Bridging environmental conflicts with social metabolism : forestry expansion and socioeconomic change

Dissertação apresentada para obtenção do Grau de Doutor em Ciências do Ambiente, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Clonal types and antimicrobial resistance profiles of methicillin-resistant Staphylococcus aureus isolates from hospitals in south Brazil

In the present study were evaluated the DNA macrorestriction profile and SCCmec types for nine multi-resistant MRSA selected. Also antimicrobial susceptibility testing by disk diffusion method was evaluated for 68 MRSA isolates against 12 antimicrobial agents. The isolates were recovered from blood culture collected from hospitalized patients in three hospitals of Porto Alegre, Brazil. PFGE and PCR for mecA and SCCmec I, II, III, IV types genes were done on selected nine isolates with susceptibility only to vancomycin, teicoplanin and linezolid. Two clone profiles, with five subtypes, were demonstrated among multi-resistant MRSA analyzed. Eight isolates showed harbor SCCmec type III and one isolate was not typeable. The knowledge of SCCmec type, clone and antimicrobial profiles among S. aureus is essential mainly to prevention and control of dissemination of the antimicrobial resistance.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

Web Processing Services for Forestry and Environmental Applications

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Optimal choice between even- and uneven-aged forestry: the case of non-industrial private forest owners

An infinite-horizon discrete time model with multiple size-class structures using a transition matrix is built to assess optimal harvesting schedules in the context of Non-Industrial Private Forest (NIPF) owners. Three model specifications accounting for forest income, financial return on an asset and amenity valuations are considered. Numerical simulations suggest uneven-aged forest management where a rational forest owner adapts her or his forest policy by influencing the regeneration of trees or adjusting consumption dynamics depending on subjective time preference and market return rate dynamics on the financial asset. Moreover she or he does not value significantly non-market benefits captured by amenity valuations relatively to forest income.

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Methicillin-susceptible Staphylococcus aureus clonal complex 398: high prevalence and geographical heterogeneity in bone and joint infection and nasal carriage.

The prevalence of clonal complex (CC) 398 methicillin-susceptible Staphylococcus aureus (MSSA) was unexpectedly high among bone and joint infections (BJIs) and nasal-colonizing isolates in France, with surprising geographical heterogeneity. With none of the major, most-known staphylococcal virulence genes, MSSA CC398 BJI was associated with lower biological inflammatory syndrome and lower treatment failure rates.

Biological comparison between three clones of Trypanosoma cruzi and the strain of origin (Bolivia) with reference to clonal evolution studies

After isolating three clones of Trypanasoma cruzi (Bolivia), we first characterized them according to parasitaemia, pleomorphism and virulence, and then histopathologically. The study's interest lies on the hypothesis that clonal evolution of T. cruzi has a major impact on biologically relevant properties of this parasite. Data obtained from the studies of parasitaemia, pleomorphism and virulence showed no differences between the groups studied. As a final point, the histopathological study shows us a muscular tissue tropism both in clones and in their mother strain (Bolivia). In this paper, we conclude that Bolivia strain and clones isolated from it, pertaining to the same major clone share similar biological properties.

Clonal heterogeneity and chromosomal instability at disease presentation in high hyperdiploid acute lymphoblastic leukemia.

Although aneuploidy has many possible causes, it often results from underlying chromosomal instability (CIN) leading to an unstable karyotype with cell-to-cell variation and multiple subclones. To test for the presence of CIN in high hyperdiploid acute lymphoblastic leukemia (HeH ALL) at diagnosis, we investigated 20 patients (10 HeH ALL and 10 non-HeH ALL), using automated four-color interphase fluorescence in situ hybridization (I-FISH) with centromeric probes for chromosomes 4, 6, 10, and 17. In HeH ALL, the proportion of abnormal cells ranged from 36.3% to 92.4%, and a variety of aneuploid populations were identified. Compared with conventional cytogenetics, I-FISH revealed numerous additional clones, some of them very small. To investigate the nature and origin of this clonal heterogeneity, we determined average numerical CIN values for all four chromosomes together and for each chromosome and patient group. The CIN values in HeH ALL were relatively high (range, 22.2-44.7%), compared with those in non-HeH ALL (3.2-6.4%), thus accounting for the presence of numerical CIN in HeH ALL at diagnosis. We conclude that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by I-FISH in HeH ALL at presentation, which would corroborate the potential role of CIN in tumor pathogenesis.

Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain rreaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Systematic survey of clonal complexity in tuberculosis at a populational level and detailed characterization of the isolates involved.

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Significant genetic and phenotypic changes arising from clonal growth of a single spore of an arbuscular mycorrhizal fungus over multiple generations.

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum.

Simultaneous detection of aneuploidies for chromosomes 4, 6, 10 and 17 by automated four colour I-FISH in high hyperdiploid acute lymphoblastic leukemia : diagnostic assessment, clonal heterogeneity and chromosomal instability in adults

SummarySimultaneous detection of aneuploidies for chromosomes 4, 6,10 and 17 by automated four color l-FISH in high hyperdiploid acute lymphoblastic leukemia: diagnostic assessment, clonal heterogeneity and chromosomal instability in adultsAnna Talamo BlandinService de Génétique Médicale, Unité de Cytogénétique du Cancer, CHUVAcute lymphoblastic leukemia (ALL) is a malignant hemopathy characterized by the accumulation of the immature lymphoid cells in the bone marrow and, most often, in the peripheral blood. ALL is a heterogeneous disease with distinct biological and prognostic entities. At diagnosis, cytogenetic and molecular findings constitute important and independent prognostic factors. High hyperdiploidy with 51-67 chromosomes (HeH), one of the largest cytogenetic subsets of ALL, in childhood particularly, is generally associated with a relatively favorable outcome. Chromosome gain is nonrandom, extracopies of some chromosome occurring more frequently than those of others. Concurrent presence of trisomy for chromosomes 4, 10 and 17 confers an especially good prognosis. The first aim of our work was to develop an automated four color interphase fluorescence in situ hybridization (l-FISH) methodology and to assess its ability to detect concurrent aneuploidies 4, 6, 10 and 17 in 10 ALL patients. Various combinations of aneuploidies were identified. All clones detected by conventional cytogenetics were also observed by l-FISH. However, in all patients, l-FISH revealed numerous additional abnormal clones, leading to a high level of clonal heterogeneity. Our second aim has been to investigate the nature and origin of this clonal heterogeneity and to test for the presence of chromosome instability (CIN) in HeH ALL at initial presentation. Ten HeH ALL and 10 non-HeH ALL patients were analysed by four colour l-FISH and numerical CIN values were determined for all four chromosomes together and for each chromosome and patient group, an original approach in ALL. CIN values in HeH ALL proved to be much higher than#iose in non-HeH ALL, suggesting that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by l-FISH. Our third aim has been to study the evolution of these cytogenetic features during the course of the disease in 10 HeH ALL patients. Clonal heterogeneity was also observed again during disease progression, particularly at relapse. Clones detected at initial presentation generally reappeared in relapse, in most cases with newly generated ones. A significant correlation between the number of abnormal clones and CIN suggested that the higher the instability, the larger the number of abnormal clones. Whereas clonal heterogeneity and its evolution most probably result from underlying chromosome instability, operating processes remain conjectural.RésuméLa leucémie lymphoblastique aiguë (LLA) est une hémopathie maligne qui résulte de l'accumulationde cellules lymphoïdes immatures dans la moelle osseuse, et, le plus souvent, dans le sangpériphérique également. La LLA est une affection hétérogène au sein de laquelle se distinguentplusieurs entités biologiques et pronostiques. Les données cytogénétiques et moléculaires font partieintégrante du diagnostic et jouent un rôle essentiel dans l'évaluation du pronostic. L'hyperdiploïdieélevée à 51-­67 chromosomes (HeH), relativement fréquente, en particulier chez l'enfant, s'associe àun pronostic favorable. Le gain de chromosomes ne relève pas du hasard, certains chromosomesétant plus fréquemment impliqués que d'autres. La présence simultanée des trisomies 4, 6, et 17s'associe à un pronostic particulièrement bon. Le premier but du travail a été de développer uneméthode d'analyse automatique par hybridation in situ fluorescente interphasique (I-­FISH) à 4couleurs et de tester sa capacité à identifier la présence simultanée d'aneuploïdies 4, 6, 10 et 17 dans10 cas de LLA. Différentes combinaisons d'aneuploïdies ont été identifiées. Tous les clones détectéspar cytogénétique conventionnelle l'ont été par I-­FISH. Or, chez tous les patients, l'I-­FISH a révélé denombreux clones anormaux additionnels générant un degré élevé d'hétérogénéité clonale. Notredeuxième but a été d'investiguer la nature et l'origine de cette hétérogénéité et de tester la présenced'instabilité chromosomique (CIN) chez les patients avec une LLA HeH en presentation initiale. DixLLA HeH et 10 LLA non-­HeH ont été analysées par I-­FISH et les valeurs de CIN numérique ont étédéterminées pour les 4 chromosomes ensemble et pour chaque chromosome et groupe de patients,approche originale dans la LLA. Ces valeurs étant beaucoup plus élevées dans la LLA HeH que dansla LLA non-­HeH, elles favorisent l'hypothèse selon laquelle la CIN serait à l'origine de l'hétérogénéitéclonale révélée par I-­FISH. Le troisième but de notre travail a été d'étudier l'évolution de cescaractéristiques cytogénétiques au cours de la maladie dans 10 cas de LLA HeH. L'hétérogénéitéclonale a été retrouvée lors de la progression de la maladie, en particulier en rechute, où les clonesanormaux détectés en présentation initiale réapparaissent, généralement accompagnés de clonesnouveaux. La corrélation existant entre nombre de clones anormaux et valeurs de CIN suggère queplus l'instabilité est élevée, plus le nombre de clones anormaux est grand. Bien que l'hétérogénéitéclonale et son évolution résultent très probablement de l'instabilité chromosomique, les processus àl'oeuvre ne sont pas entièrement élucidés.