Addition of nanopacticles in a base solvent leads to suspensions with enhanced physiochemical properties, compared to base solvent. This new type of suspensions is called nanofluids, with applications ranging from biomedicine to automotives. As a consequence extensive research is being conducted in the field, in particular, on the evaporation of these fluids as it leads to well-defined and highly ordered coffee-rings. However, the exact physics governing this phenomenon remain elusive. The goal of this experimental investigation is to elucidate how various parameters affect the progression of nanofluid coffee-stain formation. Examination of the coffee-ring structuring, produced by the free evaporation of sessile droplets containing nanoparticles, revealed an unexpected, disordered region at the exterior edge of the ring. A self-assembly mechanism with two components, particle velocity and wedge constraints, was proposed to describe the deposition of particles at contact lines of evaporating drops. Environmental pressure was used as a method to control particle crystallinity in the coffee-rings. Essentially, evaporation rate and pressure were found to be inversely proportional. Macroscopically, lowering pressure led to a transition from “stick-slip” to constant pinning. Nanoscopically, lowering pressure promoted crystallinity. Findings supported the proposed, in this thesis, particle self-assembly mechanism. Particle aspect ratio and flexibility were subsequently examined. Pinning strength was found to be a function of particle aspect ratio and rigidity, leading to constant pinning. The proposed, in this thesis, particle self-assembly mechanism was found to be applicable to a variety of aspect ratios and flexibilities. Lastly, particulate crystals grew following different pathways depending on particle flexibility.

O vírus de Leishmania 1-4 ( do inglês Leishmania RNA virus 1-4 ou LRV1-4) é um vírus da família Totiviridae, e que possui capsídeo icosaédrico e RNA dupla-fita que codifica duas proteínas (proteína capsidial e RNA polimerase). Dados recentes indicam o envolvimento do LRV1-4 na patogênese de Leishmania no hospedeiro humano, tornando seu estudo de fundamental importância para o entendimento dessa doença e de seu papel na relação parasito-hospedeiro. Há relatos sobre a purificação do vírus a partir do seu hospedeiro natural (Leishmania guyanensis) e a partir de sistemas de expressão heteróloga. Este trabalho tem por objetivo estabelecer os métodos de purificação para posteriores estudos estruturais por Microscopia Eletrônica de Transmissão por Contraste Negativo (NS-TEM) e por Crio-Microscopia Eletrônica (Cryo-EM). Os estudos aqui propostos irão permitir a construção de um modelo estrutural do capsídeo do LRV1-4 e sua identificação correta dentre os totivírus. Além das contribuições ao conhecimento da biologia/patogenia do LRV1-4 este estudo representa a primeira caracterização estrutural de um capsídeo viral realizada no Brasil, e assim um avanço importante para a área de virologia e biologia estrutural no pais. Foram realizadas ultracentrifugações biológicas, utilizando gradientes de sacarose, para a purificação do vírus a partir do extrato celular de L. guyanensis. As frações que apresentaram RNA viral foram analisadas por Microscopia Eletrônica de Transmissão (Campinas LNNano CNPEM). Além disso, foram realizadas tentativas de expressar a proteína do capsídeo (ORF2) em Leishmania tarentolae e Escherichia coli. Foram também realizados esforços para a obtenção de anticorpos a partir de peptídeos sintetizados após análise computacional da sequência de aminoácidos da proteína do capsídeo. As amostras obtidas a partir do hospedeiro natural do vírus se apresentaram heterogêneas quando analisadas por NS-TEM, de modo que não foi possível a realização de uma análise estrutural. Porém, a presença de partículas do tamanho esperado para o vírus em amostras em que foi detectado o RNA viral indicam que são necessários esforços para obtenção de uma amostra de maior pureza e homogênea. Além disso, não foi possível obter a proteína do capsídeo nos sistemas de expressão heteróloga. A presença de 25 resíduos de cisteína pode estar levando a proteína à degradação rápida em bactéria. Os experimentos de expressão em células de Leishmania ainda não foram conclusivos. Foi obtido um anticorpo anti-peptídeo que reconhece a proteína do capsídeo, tornando possíveis experimentos como imunolocalização e imunoprecipitação do vírus. / The Leishmania RNA virus 1-4 (LRV1-4) belongs to the Totiviridae family. It has an icosahedral capsid and a double-strand RNA encoding two proteins (capsid protein and RNA polymerase). Recent data indicate the involvement of LRV1-4 in the pathogenesis of Leishmania in the human host, making their study of fundamental importance for the understanding of this disease and its role in host-parasite relationship. There are reports on the purification of the virus from its natural host (Leishmania guyanensis) and from the same heterologous expression systems such as Escherichia coli.This work aims to stablish purification methods for further structural studies by Negative Stain Transmission Microscopy (NS-TEM) and Cryo-Electron Microscopy (Cryo-EM). The studies proposed here will allow the construction of a structural model of the coat protein of LRV1-4 and their correct identification amongst the Totiviridae. In addition to the contributions to the knowledge of the biology and pathogenesis of LRV1-4, this study represents the first structural characterization of a viral capsid held in Brazil and thus an important step forward for the field of virology and structural biology in the country. Sucrose for virus purification gradients were performed from the cell extract of L. guyanensis. Fractions that showed viral RNA were analised by Transmission Electron Microscopy (Campinas LNNano - CNPEM). Furthermore, attempts have been made to express the capsid protein (ORF2) in Leishmania tarentolae and Escherichia coli. There has also been made efforts to obtain antibodies from peptides synthesized accordingly to the computer analysis of the amino acid sequence of the capsid protein. The samples obtained from the natural host of the virus showed a heterogeneous distribution of particles when examined by NS-TEM so that it was not possible to perform a structural analysis. However, the presence of particles of the size expected for the virus particles in samples where the viral RNA was detected indicate that efforts are necessary to obtain a more homogeneous and pure sample. Moreover, it was not possible to obtain the capsid protein in heterologous expression systems. The presence of 25 cysteine residues could have led to the protein rapid degradation in the bacteria host. The expression experiments in Leishmania cells were not yet conclusive. It was also possible to obtain an anti-peptide antibody recognizing the capsid protein, enabling immunoprecipitation and immunolocalization experiments.

The mountain pine beetle is associated with a diverse array of fungi. Grosmannia clavigera is the most pathogenic of these fungi. A comparison was made between two methods that have been used to assess fungal pathogenicity. Results were similar for older trees inoculated with G. clavigera using either the alternating flap technique or cork borer method. Using the cork borer method, younger lodgepole pine trees were inoculated with five different isolates of G. clavigera. After a 48 week incubation period, isolates ATCC 18086, B5 and H55 had induced stronger pathogenic indicators compared to isolates KW 1407 and B20. After a 7 week incubation period, only isolate ATCC 18086 had induced stronger pathogenic indicators. Usually, this isolate grew faster at lower temperatures and in a low oxygen environment. Isolate KW 1407 consistently produced milder pathogenic indicators during both incubation periods. Among the non-pathogenic fungal associates of the mountain pine beetle, Ceratocystiopsis minuta may be considered the most important because it is the type species for the genus Ceratocystiopsis. The history of this genus is complicated because no physical specimen exists for C. minuta. The phylogeny of the genus Ceratocystiopsis was evaluated. Many isolates of C. minuta were assessed as potential epitypes. Several isolates of C. minuta from previous work were shown to be misidentified. C. minuta isolate CBS 116796 is recommended for future genetic work within the genus Ceratocystiopsis. For morphological work, using measurements from the literature is recommended since CBS 116796 did not produce fruiting bodies.

The mountain pine beetle is associated with a diverse array of fungi. Grosmannia clavigera is the most pathogenic of these fungi. A comparison was made between two methods that have been used to assess fungal pathogenicity. Results were similar for older trees inoculated with G. clavigera using either the alternating flap technique or cork borer method. Using the cork borer method, younger lodgepole pine trees were inoculated with five different isolates of G. clavigera. After a 48 week incubation period, isolates ATCC 18086, B5 and H55 had induced stronger pathogenic indicators compared to isolates KW 1407 and B20. After a 7 week incubation period, only isolate ATCC 18086 had induced stronger pathogenic indicators. Usually, this isolate grew faster at lower temperatures and in a low oxygen environment. Isolate KW 1407 consistently produced milder pathogenic indicators during both incubation periods. Among the non-pathogenic fungal associates of the mountain pine beetle, Ceratocystiopsis minuta may be considered the most important because it is the type species for the genus Ceratocystiopsis. The history of this genus is complicated because no physical specimen exists for C. minuta. The phylogeny of the genus Ceratocystiopsis was evaluated. Many isolates of C. minuta were assessed as potential epitypes. Several isolates of C. minuta from previous work were shown to be misidentified. C. minuta isolate CBS 116796 is recommended for future genetic work within the genus Ceratocystiopsis. For morphological work, using measurements from the literature is recommended since CBS 116796 did not produce fruiting bodies.

O surgimento da AIDS em meados da década de oitenta do século passado traz à tona, mais uma vez, a discussão do binarismo doença e culpa. Para o imaginário dessa época, a mancha estampada no corpo do aidético perde o caráter patológico como causa da ação virulenta do HIV e é substituída por outra mancha que vai revestir não só a pele do doente, mas, principalmente, os olhos acusatórios daqueles que vêem a realidade manchada pelo preconceito. Para tratar desse glaucoma imaginário, abordarei o símbolo da mancha nas três peças que fazem parte de minha pesquisa: Angels in America (1990) de Tony Kushner, O Homem e a Mancha (1997) de Caio Fernando Abreu e A Mancha Roxa (1988) de Plínio Marcos. O objetivo é demonstrar como a mancha é objeto/signo adequado, usado por todos e cada um dos três dramaturgos, na representação do doente de AIDS; para, finalmente, refletir no palco o esboroamento da realidade, através de seu sentido impreciso e sem uma fronteira delimitável. Em O Homem e a Mancha, por exemplo, a personagem, para se libertar da visão dessa mancha estampada em seu corpo, desenvolve um comportamento esquizofrênico, criando um mundo fictício ao seu redor e se isolando do mundo real para viver em sua ilha de solidão. Em Angels in America, o corpo lesionado de Prior Walter provoca mais estupor ao seu companheiro Louis do que ao próprio protagonista. Enquanto Louis tenta fugir da imagem emblemática da culpa, Prior se transforma, não em um marcado para a morte, mas em um escolhido, um novo eleito que desafia o Anjo, pedindo por mais vida. E por último, na peça A Mancha Roxa de Plínio Marcos, diferentemente de Caio e Kushner, o símbolo da mancha aparece de modo mais realista e, acima de tudo, de maneira mais fatalista, pois as presidiárias não estão somente presas fisicamente, mas mentalmente, sem possibilidades para escapismos imaginários / The appearance of AIDS in the first half of the 1980s brings back to surface the discussion of the illness and guilt binarism. According to the imaginary of that age, the stain stamped on the seropositive body loses its pathological caracter as a virulent action caused by the HIV, replaced by another stain, covering not only the skin of the infected but, principally, the accusing eyes of those who see reality stained by prejudice. In order to treat this imaginary glaucoma, I will analyze the metaphor of the stain in the three plays that make part of my research: Angels in America (1990) by Tony Kushner, The man and the skin lesion (1997) by Caio Fernando Abreu and The Purple stain (1988) by Plinio Marcos. It aims at showing how the stain becomes an appropriate object/sign used by the three playwrights in the representation of the seropositive; and to reflect on stage the blurring of reality, through its imprecise meaning without a delimited frontier. In The Man and the skin lesion, for example, one of the characters, in order to free himself from the vision of a stain stamped on his body, develops a sort of esquizofrenic behavior: he creates a sorrounded fictitious world, provoking his isolation from reality, so that he can inhabit on his solitude island. In Angels in America, Prior Walters skin lesion body causes much more fear to his partner Louis than to the protagonist himself. While Louis tries to escape from the emblematic imagery of guilt, Prior transforms himself not into someone marked to die, but as a chosen one, a new elect, who challenges the Angel asking for more life. And finally The Purple stain presents the metaphor of the stain in a more realistic and fatalistic way: female prisoners are not only physically imprisoned, but mentally, without the possibilities of imaginary escapisms

O surgimento da AIDS em meados da década de oitenta do século passado traz à tona, mais uma vez, a discussão do binarismo doença e culpa. Para o imaginário dessa época, a mancha estampada no corpo do aidético perde o caráter patológico como causa da ação virulenta do HIV e é substituída por outra mancha que vai revestir não só a pele do doente, mas, principalmente, os olhos acusatórios daqueles que vêem a realidade manchada pelo preconceito. Para tratar desse glaucoma imaginário, abordarei o símbolo da mancha nas três peças que fazem parte de minha pesquisa: Angels in America (1990) de Tony Kushner, O Homem e a Mancha (1997) de Caio Fernando Abreu e A Mancha Roxa (1988) de Plínio Marcos. O objetivo é demonstrar como a mancha é objeto/signo adequado, usado por todos e cada um dos três dramaturgos, na representação do doente de AIDS; para, finalmente, refletir no palco o esboroamento da realidade, através de seu sentido impreciso e sem uma fronteira delimitável. Em O Homem e a Mancha, por exemplo, a personagem, para se libertar da visão dessa mancha estampada em seu corpo, desenvolve um comportamento esquizofrênico, criando um mundo fictício ao seu redor e se isolando do mundo real para viver em sua ilha de solidão. Em Angels in America, o corpo lesionado de Prior Walter provoca mais estupor ao seu companheiro Louis do que ao próprio protagonista. Enquanto Louis tenta fugir da imagem emblemática da culpa, Prior se transforma, não em um marcado para a morte, mas em um escolhido, um novo eleito que desafia o Anjo, pedindo por mais vida. E por último, na peça A Mancha Roxa de Plínio Marcos, diferentemente de Caio e Kushner, o símbolo da mancha aparece de modo mais realista e, acima de tudo, de maneira mais fatalista, pois as presidiárias não estão somente presas fisicamente, mas mentalmente, sem possibilidades para escapismos imaginários / The appearance of AIDS in the first half of the 1980s brings back to surface the discussion of the illness and guilt binarism. According to the imaginary of that age, the stain stamped on the seropositive body loses its pathological caracter as a virulent action caused by the HIV, replaced by another stain, covering not only the skin of the infected but, principally, the accusing eyes of those who see reality stained by prejudice. In order to treat this imaginary glaucoma, I will analyze the metaphor of the stain in the three plays that make part of my research: Angels in America (1990) by Tony Kushner, The man and the skin lesion (1997) by Caio Fernando Abreu and The Purple stain (1988) by Plinio Marcos. It aims at showing how the stain becomes an appropriate object/sign used by the three playwrights in the representation of the seropositive; and to reflect on stage the blurring of reality, through its imprecise meaning without a delimited frontier. In The Man and the skin lesion, for example, one of the characters, in order to free himself from the vision of a stain stamped on his body, develops a sort of esquizofrenic behavior: he creates a sorrounded fictitious world, provoking his isolation from reality, so that he can inhabit on his solitude island. In Angels in America, Prior Walters skin lesion body causes much more fear to his partner Louis than to the protagonist himself. While Louis tries to escape from the emblematic imagery of guilt, Prior transforms himself not into someone marked to die, but as a chosen one, a new elect, who challenges the Angel asking for more life. And finally The Purple stain presents the metaphor of the stain in a more realistic and fatalistic way: female prisoners are not only physically imprisoned, but mentally, without the possibilities of imaginary escapisms

O vírus de Leishmania 1-4 ( do inglês Leishmania RNA virus 1-4 ou LRV1-4) é um vírus da família Totiviridae, e que possui capsídeo icosaédrico e RNA dupla-fita que codifica duas proteínas (proteína capsidial e RNA polimerase). Dados recentes indicam o envolvimento do LRV1-4 na patogênese de Leishmania no hospedeiro humano, tornando seu estudo de fundamental importância para o entendimento dessa doença e de seu papel na relação parasito-hospedeiro. Há relatos sobre a purificação do vírus a partir do seu hospedeiro natural (Leishmania guyanensis) e a partir de sistemas de expressão heteróloga. Este trabalho tem por objetivo estabelecer os métodos de purificação para posteriores estudos estruturais por Microscopia Eletrônica de Transmissão por Contraste Negativo (NS-TEM) e por Crio-Microscopia Eletrônica (Cryo-EM). Os estudos aqui propostos irão permitir a construção de um modelo estrutural do capsídeo do LRV1-4 e sua identificação correta dentre os totivírus. Além das contribuições ao conhecimento da biologia/patogenia do LRV1-4 este estudo representa a primeira caracterização estrutural de um capsídeo viral realizada no Brasil, e assim um avanço importante para a área de virologia e biologia estrutural no pais. Foram realizadas ultracentrifugações biológicas, utilizando gradientes de sacarose, para a purificação do vírus a partir do extrato celular de L. guyanensis. As frações que apresentaram RNA viral foram analisadas por Microscopia Eletrônica de Transmissão (Campinas LNNano CNPEM). Além disso, foram realizadas tentativas de expressar a proteína do capsídeo (ORF2) em Leishmania tarentolae e Escherichia coli. Foram também realizados esforços para a obtenção de anticorpos a partir de peptídeos sintetizados após análise computacional da sequência de aminoácidos da proteína do capsídeo. As amostras obtidas a partir do hospedeiro natural do vírus se apresentaram heterogêneas quando analisadas por NS-TEM, de modo que não foi possível a realização de uma análise estrutural. Porém, a presença de partículas do tamanho esperado para o vírus em amostras em que foi detectado o RNA viral indicam que são necessários esforços para obtenção de uma amostra de maior pureza e homogênea. Além disso, não foi possível obter a proteína do capsídeo nos sistemas de expressão heteróloga. A presença de 25 resíduos de cisteína pode estar levando a proteína à degradação rápida em bactéria. Os experimentos de expressão em células de Leishmania ainda não foram conclusivos. Foi obtido um anticorpo anti-peptídeo que reconhece a proteína do capsídeo, tornando possíveis experimentos como imunolocalização e imunoprecipitação do vírus. / The Leishmania RNA virus 1-4 (LRV1-4) belongs to the Totiviridae family. It has an icosahedral capsid and a double-strand RNA encoding two proteins (capsid protein and RNA polymerase). Recent data indicate the involvement of LRV1-4 in the pathogenesis of Leishmania in the human host, making their study of fundamental importance for the understanding of this disease and its role in host-parasite relationship. There are reports on the purification of the virus from its natural host (Leishmania guyanensis) and from the same heterologous expression systems such as Escherichia coli.This work aims to stablish purification methods for further structural studies by Negative Stain Transmission Microscopy (NS-TEM) and Cryo-Electron Microscopy (Cryo-EM). The studies proposed here will allow the construction of a structural model of the coat protein of LRV1-4 and their correct identification amongst the Totiviridae. In addition to the contributions to the knowledge of the biology and pathogenesis of LRV1-4, this study represents the first structural characterization of a viral capsid held in Brazil and thus an important step forward for the field of virology and structural biology in the country. Sucrose for virus purification gradients were performed from the cell extract of L. guyanensis. Fractions that showed viral RNA were analised by Transmission Electron Microscopy (Campinas LNNano - CNPEM). Furthermore, attempts have been made to express the capsid protein (ORF2) in Leishmania tarentolae and Escherichia coli. There has also been made efforts to obtain antibodies from peptides synthesized accordingly to the computer analysis of the amino acid sequence of the capsid protein. The samples obtained from the natural host of the virus showed a heterogeneous distribution of particles when examined by NS-TEM so that it was not possible to perform a structural analysis. However, the presence of particles of the size expected for the virus particles in samples where the viral RNA was detected indicate that efforts are necessary to obtain a more homogeneous and pure sample. Moreover, it was not possible to obtain the capsid protein in heterologous expression systems. The presence of 25 cysteine residues could have led to the protein rapid degradation in the bacteria host. The expression experiments in Leishmania cells were not yet conclusive. It was also possible to obtain an anti-peptide antibody recognizing the capsid protein, enabling immunoprecipitation and immunolocalization experiments.

The mountain pine beetle is associated with a diverse array of fungi. Grosmannia clavigera is the most pathogenic of these fungi. A comparison was made between two methods that have been used to assess fungal pathogenicity. Results were similar for older trees inoculated with G. clavigera using either the alternating flap technique or cork borer method. Using the cork borer method, younger lodgepole pine trees were inoculated with five different isolates of G. clavigera. After a 48 week incubation period, isolates ATCC 18086, B5 and H55 had induced stronger pathogenic indicators compared to isolates KW 1407 and B20. After a 7 week incubation period, only isolate ATCC 18086 had induced stronger pathogenic indicators. Usually, this isolate grew faster at lower temperatures and in a low oxygen environment. Isolate KW 1407 consistently produced milder pathogenic indicators during both incubation periods. Among the non-pathogenic fungal associates of the mountain pine beetle, Ceratocystiopsis minuta may be considered the most important because it is the type species for the genus Ceratocystiopsis. The history of this genus is complicated because no physical specimen exists for C. minuta. The phylogeny of the genus Ceratocystiopsis was evaluated. Many isolates of C. minuta were assessed as potential epitypes. Several isolates of C. minuta from previous work were shown to be misidentified. C. minuta isolate CBS 116796 is recommended for future genetic work within the genus Ceratocystiopsis. For morphological work, using measurements from the literature is recommended since CBS 116796 did not produce fruiting bodies. / Forestry, Faculty of / Graduate

Background: Blastomycosis is caused by a dimorphic fungus that can be difficult to diagnose in certain situations. The disease is sometimes serious and can be deadly. Diagnosis by fungal serology and urinary antigens is not easy to establish and unreliable. Culture is also time-consuming and is not easy to perform. Thus, documentation of such an organism on cytology offers a quick and cost-effective alternative. This report describes for the first time identification of the 'negative image' of Blastomyces budding yeast. Case: A 79-year-old man presented with a left lung nodule associated with mediastinal and hilar lymphadenopathy. Fine needle aspiration was performed, and a 'negative image' of a yeast with wide base budding was noted on Diff-Quik (DQ)-stained smears. Blastomyces species were confirmed with periodic acid-Schiff fungal stain. Additionally, the fungal capsule contained focally polarizable material on Congo red stain and lacked mucin with mucicarmine stain. Conclusion: Blastomyces yeast forms can be easily identified with DQ staining by their 'negative image'. This feature can be utilized as a quick and cost-effective cytological characteristic to further triage these specimens for confirmation. The information can be of great value to clinicians in making appropriate clinical decisions.

In the western regions of North America, mountain pine beetle, Dendroctonus ponderosae Hopk., infestations take a tremendous toll of pines , especially lodgepole pine, Pinus contorta Dougl. var. latifolia Engelm.. Mass attack by the beetles is a devastating event for the trees. As well as girdling the tree, a massive inoculation of blue stain fungus "complex" (composed of several species of Ceratocystis, numerous yeasts and other mycelial fungi) is made beneath the bark. These fungi colonize and destroy the parenchyma tissue system of the host sapwood, primarily the ray parenchyma and resin duct epithelium. A blue stain is produced in the sapwood as a consequence of destruction of the sapwood parenchyma. The stain develops inward through the sapwood, and the transpiration stream is cut off. As more and more sapwood is stained, foliar water stress begins to increase. Foliage however, remains green and apparently healthy for up to 10 months after inoculation. When spring bud break begins the year following beetle attack, terminal buds of blue-stained trees begin to expand, then abort. Soon after, the needles of these trees fade to a reddish brown color. Transpiration stream disruption was not caused by penetration of tracheids by fungal hyphae; tyloses were not observed; nor was microconidial blockage of bordered pits seen. Though resin duct epithelium was eventually destroyed, little resin soaking was observed in the initial blue stained regions. Many bordered pits of tracheids in stained regions appeared to be aspirated, suggesting introduction of embolisms.