La transplantation de sang de cordon ombilical (TSCO) constitue un traitement de choix pour une multitude de pathologies hématologiques malignes et non malignes chez l’enfant et dans certains cas l’adulte. La TSCO est associée à certaines complications, dont une reconstitution immunitaire plus lente et une incidence élevée d’infections opportunistes, notamment celles reliées au cytomégalovirus (CMV) et au virus varicella-zoster (VZV). Dans le cadre de ce travail, nous nous sommes intéressés dans un premier temps à la caractérisation de la reconstitution immunitaire spécifique au CMV et au VZV. Nos résultats ont démontré que la reconstitution de l’immunité cellulaire ne requiert ni un statut séropositif pré-transplantation ni le développement de la maladie. De plus, des reconstitutions spontanées ont été détectées chez certains patients séronégatifs vis-à-vis du CMV ou du VZV. Outre le fait qu’elle se manifeste surtout à partir de 6 mois post-transplantation, ladite reconstitution mérite le qualificatif de « protectrice » en termes de réactivations virales et du développement de signes cliniques lorsqu’une fréquence de 150 cellules produisant l’IFN-γ/million est dépassée. Toutefois, moins de 5% des patients développent une réponse T anti-VZV et anti-CMV au cours 100 premiers jours suivant la TSCO. Il est donc possible que les lymphocytes CD8+ T provenant du SCO, comparativement à leurs homologues provenant de la moelle osseuse (MO), présentent un défaut de fonctionnalité, communément appelé « épuisement clonal ». La caractérisation du répertoire de récepteurs inhibiteurs exprimés par les cellules T CD8+ suivant la TSCO ou la transplantation de moelle osseuse (TMO) a révélé une augmentation significative de la fréquence des cellules exprimant PD-1 tôt suivant la transplantation. Cette population, caractérisée majoritairement par un phénotype effecteur-mémoire (EM), démontre une perte significative de la capacité proliférative et exprime moins d'IFN-γ, d'IL-2, de TNF-α et de CD107a. Une meilleure caractérisation de la reconstitution immunitaire après TSCO permettrait, d'une part de sélectionner des biomarqueurs en vue d’une meilleure gestion des patients à risques de développer des infections virales et/ou de rechuter, et d'autre part d'améliorer leur pronostic. / Umbilical cord blood transplantation (UCBT) is a treatment of choice for a variety of hematological malignancies and non-malignant diseases in children and, in some cases, in adults. UCBT is associated with a slower immune reconstitution and a high incidence of viral infections, especially related to cytomegalovirus (CMV) and the varicella-zoster virus (VZV). As part of this work, we aimed to assess the reconstitution of CMV and VZV-specific T cell responses. Neither pre-transplant serostatus nor disease development is required for development of T cell mediated immunity. Moreover, spontaneous reconstitution detected in some patients who were seronegative for CMV or VZV. Detected especially after 6 months post-transplant, antiviral responses are protective in terms of viral reactivation and development of clinical signs, when a frequency of 150 cells producing d'IFN-γ / million is achieved. However, less than 5% of patients develop an antiviral response during the first 100 following UCBT. Compared to their bone marrow (BM) counterparts, UCB CD8+ T lymphocytes may be functionally impaired, a state commonly called « clonal exhaustion ». Characterization of the inhibitory receptors repertoire expressed by CD8+ T cells following UCBT and BMT showed a significant increase in the frequency of cells expressing PD-1 early after transplantation. This population, mainly characterized by effector phenotype, showed a significant loss of proliferative capacity and produced less IFN-γ, IL-2, TNF-α and CD107a. An improved understanding of the CD8+ T cell compartment following UCBT, as well as biomarkers related to T cell exhaustion will decrease infection, transplant related mortality and correlate with better prognosis

Le virus d’Epstein-Barr (EBV) est un herpèsvirus humain qui infecte plus de 90% de la population mondiale. Il est décrit comme associé à plusieurs pathologies cancéreuses humaines comme les carcinomes nasopharyngés et gastriques et divers lymphomes, comme le lymphome de Burkitt, les lymphomes NK/T et certains lymphomes de Hodgkin. Le lymphome T angio-immunoblastique (LTAI), un cancer des cellules T folliculaires helper TFH, contient souvent des cellules B porteuses de l’EBV. Mais jusqu’à présent le rôle de l’EBV dans la pathogenèse de cette maladie reste inconnu. Dans ce contexte, notre travail avait pour objectif de déterminer si l’EBV associé au LTAI présentait une particularité laissant envisager son rôle dans cette pathologie. Pour ce faire, nous avons étudié la séquence complète de l’EBV au sein d’échantillons de LTAI et comparé les résultats à ceux obtenus pour d’autres lymphomes (B, NK/T) ainsi qu’aux séquences publiées. Le séquençage a tout d’abord été réalisé sur 7 lignées cellulaires positives pour l’EBV, afin de valider la technique, et a ensuite été appliqué aux échantillons d’adénopathies de 40 patients atteints de syndrome lymphoprolifératif, parmi lesquels 20 souffraient de LTAI. L’enrichissement en génome viral a été réalisé par capture à l’aide de sondes spécifiques du génome de l’EBV. Ensuite les librairies ont été synthétisées et séquencées sur les plateformes Illumina MiSeq et NextSeq. Dans un deuxième temps, nous avons réalisé l’assemblage de novo des reads et déterminé la séquence complète du virus majoritaire dans chaque échantillon. Les données obtenues ont été analysées bioinformatiquement. D’une manière intéressante, le virus a été trouvé clonal ou quasi-clonal dans les LTAI alors que les lymphocytes B étaient dans la plupart des cas polyclonaux. En outre, le profil de mutations trouvé présentait des similitudes avec ce qui était trouvé pour les autres lymphomes associés à l’EBV, notamment au niveau des épitopes cibles des cellules de l’immunité suggérant un processus de sélection de la souche virale identique à celui d’une tumeur clonale associée à l’EBV. Ceci pourrait jouer un rôle important dans l’échappement au système immunitaire du virus dans ce contexte multicellulaire complexe. La présence de cellules B polyclonales avec un EBV clonal dans un compartiment T tumoral clonal pourrait relever d’une double sélection tumorale, endogène T et exogène EBV clonal, et pourrait suggérer l’existence de cross-talk entre les cellules B-T. / More than 90% of the world's population is infected by Epstein-Barr virus (EBV), a human herpesvirus. EBV is thought to be implicated in the pathogenesis of several human malignancies including epithelial tumors such as nasopharyngeal and gastric carcinomas as well as lymphoproliferative diseases such as Burkitt's lymphoma, NK/T lymphomas and some Hodgkin lymphomas. In angioimmunoblastic T-cell lymphoma (AITL), a peripheral neoplasm of follicular helper T (TFH) cells, a recurrent finding is the presence of EBV-positive B lymphocytes at the beginning of the disease. However, whether this EBV infection of B cells plays a role in AITL pathogenesis remains unclear. In this context, our work aimed to determine if the EBV associated with the AITL presented an oncogenic profile allowing us to consider its role in this pathology. To do this, we sequenced the whole EBV genomes in AIL samples and compared the results to those obtained for other lymphomas (B, NK / T) as well as to previously published sequences. Sequencing was first performed on 7 EBV-positive cell lines to validate the technique, and then was applied to lymphadenopathy specimens from 40 patients with lymphoproliferative disease, of whom 20 had AITL. Enrichment of the viral genome was performed by capture using specific EBV genome probes. The libraries were synthesized and sequenced on Illumina MiSeq and NextSeq platforms. In a second step, we performed de novo assembly and determined the sequence of the virus in each sample. The data obtained were analyzed bioinformatically. Interestingly, the virus was found to be clonal or quasi-clonal in AITL, while B cells were in some cases polyclonal. In addition, the mutational pattern was similar to other EBV-associated lymphomas, especially at the level of the target epitopes of immune cells suggesting a process of selection of the viral strain identical to that of a clone tumor associated with EBV. This could play an important role in the virus escape from the immune system in this context. The presence of polyclonal B cells with clonal EBV in a clonal tumor T cell compartment could be a dual tumor selection; or that is endogenous T and exogenous clonal EBV, and could therefore suggest the existence of a cross-talk between B-T cells.

Submitted by FERNANDO PARRA DOS ANJOS LIMA null (engfernandoparra@gmail.com) on 2016-10-31T11:47:54Z No. of bitstreams: 1 Fernando Parra A. Lima.pdf: 3667307 bytes, checksum: 0d206b9c09566cdb11de101b84976228 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-11-07T16:42:09Z (GMT) No. of bitstreams: 1 lima_fpa_dr_ilha.pdf: 3363973 bytes, checksum: d8849cdd159a11920c497d025a8ae16a (MD5) / Made available in DSpace on 2016-11-07T16:42:09Z (GMT). No. of bitstreams: 1 lima_fpa_dr_ilha.pdf: 3363973 bytes, checksum: d8849cdd159a11920c497d025a8ae16a (MD5) Previous issue date: 2016-09-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esta pesquisa é dedicada ao desenvolvimento de uma metodologia para a realização do diagnóstico de distúrbios de tensão de sistemas de distribuição de energia elétrica, baseada no uso de sistemas imunológicos artificiais (SIA). Trata-se da proposição de um novo paradigma no ambiente dos SIA que confere o aprendizado de modo contínuo (plasticidade). Esta concepção permite compor um sistema de diagnóstico apto a aprender continuamente, contemplando novos tipos de distúrbios advindos da constante evolução do setor elétrico, sem a necessidade de reiniciar o processo de aprendizado. Neste contexto, empregam-se dois algoritmos imunológicos artificiais, sendo o algoritmo de seleção negativa, responsável pelo processo de reconhecimento de padrões, e o algoritmo de seleção clonal responsável pelo processo de aprendizado. A principal aplicação deste novo método é auxiliar na operação do sistema durante distúrbios, bem como, supervisionar o sistema de proteção, e estar apto a acompanhar a evolução dos sistemas elétricos adquirindo conhecimento continuamente. Para avaliar a eficácia e o desempenho deste novo método foram realizadas simulações de distúrbios de tensão em sistemas de distribuições de energia elétrica com 5, 33, 84 e 134 barras, no software ATP/EMTP. Os resultados obtidos com esta abordagem mostram robustez e eficiência quando comparados à literatura. / This work develops a methodology to realize voltage disturbance diagnosis in electrical distribution systems, based on Artificial Immune Systems (AIS). It is a proposition of a new paradigm in AIS environment, which provides a continuous learning (plasticity). This conception allows composing a diagnosis system able to continuous learn, when new disturbances appear due to the constant evolution of the power systems, without needing to reinitialize the learning. This way, two artificial immune algorithms are used, such as the negative selection algorithm executing the pattern recognition process, and the clonal selection algorithm, executing the learning process. The main application of this new method is to aid the system operation during disturbances, as well as, supervise the system protection and be able to carry on the evolution of the electrical systems acquiring knowledge continuously. To evaluate the efficiency and the performance of this new method, voltage disturbance simulations were executed in electrical distributions systems with 5, 33, 84 and 134-bus in ATP/EMTP software. Results show robustness and efficiency when compared with those in the literature.

Orientador: Carlos Roberto Minussi / Resumo: Esta pesquisa é dedicada ao desenvolvimento de uma metodologia para a realização do diagnóstico de distúrbios de tensão de sistemas de distribuição de energia elétrica, baseada no uso de sistemas imunológicos artificiais (SIA). Trata-se da proposição de um novo paradigma no ambiente dos SIA que confere o aprendizado de modo contínuo (plasticidade). Esta concepção permite compor um sistema de diagnóstico apto a aprender continuamente, contemplando novos tipos de distúrbios advindos da constante evolução do setor elétrico, sem a necessidade de reiniciar o processo de aprendizado. Neste contexto, empregam-se dois algoritmos imunológicos artificiais, sendo o algoritmo de seleção negativa, responsável pelo processo de reconhecimento de padrões, e o algoritmo de seleção clonal responsável pelo processo de aprendizado. A principal aplicação deste novo método é auxiliar na operação do sistema durante distúrbios, bem como, supervisionar o sistema de proteção, e estar apto a acompanhar a evolução dos sistemas elétricos adquirindo conhecimento continuamente. Para avaliar a eficácia e o desempenho deste novo método foram realizadas simulações de distúrbios de tensão em sistemas de distribuições de energia elétrica com 5, 33, 84 e 134 barras, no software ATP/EMTP. Os resultados obtidos com esta abordagem mostram robustez e eficiência quando comparados à literatura. / Doutor

Streptococcus agalactiae est la première cause d’infections invasives chez le nouveau né et, malgré la mise en place de stratégies de prévention, cette bactérie reste le principal agent étiologique des infections néonatales. Les souches de séquence type 17, dites hyper-virulentes, sont particulièrement associées avec les méningites, type d’infection ayant des conséquences lourdes en terme de mortalité et morbidité. Ce clone possède des caractéristiques uniques, telle que la fixation au fibrinogène, ainsi qu’un répertoire de protéines de surface qui lui sont spécifiques. Parmi ces protéines, Srr2 appartient à une famille de larges glycoprotéines streptococcales et staphylococcales impliquées dans la pathogénicité. Un domaine central de Srr2, le domaine BR, est responsable de la fixation spécifique du fibrinogène par le clone ST-17, ainsi qu’au plasminogène et à divers composants de la matrice extracellulaire. Cette protéine promeut ainsi l’adhésion et le franchissement des barrières cellulaires. L’interaction de Srr2 avec les systèmes fibrinolytique et de coagulation de l’hôte favorise la dissémination bactérienne par l’activation de la fibrinolyse, et la persistance de la bactérie dans l’organisme par la formation d’agrégats bactériens. La liaison de Srr2 avec le fibrinogène semble également promouvoir la persistance bactérienne en favorisant l’internalisation et la survie dans les macrophages. Ainsi, la protéine Srr2 confère un avantage pour le processus infectieux du clone ST-17 dans l’hôte, et constitue une cible vaccinale intéressante pour la prévention des infections à S. agalactiae. / Streptococcus agalactiae is the leading cause of invasive infections in neonates. Despite the implementation of prevention strategies, this bacterium remains the main etiological agent of neonatal infections. Hyper-virulent sequence-type 17 strains are particularly associated with meningitis, a type of infection with serious consequences in terms of mortality and morbidity. This clone has unique characteristics, such as fibrinogen binding, and a panel of specific surface proteins. Among these proteins, Srr2 belongs to a family of large streptococcal and staphylococcal glycoproteins involved in pathogenicity. A central domain of Srr2, BR domain, is responsible for the specific binding of fibrinogen by the ST -17 clone and also binds plasminogen and various components of the extracellular matrix. Thereby, it promotes adhesion and crossing of cellular barriers. The interaction of Srr2 with fibrinolytic and coagulation systems of the host could promote bacterial spread through the activation of fibrinolysis and the persistence of the bacteria in the host by the formation of bacterial aggregates. The interaction of Srr2 with fibrinogen also seems to promote bacterial persistence in promoting the internalization and survival of the bacteria in macrophages. Thus, Srr2 confers an advantage to the infectious process of the ST- 17 clone in the host and is an attractive vaccine candidate for the prevention of S. agalactiae infections.

Background: Clonal competition in cancer describes the process in which the progeny of a cell clone supersedes or succumbs to other competing clones due to differences in their functional characteristics, mostly based on subsequently acquired mutations. Even though the patterns of those mutations are well explored in many tumors, the dynamical process of clonal selection is underexposed. Methods: We studied the dynamics of clonal competition in a BcrAbl-induced leukemia using a γ-retroviral vector library encoding the oncogene in conjunction with genetic barcodes. To this end, we studied the growth dynamics of transduced cells on the clonal level both in vitro and in vivo in transplanted mice. Results: While we detected moderate changes in clonal abundancies in vitro, we observed monoclonal leukemias in 6/30 mice after transplantation, which intriguingly were caused by only two different BcrAbl clones. To analyze the success of these clones, we applied a mathematical model of hematopoietic tissue maintenance, which indicated that a differential engraftment capacity of these two dominant clones provides a possible explanation of our observations. These findings were further supported by additional transplantation experiments and increased BcrAbl transcript levels in both clones. Conclusion: Our findings show that clonal competition is not an absolute process based on mutations, but highly dependent on selection mechanisms in a given environmental context.

Background: Clonal competition in cancer describes the process in which the progeny of a cell clone supersedes or succumbs to other competing clones due to differences in their functional characteristics, mostly based on subsequently acquired mutations. Even though the patterns of those mutations are well explored in many tumors, the dynamical process of clonal selection is underexposed. Methods: We studied the dynamics of clonal competition in a BcrAbl-induced leukemia using a γ-retroviral vector library encoding the oncogene in conjunction with genetic barcodes. To this end, we studied the growth dynamics of transduced cells on the clonal level both in vitro and in vivo in transplanted mice. Results: While we detected moderate changes in clonal abundancies in vitro, we observed monoclonal leukemias in 6/30 mice after transplantation, which intriguingly were caused by only two different BcrAbl clones. To analyze the success of these clones, we applied a mathematical model of hematopoietic tissue maintenance, which indicated that a differential engraftment capacity of these two dominant clones provides a possible explanation of our observations. These findings were further supported by additional transplantation experiments and increased BcrAbl transcript levels in both clones. Conclusion: Our findings show that clonal competition is not an absolute process based on mutations, but highly dependent on selection mechanisms in a given environmental context.

La dysbiose est une cause importante dans la survenue de maladies, favorisant la prolifération de pathogènes ou induisant l’inflammation. L’étude de ce phénomène est devenue possible grâce aux approches d’analyse génomique (AG) associé avec d’autres techniques. L’entérocolite nécrosante (ECN) et l’infection du pied diabétique (IPD) demeurent deux maladies associées à la dysbiose dans lesquels différents bactéries ont été décrites, notamment C. butyricum dans l’ECN, E. coli et S. aureus dans l’IPD. Dans le cadre de l’ECN, C. butyricum demeure l’espèce la plus fréquente chez les ECN. L’identification clusters liés géographiquement et en fonction du temps. Le portage asymptomatique est suggéré par une similarité génomique des souches patients et contrôles. La prédiction d’un gène de β-hémolysine ainsi leur effet cytotoxique sur les cellules Jurkat avait été observé. De même, sur les cellules Caco-2 malgré le KO du gène de β-hémolysine. En se basant sur l’analyse physico-chimique du surnageant bactérien, nous avons suggéré que la fraction cytotoxique est protéique. La purification de la fraction cytotoxique a permis de trouver une protéine codant pour PspC family possédant un domain conservé commun avec celui de la toxine A/B. L’inactivation du gène codant pour cette protéine n’a pas supprimé l’effet cytotoxique, suggérant la présence d’une combinaison gènes. En parallèle, nous avons ciblé l’impact de C. neonatale par qRPC spécifique rpoB. Cette espèce était plus fréquente chez les patients, ainsi de clones géographiques ont été identifiées. Enfin, des SNPs ont été observés dans des gènes de virulence dans le cas des E. coli et S. aureus isolés de l’IPD. / Dysbiosis remains a main cause during the establishment of several diseases, by promoting bacterial translocation, leading to inflammation process. Specific microorganisms were involved in the pathogenesis of dysbiosis-associated diseases, notably necrotizing enterocolitis (NEC) and diabetic foot (DF). This was possible by the implication of whole-genome analysis (WGA) in association with other techniques. In case of NEC, C. butyricum was significantly associated with in NEC; tested on a South-East French cohort. Geographical and/or temporal clusters were identified, thus genomic relationship between NEC-associated isolates and controls, suggesting the presence of asymptomatic carriage. Genes encoding for β-hemolysin was detected and C. butyricum supernatant exhibited cytotoxic effect on Jurkat cells. Cytotoxic effect was also presented on Caco-2 cells. Supernatant of β-hemolysin-mutant C. butyricum showed enterotoxic effect. Basing on physico-chemical data, we assumed that the evaluated fraction was a protein. Proteomics analysis revealed that PspC family was the cytotoxic protein. This protein owned a glucan-binding domain, shared by C. difficile toxin A/B. The KO of PspC gene was enterotoxic, suggesting by this the existence of a combination of genes. In parallel, a specific rpoB-based qPCR was developed to identify C. neonatale. We found that, C. neonatale was more prevalent in NEC than in controls. Although co-identified in association with C. butyricum. C. neonatale clones were distinguished especially in strains isolated from the same hospital. Regarding to DF infection, SNPs were identified within S. aureus and E. coli genomes, especially in virulent genes.

A produtividade florestal é definida pelo ambiente de produção em que o povoamento está inserido, pela implantação de materiais genéticos superiores e pelo seu correto manejo silvicultural. A correta prescrição de recomendações silviculturais e a execução das operações de maneira padronizada tem por objetivo eliminar ou minimizar as restrições ao crescimento para cada árvore. Apesar das melhorias silviculturais que ocorreram nas últimas décadas, sabe-se que ainda é possível ganhos em produtividade através do monitoramento, detecção e correção precoce de desvios na qualidade silvicultural. Assim, o objetivo deste trabalho foi investigar um índice para o monitoramento da qualidade silvicultural utilizando uma rede de inventário florestal. Para isto, foram realizadas três etapas de trabalho: i) Definição de índices de uniformidade das variáveis dendrométricas; ii) Padronização dos chamados intervalos ótimos de uniformidade (IOU) e iii) Validação da metodologia via sua aplicação numa rede de inventário florestal para um único clone comercial. Na primeira etapa utilizaram-se três ensaios da rede BEPP (Brasil Eucalyptus Produtividade Potencial) com diferentes níveis de produtividade para estabelecer os índices adequados em caracterizar a uniformidade silvicultural. Na segunda etapa utilizaram-se cinco testes clonais de Eucalyptus no Estado de São Paulo para validar o conceito de Intervalo Ótimo de Uniformidade. Na última etapa, os índices e conceito do IOU foram aplicados em escala comercial em uma rede de parcelas de inventário florestal instalada em 12.000 hectares de plantios clonais de Eucalyptus no Nordeste do Estado de São Paulo, com cerca de 2 anos de idade, e plantados no período de 1995 a 2009. Na fase de definição, o índice que representa a porcentagem do volume total existente em 50% das menores árvores plantadas (o que inclui as falhas de plantio) (PV50) se mostrou como o que melhor se adequou à proposta do trabalho, pois possui limites finitos (50% a 0%), contempla as falhas de plantio como parte do índice de uniformidade do povoamento e, indiretamente, representa a distribuição de classes de crescimento. As uniformidades aos 5, 9, 12 e 24 meses foram fortemente correlacionadas à uniformidade aos 6 anos (r² > 0,74) mostrando a possibilidade de monitoramento precoce para detecção de desvios de qualidade na silvicultura. Além disso, o índice PV50 inicial foi altamente correlacionado com a produtividade final (p < 0,001). Na fase de padronização, não se detectou diferença estatística (Tukey, 5%) do PV50 entre os testes clonais a despeito de suas distintas produtividades, evidenciando que o índice de uniformidade pode ser generalizado, independentemente da produtividade do sítio. O IOU do PV50 foi de 34 a 50 %, ou seja, parcelas amostrais que possuírem o PV50 dentro deste intervalo podem ser consideradas satisfatoriamente uniformes. Na etapa de validação, quando o conceito foi aplicado em escala comercial observou-se uma forte evolução temporal do PV50. Nos plantios realizados em 1995 a média do índice foi de 29% e elevou-se para 42% em 2009. O percentual de parcelas dentro do intervalo ótimo de uniformidade tendeu claramente a se elevar ao longo deste período, devendo estar relacionada com as melhorias nas principais operações silviculturais e seu monitoramento através do controle de qualidade. / Forest productivity is defined by the environment the population is inserted, by the implementation of genetically superior material and by the correct silvicultural management applied at this material. The correct prescription of technical recommendations and the execution of those operations according to acceptable quality standard are intended to eliminate or minimized the growth constrains. Despite the silvicultural improvements that have occurred in recent decades, it is known that it is still possible to obtain gains in productivity through monitoring, early detection and correction of deviations in the silviculture. The objective of this study was to investigate an index for monitoring the silvicultural quality using forest inventory networks. For this, there were three stages of work: i) Definition of uniformity indexes of dendrometric variables; ii) Standardization of so-called optimal range of uniformity (ORU) and validation of the methodology through its application in a network of forest inventory for a single commercial clone. In the first stage we used three tests of the network BEPP (Brazil Eucalyptus Potential Productivity) with different levels of productivity to establish the appropriate indexes to characterize the silvicultural uniformity. In the second stage we used five clonal test of Eucalyptus in Sao Paulo state to validate the concept of Optimum Range of Uniformity. In the last step, the indexes and the concept of IOU were applied on a commercial scale in a network of forest inventory plots installed in 12.000 hectares of Eucalyptus clonal plantations in northeastern of São Paulo state, with about 2 years old, and planted within 1995 to 2009. In the definition phase, the index that represents the percentage of the total volume of 50% of smaller trees planted (which includes the planting holes) (PV50) was shown as the best adapted to the purpose of this study because it has finite limits (50% to 0%), includes the holes of planting and indirectly represents the distribution of classes of growth. The uniformity at 5, 9, 12 and 24 months were strongly correlated to uniformity to 6 years (r² > 0.74) showing the possibility of early monitoring for detection of quality deviations in forestry. Furthermore, the initial rate PV50 was highly correlated with the final yield (p <0.001). At the stage of standardization, there was no statistical difference (Tukey, 5%) of PV50 among clonal tests despite their different yields, showing that the uniformity index can be generalized, irrespective of the productivity of the site. The ORU of PV50 was from 37 to 50%, i.e., sample plots which have the PV50 within this range can be considered satisfactory \"uniform\". In the validation phase, when the concept was applied on a commercial scale there was a strong temporal evolution of PV50. In the plantations made in 1995 the average of PV50 was 29% and increased to 42% in 2009. The percentage of plots within the optimal range of uniformity clearly tended to rise over this period and could be related to major improvements in forestry operations and their monitoring through quality control.

Taxonomic delimitation of rare species is vital for accurate assessments of diversity and for their conservation. Cycladenia humilis, the sole species of Cycladenia, is an enigmatic perennial widely dispersed across the western United States. Within this species there are three currently recognized varieties: C. humilis var. humilis in Northern California, C. humilis var. venusta in Southern California, and C. humilis var. jonesii in Utah and Northern Arizona. Some populations occur geographically in areas between the typical distribution of each variety and the presently accepted taxonomy inadequately addresses these populations. Using five nDNA regions, we seek to clarify relationships between current varieties and assess the pattern of variation throughout the species. Analyses including K-means clustering, principle component analysis, fields for recombination, AMOVA, and ecological niche modeling were applied. Results indicate significant genetic structure between varieties and supports recognition of C. jonesii at the species level as distinct from C. humilis. Well defined intraspecific groupings are evident in the data, with evidence supporting the recognition of an additional variety in C. humilis, and two varieties in C. jonesii. Haplotype diversity and relationships between metapopulation clusters inform conservation efforts regarding diversity within Cycladenia and offer insights into the historical demography of this genus.