Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions. In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

Uma evidência contundente da ocupação pré-histórica na Amazônia são os solos de cor escura com material arqueológico, conhecidos regionalmente como Terra Preta de Índio (TPI). Apesar de amplamente estudados, alguns de seus atributos permanecem ainda pouco conhecidos, especialmente os micromorfológicos, mineralógicos e geoquímicos. Esses estudos podem identificar os processos envolvidos na gênese e evolução desses solos, e quando empregados em conjunto com estudos fitolíticos, podem auxiliar na elucidação das suas formas de uso pretéritas. O objetivo desse estudo foi obter uma aproximação da hierarquia dos processos envolvidos na gênese desses solos e as suas prováveis formas de uso em tempos pré-colombianos. A pesquisa foi conduzida no Campo Experimental do Caldeirão, Iranduba - AM. Foram estudados dois perfis com TPI (P1 e P2) e um solo adjacente com horizonte A moderado (P3). Em cada horizonte foram coletadas amostras deformadas para análises físico-químicas, mineralógicas e geoquímicas e a cada 5 cm de profundidade para análise fitolítica e isotópica. Lâminas delgadas de amostras indeformadas de horizontes selecionados foram confeccionadas e descritas em sua micromorfologia, com posterior exame em microscópio eletrônico de varredura com microanálise química. A idade dos solos foi estabelecida com base em datações 14C de carvões. A microestrutura granular das TPI é de origem zoogenética e geoquímica. A gênese dos horizontes antrópicos envolveu: i) a ação do homem descartando e queimando resíduos (antropização); ii) espessamento do horizonte A e escurecimento dos horizontes subsuperficiais por bioturbação (cumulização e melanização); iii) dispersão e translocação de colóides (argiluviação); iv) condições pedoambientais diferentes das atuais (pedorrelíquia - nódulos ferruginosos). Revestimentos de argila com extinção forte, contínua e estriada nas cerâmicas indica que o processo de argiluviação é atual. O processo de elutriação predomina no solo não antrópico. A degradação dos nódulos de ferro na TPI favorece a xantização e atua como fonte de argila (pedoplasmação). Arecaceae e Cyperaceae são mais abundantes nos horizontes antrópicos, notadamente nos níveis com maior quantidade de cerâmica. A ausência de fitólitos de plantas domesticadas indica que a formação das TPI não está relacionada com práticas agrícolas. As evidências fitolíticas demonstram que as atividades antrópicas ocorreram de forma mais intensa no P1. A rápida ciclagem de silício, evidenciada pela presença de fitólitos com silicificação incompleta, favorece a estabilidade da mineralogia caulínitica. VHE, ilita e variscita-estrengita ocorrem somente nos perfis com TPI. P2O5-CaO-K2O-NaO-Cs-Co-Zn-Cu-Ba-Rb-Ni representa a assinatura geoquímica das TPI. A presença de variscita-estrengita, tridimita e maghemita nas TPI, notadamente nas cerâmicas, confirma a formação de minerais em decorrência das práticas antrópicas. As cerâmicas apresentam predominantemente cauixi (Tubella reticulata e Parnula betesil) e cariapé (Licania utilis). A presença comum de micas primárias nesses artefatos sugere material alóctone em seu fabrico. As TPI resultam da adição de artefatos arqueológicos e melanização de horizontes pedogenéticos não antropizados. Tais atividades enriqueceram em nutrientes e alteraram a assinatura geoquímica do solo, assim como promoveram a formação de minerais. Essa antropização acelerou os processos de argiluviação e de degradação de petroplintitas. No decorrer de sua evolução, foram utilizados e adicionados resíduos de plantas, destacadamente de palmeiras e Cyperaceae. / A remarkable evidence of human occupation in Amazonian region is the existence of soils with dark colors and presence of ceramic materials, known as Indigenous Dark Earth (IDE). Despite of widely studied some of their features are still poorly understood, mainly that related to micromorphology, mineralogy and geochemical aspects. Such approach, in combination to phytolytic studies, is able to identify soil genesis processes and unravel the comprehension of occupation mechanisms of human. The aim of this study was to establish the hierarchy of these processes and their association with ancient activities of pre-Columbian populations. The research was carried out in the experimental site of Caldeirão, Iranduba city (Amazon state, Brazil). Two pedons containing surface anthropogenic horizons (P1 and P2) were directly compared to a non-anthropogenic soil (P3). In each soil horizon disturbed soil samples were sampled in order to perform physical, chemical, mineralogical and geochemical analyses. For phytolitic analyses samples were taken each 5 cm of depth. Micromorphological samples were studied in thin sections in the optical microscope and further analyzed in Scanning Electron Microscopy (SEM). The chronology was accomplished after 14C dating. The microaggregates in anthropogenic horizon are related to geochemical and biological processes. The genesis of IDE implicates in the following mechanisms: i) disposal and burning of residues by humans (anthropization); ii) deepening A horizons and darkening subsurface horizons by bioturbation (cumulization and melanization processes); iii) dispersion and migration of colloidal particles leading to argiluviation process; iv) different condictions of environment that not occur nowdays (pedorelict - ferruginous nodules). Clay coatings with extinction bands and continuous orientation in the ceramic artifacts suggest a current argiluviation process. The degradation of Fe nodules enhances the xantization process also providing clay (source of clay). The prevalent soil genesis in non-anthropic soil is the elutriation. The degradation of Fe nodules in the IDE enhances the xantization process also providing clay (pedoplasmation). The number of phytoliths of Arecaceae and Cyperaceae is higher in IDE than non-IDE, mainly in the horizons with more ceramics. The phytolitic evidence demonstrate that activities anthropic was more intense in the P1. The rapid Si cycling, highlighted by the presence of phytoliths without complete silicification, contribute to stability of kaolinitic mineralogy. HIV, illite and variscite-strengite are constrained to IDE pedons. P2O5-CaO-K2O-NaO-Cs-Co-Zn-Cu-Ba-Rb-Ni represents the geochemical signature of IDE. The presence of maghemite, variscite-strengite and tridimite strengthen a mineral forming process linked to human activity. In ceramic materials there is a prevalence of phytoliths from cauixi (Tubella reticulata and Parnula betesil) and cariapé (Licania utilis). The presence of mica suggests an alloctone material for their manufacturing. Hence the anthropic horizons result from the addition of archeological artifacts and melanization of non-anthropic horizons. These activities chemically enriched and modify the geochemical signature of soil, as soon as promoted formation of minerals. The anthropic activities conducted the argiluviation and degradation of Fe nodules. During their evolution there was a clear addition of plant residues, notably related to palm trees and Cyperaceae species.

Uma evidência contundente da ocupação pré-histórica na Amazônia são os solos de cor escura com material arqueológico, conhecidos regionalmente como Terra Preta de Índio (TPI). Apesar de amplamente estudados, alguns de seus atributos permanecem ainda pouco conhecidos, especialmente os micromorfológicos, mineralógicos e geoquímicos. Esses estudos podem identificar os processos envolvidos na gênese e evolução desses solos, e quando empregados em conjunto com estudos fitolíticos, podem auxiliar na elucidação das suas formas de uso pretéritas. O objetivo desse estudo foi obter uma aproximação da hierarquia dos processos envolvidos na gênese desses solos e as suas prováveis formas de uso em tempos pré-colombianos. A pesquisa foi conduzida no Campo Experimental do Caldeirão, Iranduba - AM. Foram estudados dois perfis com TPI (P1 e P2) e um solo adjacente com horizonte A moderado (P3). Em cada horizonte foram coletadas amostras deformadas para análises físico-químicas, mineralógicas e geoquímicas e a cada 5 cm de profundidade para análise fitolítica e isotópica. Lâminas delgadas de amostras indeformadas de horizontes selecionados foram confeccionadas e descritas em sua micromorfologia, com posterior exame em microscópio eletrônico de varredura com microanálise química. A idade dos solos foi estabelecida com base em datações 14C de carvões. A microestrutura granular das TPI é de origem zoogenética e geoquímica. A gênese dos horizontes antrópicos envolveu: i) a ação do homem descartando e queimando resíduos (antropização); ii) espessamento do horizonte A e escurecimento dos horizontes subsuperficiais por bioturbação (cumulização e melanização); iii) dispersão e translocação de colóides (argiluviação); iv) condições pedoambientais diferentes das atuais (pedorrelíquia - nódulos ferruginosos). Revestimentos de argila com extinção forte, contínua e estriada nas cerâmicas indica que o processo de argiluviação é atual. O processo de elutriação predomina no solo não antrópico. A degradação dos nódulos de ferro na TPI favorece a xantização e atua como fonte de argila (pedoplasmação). Arecaceae e Cyperaceae são mais abundantes nos horizontes antrópicos, notadamente nos níveis com maior quantidade de cerâmica. A ausência de fitólitos de plantas domesticadas indica que a formação das TPI não está relacionada com práticas agrícolas. As evidências fitolíticas demonstram que as atividades antrópicas ocorreram de forma mais intensa no P1. A rápida ciclagem de silício, evidenciada pela presença de fitólitos com silicificação incompleta, favorece a estabilidade da mineralogia caulínitica. VHE, ilita e variscita-estrengita ocorrem somente nos perfis com TPI. P2O5-CaO-K2O-NaO-Cs-Co-Zn-Cu-Ba-Rb-Ni representa a assinatura geoquímica das TPI. A presença de variscita-estrengita, tridimita e maghemita nas TPI, notadamente nas cerâmicas, confirma a formação de minerais em decorrência das práticas antrópicas. As cerâmicas apresentam predominantemente cauixi (Tubella reticulata e Parnula betesil) e cariapé (Licania utilis). A presença comum de micas primárias nesses artefatos sugere material alóctone em seu fabrico. As TPI resultam da adição de artefatos arqueológicos e melanização de horizontes pedogenéticos não antropizados. Tais atividades enriqueceram em nutrientes e alteraram a assinatura geoquímica do solo, assim como promoveram a formação de minerais. Essa antropização acelerou os processos de argiluviação e de degradação de petroplintitas. No decorrer de sua evolução, foram utilizados e adicionados resíduos de plantas, destacadamente de palmeiras e Cyperaceae. / A remarkable evidence of human occupation in Amazonian region is the existence of soils with dark colors and presence of ceramic materials, known as Indigenous Dark Earth (IDE). Despite of widely studied some of their features are still poorly understood, mainly that related to micromorphology, mineralogy and geochemical aspects. Such approach, in combination to phytolytic studies, is able to identify soil genesis processes and unravel the comprehension of occupation mechanisms of human. The aim of this study was to establish the hierarchy of these processes and their association with ancient activities of pre-Columbian populations. The research was carried out in the experimental site of Caldeirão, Iranduba city (Amazon state, Brazil). Two pedons containing surface anthropogenic horizons (P1 and P2) were directly compared to a non-anthropogenic soil (P3). In each soil horizon disturbed soil samples were sampled in order to perform physical, chemical, mineralogical and geochemical analyses. For phytolitic analyses samples were taken each 5 cm of depth. Micromorphological samples were studied in thin sections in the optical microscope and further analyzed in Scanning Electron Microscopy (SEM). The chronology was accomplished after 14C dating. The microaggregates in anthropogenic horizon are related to geochemical and biological processes. The genesis of IDE implicates in the following mechanisms: i) disposal and burning of residues by humans (anthropization); ii) deepening A horizons and darkening subsurface horizons by bioturbation (cumulization and melanization processes); iii) dispersion and migration of colloidal particles leading to argiluviation process; iv) different condictions of environment that not occur nowdays (pedorelict - ferruginous nodules). Clay coatings with extinction bands and continuous orientation in the ceramic artifacts suggest a current argiluviation process. The degradation of Fe nodules enhances the xantization process also providing clay (source of clay). The prevalent soil genesis in non-anthropic soil is the elutriation. The degradation of Fe nodules in the IDE enhances the xantization process also providing clay (pedoplasmation). The number of phytoliths of Arecaceae and Cyperaceae is higher in IDE than non-IDE, mainly in the horizons with more ceramics. The phytolitic evidence demonstrate that activities anthropic was more intense in the P1. The rapid Si cycling, highlighted by the presence of phytoliths without complete silicification, contribute to stability of kaolinitic mineralogy. HIV, illite and variscite-strengite are constrained to IDE pedons. P2O5-CaO-K2O-NaO-Cs-Co-Zn-Cu-Ba-Rb-Ni represents the geochemical signature of IDE. The presence of maghemite, variscite-strengite and tridimite strengthen a mineral forming process linked to human activity. In ceramic materials there is a prevalence of phytoliths from cauixi (Tubella reticulata and Parnula betesil) and cariapé (Licania utilis). The presence of mica suggests an alloctone material for their manufacturing. Hence the anthropic horizons result from the addition of archeological artifacts and melanization of non-anthropic horizons. These activities chemically enriched and modify the geochemical signature of soil, as soon as promoted formation of minerals. The anthropic activities conducted the argiluviation and degradation of Fe nodules. During their evolution there was a clear addition of plant residues, notably related to palm trees and Cyperaceae species.

The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.

Neurogenesis primarily occurs during embryonic development in decapod crustaceans, ending when the embryonic precursor cells die. However, areas in the central olfactory pathway are exceptions to this. Here, neurons are produced throughout the animal’s life from precursor cells in the neurogenic niche. Cells within the niche divide and migrate to clusters in the olfactory pathway where they eventually differentiate into neurons. The number of cells in the niche correlates with the total number of hemocytes, which have been suggested to be a source of adult-born neurons. Hemocytes are further an important part of the innate immunity since containing the compounds of the proPO-system needed for the melanization reaction. The purpose of this study was to find out whether the melanization reaction matters when it comes to neural injuries. Neural injury was induced by cutting of the first pair of antennae, where neurons are connected to the olfactory pathway. Hemocytes in the hemolymph were counted and characterized and phenoloxidase activity in the brain was measured before and after neural injury. mRNA expression was measured for prophenoloxidase, the neurogenic niche marker glutamine synthetase 2 as well as for astakine 1. Astakine 1 protein had been found in increased levels after neural injury in previous studies. Significant differences were detected for number of hemocytes in injured crayfish and for glutamine synthetase 2 and prophenoloxidase in control crayfish. However, these findings did not provide strong enough evidence to suggest that the melanization reaction plays a role after neural injury. More research is still needed, perhaps by studying the distribution of hemocytes in the brain at different times post-injury by histological sectioning.

A radiação solar é um dos principais fatores responsáveis pelo controle das populações de fungos no meio ambiente. A inatividade de estruturas de dispersão e de infecção pela radiação UVA e UVB é ainda mais importante no controle de fungos que se dispersam pelo ar e que podem infectar o hospedeiro, quando inalados, como é o caso de espécies do gênero Cryptococcus. C. neofarmans é capaz de produzir melanina na presença de substratos exógenos, como a L-dopa. A melanização é capaz de proteger o fungo contra diversos fatores ambientais bióticos e abióticos. Neste trabalho, foi avaliado o efeito de exposições a uma intensidade ambiental de radiação UVB (I.000mW m-2) na sobrevivência de células melanizadas e não melanizadas de quatro linhagens de Cryptococcus neoformans e de quatro linhagens de Cryptococcus laurentii. Foi determinada a sobrevivência relativa (sobrevivência das células expostas em relação à das células não expostas à radiação de células com 2, 4, 6 e 8 dias, crescidas em meio com e sem L-dopa, após exposição às doses de 1.8 e 3.6 kJ m-2. Tanto a irradiância como as doses, são observadas no meio ambiente, mesmo em regiões temperadas. Foram observadas diferenças na tolerância à radiação UVB, tnato entre as linhagens de C. neoformans, como entre as linhagens de C. laurentii. As linhagens de C. neoformans, foram observbadas diferenças na tolerância à radiação ao longo do desenvolvimento, tanto nas células melanizadas como nas não melanizadas. Na maioria das situações (linhagem, tempo de crescimento, dose de UVB), não houve diferença significativa entre a tolerância das células melanizadas e não melanizadas e, quando isso ocorreu, a diferença foi menor do que a observada anteriormente com UVC. E, C laurentii, não houve diferença na tolerância à radiação ao longo do desenvolvimento das células melanizadas e não melanizadas. Também não foram observadas diferenças significativas entre a tolerância das células melanizadas e não melanizadas em nenhuma das linhagens. / Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. The inactivation of dispersal and infection structures by UVA and UVB radiation is even more important for the control of fungi that disperse through the air and that can produce melanin in the presence of oxogeneous substrates such as L-dopa. Melanization can protect the fungus against various biotic and abiotic factors. In the present study, we investigated the effect of exposure to an environmental intensity of UVB radiation of 1,000 mWm-2 on the survival of melanized and non-melanized cells of four strains of Cryptococcus neoformans and of four strains of Cryptococcus laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of 2, 4, 6 and 8 day cells grown on medium with and without L-dopa was determinated after exposure to doses of 1.8 and 3.6kJ m-2. Both irradiance and these doses are observed in the environment even in temperate regions. Difference in tolerance to UVB radiation were observed both in the C. neoformans and C. laurentii strains. The C. neofarmans strains were more sensitive to radiation than the C. laurentii strains. In C. neoformans, diferences in tolerance to radiation were observed during development both in melanized and non-melanized cells. For most features (strains, time of growth and UVB dose) there was no difference in tolerance between melanized and non-melanized cell and when differences occurred they were smaller than those previously observed with UVC. In C. laurentii, there was no difference in tolerance to radiation during development between melanized and non-melanized cells., Also, no significant differences in tolerance were observed between melanized and non-melanized cells of any strain

A radiação solar é um dos principais fatores responsáveis pelo controle das populações de fungos no meio ambiente. A inatividade de estruturas de dispersão e de infecção pela radiação UVA e UVB é ainda mais importante no controle de fungos que se dispersam pelo ar e que podem infectar o hospedeiro, quando inalados, como é o caso de espécies do gênero Cryptococcus. C. neofarmans é capaz de produzir melanina na presença de substratos exógenos, como a L-dopa. A melanização é capaz de proteger o fungo contra diversos fatores ambientais bióticos e abióticos. Neste trabalho, foi avaliado o efeito de exposições a uma intensidade ambiental de radiação UVB (I.000mW m-2) na sobrevivência de células melanizadas e não melanizadas de quatro linhagens de Cryptococcus neoformans e de quatro linhagens de Cryptococcus laurentii. Foi determinada a sobrevivência relativa (sobrevivência das células expostas em relação à das células não expostas à radiação de células com 2, 4, 6 e 8 dias, crescidas em meio com e sem L-dopa, após exposição às doses de 1.8 e 3.6 kJ m-2. Tanto a irradiância como as doses, são observadas no meio ambiente, mesmo em regiões temperadas. Foram observadas diferenças na tolerância à radiação UVB, tnato entre as linhagens de C. neoformans, como entre as linhagens de C. laurentii. As linhagens de C. neoformans, foram observbadas diferenças na tolerância à radiação ao longo do desenvolvimento, tanto nas células melanizadas como nas não melanizadas. Na maioria das situações (linhagem, tempo de crescimento, dose de UVB), não houve diferença significativa entre a tolerância das células melanizadas e não melanizadas e, quando isso ocorreu, a diferença foi menor do que a observada anteriormente com UVC. E, C laurentii, não houve diferença na tolerância à radiação ao longo do desenvolvimento das células melanizadas e não melanizadas. Também não foram observadas diferenças significativas entre a tolerância das células melanizadas e não melanizadas em nenhuma das linhagens. / Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. The inactivation of dispersal and infection structures by UVA and UVB radiation is even more important for the control of fungi that disperse through the air and that can produce melanin in the presence of oxogeneous substrates such as L-dopa. Melanization can protect the fungus against various biotic and abiotic factors. In the present study, we investigated the effect of exposure to an environmental intensity of UVB radiation of 1,000 mWm-2 on the survival of melanized and non-melanized cells of four strains of Cryptococcus neoformans and of four strains of Cryptococcus laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of 2, 4, 6 and 8 day cells grown on medium with and without L-dopa was determinated after exposure to doses of 1.8 and 3.6kJ m-2. Both irradiance and these doses are observed in the environment even in temperate regions. Difference in tolerance to UVB radiation were observed both in the C. neoformans and C. laurentii strains. The C. neofarmans strains were more sensitive to radiation than the C. laurentii strains. In C. neoformans, diferences in tolerance to radiation were observed during development both in melanized and non-melanized cells. For most features (strains, time of growth and UVB dose) there was no difference in tolerance between melanized and non-melanized cell and when differences occurred they were smaller than those previously observed with UVC. In C. laurentii, there was no difference in tolerance to radiation during development between melanized and non-melanized cells., Also, no significant differences in tolerance were observed between melanized and non-melanized cells of any strain