x Sertoli (0,96). Conclui-se ser válido o emprego da técnica AgNOR para avaliar o potencial proliferativo das células saudáveis em testículos de ovinos com prática execução e baixo custo.

Made available in DSpace on 2016-07-18T17:53:10Z (GMT). No. of bitstreams: 1 daniel.pdf: 186929 bytes, checksum: 3b546022c9b10042ce06a35c124600cd (MD5) Previous issue date: 2012-04-13 / The silver staining technique for AgNOR nucleolar organizer regions (NORs) is characterized by marking proteins linked to the ribosomal ribonucleic acid, evaluating cell proliferation. The aim was to study the validity of the AgNOR staining technique in the testicular cell proliferation in crossbred ovine. Was used treis pairs forty crossbred sheep testes between six and ten months old.Wilcoxon and Spearman tests were used with a significance level of 5%. The mean NORs count in cells of the gonads right and left were respectively: spermatogonia (8.77±1.14 and 9.04±0.96), spermatocyte (4.99±2.00 and 6.20±2.07, P<0.05), Leydig (8.05±2.82 and 7.89±2.29) and Sertoli cells (8.07±1.88 and 7.61±2.16; P<0.05). There was a correlation between the mean values for right and left sides for the number of NORs (P<0.05) between Leydig x spermatocytes (0.49); spermatocytes x Sertoli (0.49) and Sertoli x Leydig (0.96). The study demonstrates that the AgNOR staining technique is indicated to evaluate the cell proliferative potential in ovine testis with practical implementation and low cost. / A coloração pela prata das regiões organizadoras de nucléolos (NORs) é caracterizada por marcar proteínas ligadas ao ácido ribonucléico ribossômico, avaliando a proliferação em células normais ou neoplásicas. O objetivo do trabalho foi estudar em testículos de ovinos obtidos em matadouro a validade do uso da técnica de coloração AgNOR na proliferação de células saudáveis da linhagem espermatogênica. Utilizou-se 43 pares de testículos de ovinos mestiços entre seis e 10 meses de idade. Testes de Wilcoxon e Spearman foram empregados, com nível de 5%. As médias das NORs nas células das gônadas direita e esquerda foram respectivamente: espermatogônia (8,77±1,14 e 9,04±0,96), espermatócito (4,99±2,00 e 6,20±2,07; P<0,05), Leydig (8,05±2,82 e 7,89±2,29) e Sertoli (8,07±1,88 e 7,61±2,16; P<0,05). Houve correlação (P<0,05) entre os lados para o número de NORs: espermatócito x Leydig (0,49); espermatócito x Sertoli (0,49) e Leydig x Sertoli (0,96). Conclui-se ser válido o emprego da técnica AgNOR para avaliar o potencial proliferativo das células saudáveis em testículos de ovinos com prática execução e baixo custo.

O objetivo deste estudo foi avaliar o processo de aquisição de anticorpos em cordeiros recém-nascidos aleitados com colostro bovino e ovino, bem como a taxa de proliferação celular no epitélio intestinal. Este estudo contribui com informações sobre a aquisição de imunidade passiva nesta espécie, com o conhecimento do desenvolvimento e maturação do trato gastrintestinal no período neonatal, e para a avaliação de uma alternativa de manejo de colostro para estes pequenos ruminantes. Foram utilizados 30 cordeiros recém-nascidos. Às 0 e 6 horas de vida, 12 animais receberam 250 mL de colostro bovino (grupo CB) e outros 12 animais receberam 250 mL de colostro ovino (grupo CO). Amostras de sangue foram coletadas às 0, 6, 24, e 72 horas de vida para quantificação de imunoglobulina G (IgG) e proteína total sérica (PT). Seis animais foram sacrificados aleatoriamente, logo após o nascimento, sem ingestão de colostro, constituindo o grupo controle. Os demais grupos foram abatido às 24 e 72 horas. Amostras do intestino delgado foram coletadas para a quantificação da taxa de divisão celular nas criptas intestinais. O delineamento experimental adotado foi inteiramente casualizado, sendo as variáveis séricas analisadas como medidas repetidas no tempo. Para a variável histológica foi considerado um arranjo fatorial 2 X 2 + 1, tendo como efeitos principais o colostro fornecido, as idades de abate e o grupo controle. As concentrações de IgG sérica às 6, 24 e 72 horas foram significativamente superiores para o grupo CB (16,58±6,19; 34,12±5,67 e 28,77±5,45 mg mL-1) comparado com CO (10,76±6,08, 20,77±6,53 e 20,25±7,3 mg mL-1). A eficiência aparente de absorção (EAA) da IgG mostrou-se inferior no grupo CB (15,06±4,97%) em relação aos animais do grupo CO (25,70±13,08%). O grupo CB apresentou às 24 e 72 horas maiores (P<0,05) valores de PT (7,29±0,87 e 6,89±0,30 g 100mL-1) em relação ao grupo CO (5,73±1,35 e 5,69±0,57 g 100mL-1). Ao nascimento, os animais apresentaram 32,52%, 45,47% e 30,60% de células em divisão para as regiões do duodeno, jejuno médio e íleo, respectivamente. Às 24 horas, os animais do grupo CO apresentaram menor (P<0,0001) porcentagem de células em mitose no duodeno (42,12%) e no íleo (35,66%) em relação aos animais CB, 46,44% e 39,74%, respectivamente. Às 72 horas, foi observada uma porcentagem menor (P<0,0001) de células em divisão nas criptas do duodeno dos animais CO (36,28%), comparados com o grupo CB (43,18%). Não foi observada diferença significativa entre os tratamentos na porcentagem de células mitóticas nas criptas do jejuno às 24 e 72 horas, bem como nas criptas do íleo às 72 horas (P>0,05). Independente do tratamento, o jejuno foi o segmento com maior (P<0,0001) porcentagem de células mitóticas em todos os períodos. Os valores superiores na taxa de divisão celular no grupo CB indicam que o colostro bovino, provavelmente pela elevada concentração de fatores bioativos e de anticorpos, influencia positivamente o processo de renovação epitelial e que o mesmo pode ser utilizado como fonte alternativa de IgG para cordeiros recém-nascidos. / The objective of this study was to evaluate the antibody acquisition mechanism in newborn lambs fed bovine or ovine colostrum as well as the cell proliferation rate in the intestine epithelium. This study contributes with information about passive immunity acquisition in this specie, with knowledge about development and maturation of the small ruminant intestine tract, and for the evaluations of colostrums management alternative to these small ruminant. Thirty newborn lambs were used. At 0 and 6 hours of life, 12 animals received 250 mL of bovine colostrum (BC group) and another 12 animals received 250 mL of ovine colostrum (OC group). Blood samples were collected at 0, 6, 24, e 72 hours of life for immunoglobulin G (IgG) and total serum protein (TP) quantification. Six animals were randomly slaughtered just after birth, without colostrum intake, constituting the control group. The other groups were randomly slaughtered at 24 and 72 hours. Samples of the small intestine were collected for quantification of cellular division rate in intestinal crypts. A completely randomized desining was used, with the serum variables analyzed as repeated measures on time. For the histological variable it was considered a 2 X 2 + 1 factorial arrangement, having as the main factors colostrum supply, slaughter date and the control group. The IgG serum concentration at 6, 24 and 72 hours were significantly higher for the BC group (16,58±6,19; 34,12±5,67 and 28,77±5,45 mg mL-1) compared with the OC group (10,76±6,08, 20,77±6,53 and 20,25±7,3 mg mL-1). The apparent efficiency of IgG absorption (AEA) were lower for the BC group (15,06±4,97%) in relation to the animals from the OC group (25,70±13,08%). The BC group showed at 24 and 72 hours higher (P<0,05) TP values (7,29±0,87 and 6,89±0,30 g 100mL-1) in relation to the OC group (5,73±1,35 and 5,69±0,57 g 100mL-1). At birth, the animals showed 32,52%, 45,47% and 30,60% cells in division for duodenum, jejunum and ileum, respectively. At 24 hours, the animals from the OC group showed lower (P<0,0001) percentage of cells in mitosis in the duodenum (42,12%) and ileum (35,66%) in relation to the BC animals, 46,44% and 39,74%, respectively. At 72 hours, it was observed lower percentage (P<0,0001) of cells in division in the duodenum crypts of the OC animals (36,28%) compared with the BC group (43,18%). It was not observed significantly difference between treatment in mitotic cell percentage of jejunum crypts at 24 and 72 hours as well as in ileum crypts at 72 hours. Independent of the treatment the jejunum was the segment with higher mitotic cells percentage in all periods. The highest values in cellular division rate in the BC group, probably due to high concentrations of bioactive factors and antibodies, indicates that bovine colostrum influences positively the epithelium renovation process and that it can be used as an alternative source of IgG for newborn lambs.

Les gliomes sont des tumeurs qui seforment par prolifération anormale de cellules dansle tissu cérébral. La dangerosité de ces tumeursréside dans le fait que la plupart des gliomes sontinvasifs : les cellules tumorales migrent dans le tissusain autour de la tumeur. Ces cellules tumoralesisolées provoquent des récidives quasi systématiquesaprès traitement (chirurgie, chimiothérapie,radiothérapie), rendant ces tumeurs incurablesactuellement et conduisant au décès du patient. Il estimportant d'associer des études fondamentales pourmieux comprendre leur évolution dès l'origine et desétudes plus appliquées en développant de nouveauxsubstrats pour reproduire in vitro leur évolution. Lescellules progénitrices des oligodendrocytes (OPC)représentent la plus grande population de cellules enprolifération et la plus largement distribuée dans lecerveau adulte, ce qui en fait un suspect idéal del’origine des gliomes. A partir de donnéesexpérimentales de la littérature sur la dynamique invivo de ces cellules, un modèle mathématiquereproduisant cette dynamique dans un tissu sain a étédéveloppé.Ce modèle montre également que les OPC pourraientêtre à l’origine de toutes les formes de gliomerencontrées aussi bien de bas grade que de hautgrade. Par ailleurs, l’approche expérimentale utiliséevisait à développer un substrat de culture cellulaireadapté à l’étude des gliomes in vitro. Ainsi, unhydrogel biocompatible, minimaliste et contrôlable aété élaboré. Celui-ci mime l’élasticité de la matriceextracellulaire (MEC) cérébrale avec une rigidité del’ordre de 200 Pa et l’effet adhésif des molécules dela MEC impliqué dans l’adhésion et la proliférationdes cellules tumorales. Grâce à ses propriétés,l’hydrogel favorise la survie de près de 90% desmodèles cellulaires de gliome utilisés dans notreétude et supporte la croissance en trois dimensionsd’agrégats multicellulaires semblables à lamorphologie de micro-tumeurs in vivo. Le modèled’hydrogel est donc validé pour favoriser la viabilitéet la prolifération cellulaires. Les perspectives detravail futures porteront sur l'optimisation de sacomposition pour mimer de manière encore plusréaliste la croissance tumorale in vivo. / Gliomas are brain tumors arising fromanomalous cell proliferation into the brain tissue.The hazard of these tumors resides in their invasiveability : tumor cells migrate into the healthy tissuesurrounding the tumor. These isolated cells causequasi systematic recurrences after treatment(surgery, chemotherapy, radiotherapy) making thesetumors currently incurable and leading to patientdeath. Hence, it is important to associatefundamental studies for better understanding of theirevolution from their origin with more appliedstudies developing new substrates for reproducingtheir evolution in vitro. Oligodendrocyte progenitorcells (OPC) are the most widely spread proliferatingpopulation in the adult brain, which makes them themain suspect of causing gliomas origin. Fromexperimental data in the literature about in vivodynamic of OPC, a mathematical model that depictsthis dynamic into a healthy tissue has beendeveloped.This model also shows that OPC could be at theorigin of all glioma forms from low to high grade.Furthermore, the experimental approach used aimedat designing a cell culture substrate adapted toglioma studies in vitro. Thus, a biocompatible,minimalistic and controllable hydrogel has beenperformed. It mimics brain extracellular matrix(ECM) elasticity around 200 Pa and the adhesiveeffect of ECM molecules involved in tumor celladhesion and proliferation. Due to these properties,the hydrogel contributes to around 90% of gliomacell models survival used in our study and promotesmulticellular aggregates growth in three dimensionsthat look like in vivo microtumors morphology. Thishydrogel model is thus validated for cell viabilityand proliferation. Future works will be devoted tothe optimization of its composition for bettermimicking of tumor growth in vivo in a morerealistic manner.

Indiana University-Purdue University Indianapolis (IUPUI) / Type II nephronophthisis (NPHP2) is an autosomal recessive renal cystic disorder characterized by mutations in the inversin gene. Humans and mice with mutations in inversin have enlarged cystic kidneys. Increased kidney size in NPHP2 may involve altered cell growth, apoptosis, electrolyte transport and fluid accumulation in the cysts. To test this hypothesis, histology and transcriptome analysis were performed on one-day old wild-type and inv/inv mice to uncover molecular pathways altered in the mutant mice. Histology of inv/inv mice kidneys showed dilated cystic tubules compared to wild type. Pathway analysis of transcriptome data showed that inversin exerts its effects on kidneys, at least in part, through the transcriptional regulation of genes implicated in inflammation, immune response, cellular metabolism, cell cycle and ion transport. Genes involved in inflammation or immune response were upregulated whereas the genes involved in cell cycle progression and ion transport were downregulated. To validate the array findings from inv/inv mice kidneys, functional consequence of inversin loss on transepithelial ion transport was measured by electrophysiological techniques in shRNA mediated inversin-depleted renal cell type isolated from mouse cortical collecting duct (mCCD). Depletion of inversin decreased vasopressin-induced Na+ absorption, but did not alter Cl- secretion in mCCD cells. Addition of amiloride, a specific blocker of the epithelial sodium channel (ENaC), abolished basal ion transport in both inversin knockdown and control cells indicating ENaC involvement. Loss of inversin decreased Na+ absorption and this effect, in part, was mediated by transcriptional and post-translational regulation of ENaC mediators. To better understand inversin function in renal cells, transcriptome analysis was performed in control and inversin-depleted mCCD cells. Pathway analysis showed that inversin-depletion altered the genes represented in cell cycle, cellular assembly and organization, DNA replication, cell proliferation and ion transport in this isolated renal cell type. In concordance with the array data from inv/inv mice kidneys, a decrease in the expression of cell cycle, ion transport and apoptotic genes were observed accompanied by an upregulation of genes implicated in inflammatory or immune response indicating a direct effect of inversin on renal cells. Together, this study utilized a combination of transcriptome and functional analyses to unravel the role of inversin in renal cells. These data demonstrate that loss of inversin can cause a delay in cell cycle progression with a decrease in cell proliferation and apoptosis which in turn can perturb the development of the renal tubule. Also, a decrease in Na+ reabsorption together with differential regulation of other ion transporters can result in altered electrolyte transport contributing to cystogenesis, cyst growth, fluid accumulation and cyst expansion in NPHP2.

Indiana University School of Dentistry / Introduction: The goal of nonsurgical root canal therapy is to reduce the bacterial load within an infected root canal system, and the subsequent objective is to prevent or treat apical periodontitis. Clinical studies have shown more expedient healing of apical periodontitis treated with electromagnetic wave stimulation (EMS) as compared to apical periodontitis not treated with EMS. Stimulation of osteoblasts and growth factors has been shown when EMS was applied to rat calvaria, resulting in increased bone healing. Objective: The purpose of this vitro study was to evaluate the effects of EMS on the proliferation and differentiation of osteoblasts. Using primary neonatal calvaria osteoblast-lineage cells, the effects of different EMS regimens on proliferation, alkaline phosphatase (ALP) activity, and mineral deposition were determined. Materials and Methods: EMS regimen included currents of 0mA, 0.1mA, 1mA, and 10mA delivered for five consecutive 1s pulses per day for one, two, and three days. Cell proliferation was assayed after 1 or 2 days using an MTS assay. Alkaline phosphatase activity and mineral deposition were assayed after culturing the cells in osteogenic media containing ascorbic acid and -glycerol phosphate for 7 days. Comparisons were performed using analysis of variance, with a 5% significance level. Results: There was no statistically significant differences noted in MTS proliferation and mineral deposition between the experiment EMS treatment groups of 0.1, 1.0, and 10.0 mA compared to the control group of 0 mA current on calvaria-derived osteoblast. While there were no statistically significant differences noted in ALP activity in the 0.1, and 1.0 mA EMS groups, compared to 0 mA control, alkaline phosphatase activity was significantly increased in the 10 mA EMS group. Conclusion: There was no significant differences in MTS proliferation and mineral deposition of the EMS group compared to the control group. However, 10 mA EMS favored increased ALP activity suggesting EMS can promote matrix maturation by osteoblasts. Additional in vitro experimental studies, including different stem cell populations, culture duration and EMS treatment regimens are needed to understand the mechanism of action of EMS for future applications in regenerative endodontics.

Background: Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types and possess immunomodulatory and anti-inflammatory effects, making them interesting candidates for therapeutic applications. MSCs are present in small quantities in tissues like bone marrow and therefore need to be expanded while preserving their essential characteristics. They should adhere to plastic, differentiate into osteocytes, adipocytes and chondrocytes and express specific cell surface markers. Currently, the “golden standard” culture media supplement is fetal bovine serum (FBS). However, there is a potential contamination risk of MSCs by xenogeneic and zoonotic infectious agents, which can trigger an immune response. As an alternative, xeno-free serum supplements derived from human sources, e.g., human serum (HS) can be used.  Aim: This study aimed to identify and characterize human bone marrow derived MSCs and examine the effects different supplements have on the cells.  Methods: MSCs were cultured in 10% FBS, 2% FBS and 10% HS for 20 -21 days. Differentiation was induced and the potential was detected with immunocytochemistry. Cell surface markers CD73, CD90, CD105 and CD45 were identified with flow cytometry.  Results and Conclusion: There was no significant difference in morphology, differential potential or immunophenotype between the different serum conditions. However, HS-supplemented culture media resulted in a significantly higher number of cells with 1 x 107 cells after 20 days without affecting their differentiation potential and immunophenotype in comparison to 10% FBS with 2.2 x 106 cells (p=0.0004). MSCs cultured in 2% FBS resulted in the least number of cells (9.9 x 105) after 21 days of expansion.

No description available.