Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados / Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)

A indústria sucroenérgetica gera grande quantidade de resíduos, sendo a torta de filtro e as cinzas os principais resíduos sólidos. Uma das tecnologias desenvolvidas para o manejo destes resíduos é a compostagem. A aplicação do composto tem mostrado efeitos positivos na cultura da cana-de-açúcar no que diz respeito à fertilização fosfatada. Os objetivos do presente trabalho foram: I. Observar o comportamento das comunidades bacterianas durante a compostagem e o efeito da inoculação de estirpes bacterianas mobilizadoras de fósforo sobre a disponibilidade de fósforo no composto final; II. Avaliar o efeito da aplicação de diferentes fontes de P e de bactérias mobilizadoras de fósforo no desenvolvimento de plantas de cana-de-açúcar e o efeito sobre as comunidades bacterianas do solo. Nesse sentido, pilhas de compostagem foram instaladas na Usina São José da Estiva em Novo Horizonte, SP. Os tratamentos consistiram de pilhas com e sem rocha fosfática; e pilhas com e sem a inoculação das estirpes Pseudomonas aeruginosa PSBR12 e Bacillus sp. BACBR1. Amostragens foram realizadas quinzenalmente durante 60 dias. Adicionalmente determinou-se a atividade enzimática, e parâmetros químicos do composto. A comunidade bacteriana foi acessada por meio da técnica independente de cultivo T-RFLP (restriction fragment length polymorphism) e sequenciado por meio da plataforma de nova geração MiSeqTM System (Illumina). Com o objetivo de avaliar o efeito do composto e da inoculação de bactérias mobilizadoras de P no desenvolvimento da cana-de-açúcar foi instalado um experimento em casa de vegetação com plantas de cana de açúcar, utilizando-se composto, rocha fosfática e super fosfato triplo como fontes de P, além da inoculação dos seguintes: Inoculante 1: Pseudomonas sp. PSBR10, Azotobacter sp. AZTBR19, Rhizobium sp. RIZBR01; e o Inoculante 2: Bacillus simplex BACBR04, Bacillus sp. BACBR06, Rhizobium sp. RIZBR01. O experimento foi conduzido durante 75 dias. Ao fim do experimento foram analisados os seguintes parâmetros: biomassa seca, acúmulo de P, N, e K na parte aérea, e fosfatases no solo. A estrutura das comunidades bacterianas no solo foram avaliadas por meio do sequenciamento na plataforma Illumina. A aplicação de bactérias mobilizadoras de P durante a compostagem diminuiu o P ligado ao Ca. A mudança nas comunidades bacterianas durante a compostagem foi temporal, no início dominaram membros da ordem Lactobacillales, após 15 dias as ordens Bacillales e Clostridiales passaram a dominar o processo. As comunidades bacterianas são influenciadas principalmente pelos parâmetros pH, temperatura e umidade. Quanto ao experimento em casa de vegetação, o uso dos inoculantes (principalmente o inoculante 2) aumentou o acúmulo de P, N e K na parte aérea nos tratamentos que receberam composto e super fosfato triplo como fonte de P. A aplicação dos inoculantes e a adição do composto modificou a estrutura das comunidades bacterianas do solo; essa alteração quando os inoculantes são aplicados pode estar relacionada com o incremento no acúmulo de nutrientes. O uso de bactérias mobilizadoras de P é uma tecnologia potencial para o uso na agricultura, tanto na compostagem de resíduos da indústria sucroenergética com rocha fosfática, como no aumento da eficiência da fertilização fosfatada na cana-de-açúcar. / Sugarcane industry generates large amounts of waste, filter cake and ashes being the main solid wastes. One of the technologies developed for the management of this waste is composting. The application of compost has shown positive effects on the sugarcane culture with regard to phosphorus fertilization. The objectives of this study are: I. To study the diversity of bacterial communities during composting and the effect of inoculation of phosphorus mobilizing bacterial strains on the phosphorus availability in the final compost; II. To evaluate and compare the effects of the application of different sources of phosphorus and P mobilizing bacteria on the development of sugarcane and the effect on soil bacterial communities. Therefore, compost piles were installed at Usina São José da Estiva in Novo Horizonte, Brazil. Treatments consisted of piles with and without rock phosphate and with and without inoculation of Pseudomonas aeruginosa PSBR12 and Bacillus sp. BACBR1 strains. Samples were taken every two weeks for 60 days. In addition, the enzymatic and chemical composition of the compost was determined. The bacterial community was assessed through T-RFLP (restriction fragment length polymorphism) and was sequenced through the new generation platform MiSeqTM System (Illumina). The abundance of bacteria was evaluated by qPCR. In order to evaluate the effect of compost and of inoculating P mobilizing bacteria on sugarcane development, a greenhouse experiment was installed with sugarcane using compost, rock phosphate and triple superphosphate as P sources, besides inoculations, as follows: Inoculant 1: Pseudomonas sp. (PSBR10) Azotobacter sp. (AZTBR19), Rhizobium sp. (RIZBR01); and Inoculant 2: Bacillus simplex (BACBR04), Bacillus sp. (BACBR06), Rhizobium sp. (RIZBR01). The experiment was conducted during 75 days. At the end of the experiment the following parameters were analyzed: dry plant biomass, accumulation of P, N, and K in the shoot, and phosphatases in soil. Bacterial soil communities were sequenced through the new generation Illumina. The application of P mobilizing bacteria during composting decreased the Ca-linked P. Changes of the bacterial communities during composting were temporal. In the beginning, members of the order Lactobacillales were dominant and after 15 days a succession of bacterial communities occurred, when Bacillales and Clostridiales began to dominate. Bacterial communities are mostly influenced by the parameters pH, temperature and humidity. Moreover, in the greenhouse experiment, the use of inoculants (mainly inoculant 2) increased the accumulation of P, N and K in shoots in the treatments that received compost and triple superphosphate as P source. Inoculant application and compost addition modified soil bacterial community structures. Changes in the soil microbiome when inoculant was applied may be related to the increase in nutrients accumulation. The use of P mobilizing bacteria is a potential technology for use in agriculture, when composting waste from the sugarcane industry with rock phosphate or when increasing P fertilization efficiency in sugarcane in the field.

A indústria sucroenérgetica gera grande quantidade de resíduos, sendo a torta de filtro e as cinzas os principais resíduos sólidos. Uma das tecnologias desenvolvidas para o manejo destes resíduos é a compostagem. A aplicação do composto tem mostrado efeitos positivos na cultura da cana-de-açúcar no que diz respeito à fertilização fosfatada. Os objetivos do presente trabalho foram: I. Observar o comportamento das comunidades bacterianas durante a compostagem e o efeito da inoculação de estirpes bacterianas mobilizadoras de fósforo sobre a disponibilidade de fósforo no composto final; II. Avaliar o efeito da aplicação de diferentes fontes de P e de bactérias mobilizadoras de fósforo no desenvolvimento de plantas de cana-de-açúcar e o efeito sobre as comunidades bacterianas do solo. Nesse sentido, pilhas de compostagem foram instaladas na Usina São José da Estiva em Novo Horizonte, SP. Os tratamentos consistiram de pilhas com e sem rocha fosfática; e pilhas com e sem a inoculação das estirpes Pseudomonas aeruginosa PSBR12 e Bacillus sp. BACBR1. Amostragens foram realizadas quinzenalmente durante 60 dias. Adicionalmente determinou-se a atividade enzimática, e parâmetros químicos do composto. A comunidade bacteriana foi acessada por meio da técnica independente de cultivo T-RFLP (restriction fragment length polymorphism) e sequenciado por meio da plataforma de nova geração MiSeqTM System (Illumina). Com o objetivo de avaliar o efeito do composto e da inoculação de bactérias mobilizadoras de P no desenvolvimento da cana-de-açúcar foi instalado um experimento em casa de vegetação com plantas de cana de açúcar, utilizando-se composto, rocha fosfática e super fosfato triplo como fontes de P, além da inoculação dos seguintes: Inoculante 1: Pseudomonas sp. PSBR10, Azotobacter sp. AZTBR19, Rhizobium sp. RIZBR01; e o Inoculante 2: Bacillus simplex BACBR04, Bacillus sp. BACBR06, Rhizobium sp. RIZBR01. O experimento foi conduzido durante 75 dias. Ao fim do experimento foram analisados os seguintes parâmetros: biomassa seca, acúmulo de P, N, e K na parte aérea, e fosfatases no solo. A estrutura das comunidades bacterianas no solo foram avaliadas por meio do sequenciamento na plataforma Illumina. A aplicação de bactérias mobilizadoras de P durante a compostagem diminuiu o P ligado ao Ca. A mudança nas comunidades bacterianas durante a compostagem foi temporal, no início dominaram membros da ordem Lactobacillales, após 15 dias as ordens Bacillales e Clostridiales passaram a dominar o processo. As comunidades bacterianas são influenciadas principalmente pelos parâmetros pH, temperatura e umidade. Quanto ao experimento em casa de vegetação, o uso dos inoculantes (principalmente o inoculante 2) aumentou o acúmulo de P, N e K na parte aérea nos tratamentos que receberam composto e super fosfato triplo como fonte de P. A aplicação dos inoculantes e a adição do composto modificou a estrutura das comunidades bacterianas do solo; essa alteração quando os inoculantes são aplicados pode estar relacionada com o incremento no acúmulo de nutrientes. O uso de bactérias mobilizadoras de P é uma tecnologia potencial para o uso na agricultura, tanto na compostagem de resíduos da indústria sucroenergética com rocha fosfática, como no aumento da eficiência da fertilização fosfatada na cana-de-açúcar. / Sugarcane industry generates large amounts of waste, filter cake and ashes being the main solid wastes. One of the technologies developed for the management of this waste is composting. The application of compost has shown positive effects on the sugarcane culture with regard to phosphorus fertilization. The objectives of this study are: I. To study the diversity of bacterial communities during composting and the effect of inoculation of phosphorus mobilizing bacterial strains on the phosphorus availability in the final compost; II. To evaluate and compare the effects of the application of different sources of phosphorus and P mobilizing bacteria on the development of sugarcane and the effect on soil bacterial communities. Therefore, compost piles were installed at Usina São José da Estiva in Novo Horizonte, Brazil. Treatments consisted of piles with and without rock phosphate and with and without inoculation of Pseudomonas aeruginosa PSBR12 and Bacillus sp. BACBR1 strains. Samples were taken every two weeks for 60 days. In addition, the enzymatic and chemical composition of the compost was determined. The bacterial community was assessed through T-RFLP (restriction fragment length polymorphism) and was sequenced through the new generation platform MiSeqTM System (Illumina). The abundance of bacteria was evaluated by qPCR. In order to evaluate the effect of compost and of inoculating P mobilizing bacteria on sugarcane development, a greenhouse experiment was installed with sugarcane using compost, rock phosphate and triple superphosphate as P sources, besides inoculations, as follows: Inoculant 1: Pseudomonas sp. (PSBR10) Azotobacter sp. (AZTBR19), Rhizobium sp. (RIZBR01); and Inoculant 2: Bacillus simplex (BACBR04), Bacillus sp. (BACBR06), Rhizobium sp. (RIZBR01). The experiment was conducted during 75 days. At the end of the experiment the following parameters were analyzed: dry plant biomass, accumulation of P, N, and K in the shoot, and phosphatases in soil. Bacterial soil communities were sequenced through the new generation Illumina. The application of P mobilizing bacteria during composting decreased the Ca-linked P. Changes of the bacterial communities during composting were temporal. In the beginning, members of the order Lactobacillales were dominant and after 15 days a succession of bacterial communities occurred, when Bacillales and Clostridiales began to dominate. Bacterial communities are mostly influenced by the parameters pH, temperature and humidity. Moreover, in the greenhouse experiment, the use of inoculants (mainly inoculant 2) increased the accumulation of P, N and K in shoots in the treatments that received compost and triple superphosphate as P source. Inoculant application and compost addition modified soil bacterial community structures. Changes in the soil microbiome when inoculant was applied may be related to the increase in nutrients accumulation. The use of P mobilizing bacteria is a potential technology for use in agriculture, when composting waste from the sugarcane industry with rock phosphate or when increasing P fertilization efficiency in sugarcane in the field.

No description available.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 µm²) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

Triops cancriformis (Crustacea: Notostraca) occurs in ephemeral habitats like rain pools or floodplain pools distributed over a large geographical range. The named habitats are disturbed by human impacts and, consequently, T. cancriformis is endangered throughout its distribution range. In the present thesis the populated habitats and threats are characterised and further morphological and genetic variations detected among and within European populations are reported. On the basis of recent investigations it is shown that T. cancriformis subspecies separation is hampered by an individual variability which points to the necessity of species revision. The analysis of mitochondrial gene sequence data suggests that the species has colonised most of Europe very recently. The advantage of a complex reproductive strategy in T. cancriformis in this process is discussed. The population structure resolved with nuclear DNA markers highlights that there is low allelic diversity among and within populations compared to other Branchiopoda (Daphnia). By means of the present study it can be shown that habitat conservation is most important to protect T. cancriformis.

Since the human inclination to estimate and trace natural diversity, usable species definitions as well as taxonomical systems are required. As a consequence, the first proposed classification schemes assigned the filamentous and parenchymatous taxa to the green algal order Chaetophorales sensu Wille. The introduction of ultrastructural and molecular methods provided novel insight into algal evolution and generated taxonomic revisions based on phylogenetic inference. However, until now, the number of molecular phylogenetic studies focusing on the Chaetophorales s.s. is surprisingly low. To enhance knowledge about phylogenetic relationships among taxa within the order, the nuclear?encoded SSU rDNA sequences from 30 strains covering all three chaetophoralean families have been investigated. All revealed monophyletic groupings were further screened for molecular non-homoplasious synapomorphies within the Viridiplantae. To address the question of the correspondence between morphological characters traditionally used for taxonomical delimitation of the Chaetophorales and the tree topology favored by molecular data, the list of morphological/ ultrastructural/ecological characters was elaborated and further analyzed. In addition, to obtain a close-up view into the evolution of Compensatory Base Changes (CBCs) of the second internal transcribed spacer (ITS2) which is currently often used to delimit putative biological species, 86 newly obtained/published sequences of ITS2 for five families of the order Ulvales were analyzed. Furthermore, a detailed comparative study of all ITS2 substitutions has been done. Subsequently all revealed CBCs and hemi- CBCs have been mapped upon the ITS2 phylogenetic tree topology. Finally, CBCs/hCBCs taxonomic inference in the Ulvales has been discussed.

This thesis reviews knowledge about diversity of blood parasites of fish. Blood smears of fish used in this study were obtained in Billefjorden (Svalbard archipelago, Arctic). Desseria myoxocephali, the type species of Desseria, is the only one parasite that was found in the smears. A partial 18S rDNA sequence of D. myoxocephali was prepared and phylogenetic analyses were computed. D. myoxocephali forms a lineage together with Dactylosoma ranarum and Babesiosoma stableri within adeleorinid clade.

Due to their phylogenetic position, Psychidae play an important role in the investigation of the W chromosome origin in Lepidoptera. Several species of Psychidae were tested for the presence of sex-chromatin and investigated via comparative genomic hybridization. Furthermore, odd chromosome numbers and a Z univalent were observed in females. Overall, this study brings tangible evidence for the absence of the W chromosome in Psychidae, thus contributes to complex knowledge of the W chromosome evolution. In addition, karyotypes of the given species were analyzed using 18S rDNA and histone H3 probes. The results indicate relative stability of their karyotypes.

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados / Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)