Application of ionic liquids and enzymes for the removal of proteinaceous layers from polychrome of works of art and evaluation of the cleaning effectiveness

Dissertação Apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Ciências da Conservação, especialização em Pintura

Cryptococcus neoformans isolados de pacientes com AIDS

Criptococose é considerada a infecção fúngica sistêmica oportunista mais comum em pacientes com AIDS. Nestes pacientes tem predominado como agente etiológico Cryptococcus neoformans var. neoformans, e muito raramente relata-se C. neoformans var. gattiii, mesmo nas regiões onde se verifica a sua prevalência. Foram estudados 50 pacientes com lesões de criptococose meningoencefálica associada com AIDS. Os isolados foram identificados através de características microscópicas e macroscópicas exibidas em meios de ágar Sabouraud, ágar niger e Christensen. As variedades de C. neoformans foram determinadas pela reação de coloração obtida em meio de L-canavanina glicina-azul de bromotimol (CGB). Em todos os pacientes examinados foram isolados C. neoformans, sendo identificados C. neoformans var. neoformans em 47 isolados e C. neoformans var. gattii em 3. Os resultados encontrados mostram que a criptococose em pacientes com AIDS pode também ser causada por C. neoformans var. gatti, apesar de haver predominância de C. neoformans var neoformans nesta população.

Design and characterization of Chitin- Glucan polymeric structures for wound dressing materials

The main objective of this work was the development of polymeric structures, gel and films, generated from the dissolution of the Chitin-Glucan Complex (CGC) in biocompatible ionic liquids for biomedical applications. Similar as chitin, CGC is only soluble in some special solvents which are toxic and corrosive. Due to this fact and the urgent development of biomedical applications, the need to use biocompatible ionic liquids to dissolve the CGC is indispensable. For the dissolution of CGC, the biocompatible ionic liquid used was Choline acetate. Two different CGC’s, KiOnutrime from KitoZyme and biologically produced CGC from Faculdade de Ciencias e Tecnologia (FCT) - Universidade Nova de Lisboa, were characterized in order to develop biocompatible wound dressing materials. The similar result is shown in term of the ratio of chitin:glucan, which is 1:1.72 for CGC-FCT and 1:1.69 for CGC-Commercial. For the analysis of metal element content, water and inorganic salts content and protein content, both polymers showed some discrepancies, where the content in CGC-FCT is always higher compared to the commercial one. The different characterization results between CGC-FCT and CGC-Commercial could be addressed to differences in the purification method, and the difference of its original strain yeast, whereas CGC-FCT is derived from P.pastoris and the commercial CGC is from A.niger. This work also investigated the effect of biopolymers, temperature dissolution, non-solvent composition on the characteristics of generated polymeric structure with biocompatible ionic liquid. The films were prepared by casting a polymer mixture, immersion in a non-solvent, followed by drying at ambient temperature. Three different non-solvents were tested in phase inversion method, i.e. water, methanol, and glycerol. The results indicate that the composition of non-solvent in the coagulation bath has great influence in generated polymeric structure. Water was found to be the best coagulant for producing a CGC polymeric film structure. The characterizations that have been done include the analysis of viscosity and viscoelasticity measurement, as well as sugar composition in the membrane and total sugar that was released during the phase inversion method. The rheology test showed that both polymer mixtures exhibit a non- Newtonian shear thinning behaviour. Where the viscosity and viscoelasticity test reveal that CGCFCT mixture has a typical behaviour of a viscous solution with entangled polymer chains and CGCCommercial mixture has true gel behaviour. The experimental results show us that the generated CGC solution from choline acetate could be used to develop both polymeric film structure and gel. The generated structures are thermally stable at 100° C, and are hydrophilic. The produced films have dense structure and mechanical stabilities against puncture up to 60 kPa.

Isolamento de fungos filamentosos em água utilizada em uma unidade de hemodiálise

A despeito da relativa freqüência de infecções fúngicas oportunísticas em pacientes sob hemodiálise, os reservatórios ambientais destes permanecem desconhecidos, embora alguns estudos recentes tenham correlacionado o suprimento de água como fonte desses microrganismos. O objetivo deste trabalho foi monitorar a qualidade micológica do sistema hídrico de uma Unidade de Hemodiálise, do interior do Estado de São Paulo, Brasil, no período entre abril e julho de 2006. Foram coletadas amostras (15), de 1000mL em 7 pontos de distribuição de água empregando-se técnica da membrana filtrante (0,45µm). Foram isolados 116 fungos filamentosos, dos quais 47 (40,5%) Trichoderma sp, 29 (25%) Cladosporium sp, 16 (13,8%) Aspergillus sp e 11 (9,5%) Fusarium sp. Mediante os resultados, sugerimos que suprimentos de água para Unidades de Hemodiálise devam ser monitorados também quanto ao aspecto micológico, adotando-se medidas profiláticas eficazes que minimizem a exposição destes pacientes imunodeficientes a fontes aquáticas ambientais contaminadas.

Aspergilose pulmonar em paciente imunocompetente e previamente sadio

A aspergilose pulmonar compreende uma das formas de infecção por fungo do gênero Aspergillus, tendo diversos modos de apresentação clínica a depender da imunidade e comorbidades. O objetivo deste trabalho é relatar um caso de paciente, imunocompetente e previamente hígido, que desenvolveu uma forma de aspergilose pulmonar crônica e fazer uma breve revisão sobre o assunto.

Different culture media containing methyldopa for melanin production by Cryptococcus species

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.

Diversity and dynamics of airborne fungi in São Luis, State of Maranhão, Brazil

Introduction: This study aimed to identify airborne fungi in São Luis, Maranhão, Brazil, to determine the prevalent genera and to correlate these genera with the area and season. Methods: In total, 1,510 colony-forming units (CFUs) of airborne fungi were isolated from the north, south, east and west sides and from the center of the city from January to December 2007. The samples were collected on Petri dishes that were exposed to the fungi by the gravitational method. Results: Twenty genera of fungi were isolated; the most common were Aspergillus (33.5%), Penicillium (18.8%), Cladosporium (14.2%), Curvularia (10.6%) and Fusarium (7.6%). The CFUs of the fungi were statistically significant (p < 0.0001). Fungal biological diversity was present all year, without any large seasonal variations but with slight increases in May, August and September. Conclusions: The fungal genera identified in this study were correlated with natural systems and could be useful when evaluating the impact of environmental changes on the region.

Invasive aspergillosis in a user of inhaled cocaine: rhinosinusitis with bone and cartilage destruction

Aspergillosis is an infection caused by saprophytic fungi of the genus Aspergillus, which typically occurs in immunosuppressed individuals, but has also been reported in immunocompetent patients. The main routes of entry are the respiratory tract, skin, cornea, and ear, and the infection may be localized or disseminated by contiguity or vascular invasion. We report a severe case of rhinosinusitis with cutaneous involvement, caused by invasive aspergillosis, in an immunocompetent user of inhaled cocaine. Invasive aspergillosis related to cocaine abuse has not yet been reported in the literature. After itraconazole treatment and surgical debridement, complete clinical remission was achieved. Nasal reconstruction with a skin graft over a silicone prosthesis resulted in a satisfactory esthetic outcome.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Fungos contaminantes e produtores de aflatoxinas em castanha do Pará (Bertholletia excelsa HUMB. & BONPL 1808)

Cem amostras de castanhas do Para, procedentes dos Estados do Amazonas e Sao Paulo, foram analisadas micológico e toxicologicamente para aflatoxinas B1 e G1. Isolou-se 312 colônias de fungos, sendo 91 do gênero Aspergillus, 83 do gênero Penicillium e as restantes incluídas em 23 gêneros diferentes. Entre as amostras de Aspergillus, 26 foram aflatoxigênicos, distribuídos em 3 espécies: Aspergillus flavas Link (18); Aspergillus parasiticus Speare (7) e Aspergillus fresenii Subram (1). Dos 100 extratos de castanhas analisados 3 foram positivos para aflatoxina.

Seleção de fungos produtores de β-D-frutosiltransferase por fermentação em estado sólido

A enzima β-D-frutosiltransferase é responsável pela síntese de FOS (frutooligossacarídeos) a partir de sacarose por reação de transfrutosilação é produzida por diferentes micro-organismos, principalmente por fungos filamentosos. O objetivo deste trabalho foi selecionar a melhor linhagem fúngica produtora da -D-frutosiltransferase por fermentação em estado sólido, bem como o método de extração. A fermentação em estado sólido utilizando o substrato farelo de trigo umedecido com solução de sacarose atingindo 70% de umidade na concentração de esporos de 107 no tempo de 96 horas de crescimento. Todas as linhagens manipuladas apresentaram atividade hidrolítica, no entanto apenas uma linhagem não demonstrou atividade transfrutosilação. O isolado SIS 14 que pertence ao gênero Aspergillus sp. destacou-se pelos maiores valores em atividade no método de extração utilizando água destilada, apresentando 300,90 U/mL na atividade de transfrutosilação e na atividade hidrolítica de 155,74 U/mL. Contudo, pode-se perceber que dos solventes estudados a água destilada foi melhor obtendo o valor em atividade de transfrutosilação, como também a linhagem SIS 14 é promissora para a produção da β-D-frutosiltransferase.

Aspergilli and Penicillia related to the surface of ripening italian grana type cheese and its ripening environment

Information available on the mycoflora associated to ripening Italian “grana type” cheese is very poor. Recently, ochratoxin A (OTA) was detected in samples of packed grated cheese [1]; therefore, the need of information to perform a risk management was highlighted. Moreover, sterigmatocystin (STC) has been reported in cheese and it is considered an emerging problem. Despite the fact that both of them are mycotoxins included in group 2B by IARC [2,3], no European regulation exists. So, the main goal of this work is to give for the first time a general overview about Penicillia and Aspergilli growing on the surface of ripening “grana type” cheese, with particular attention on mycotoxigenic species. To perform this, in 2013 and 2014 crust samples were scratched from ripening grana cheese wheels and also Potato Dextrose Agar plates were exposed to monitor ripening house air. Then, 140 fungal isolates were randomly chosen, purified and monosporic colonies were obtained for their identification at specie level. A polyphasic approach is followed, based on morphological characterisation, toxic extrolites profiling and gene sequencing. The identification is still in progress, but the first results based on the morphological approach showed the presence of mycotoxigenic Aspergilli (Aspergillus flavus and A. versicolor) and various Penicillium species; among them Penicillium chrysogenum, P. implicatum and P. solitum were identified. Only P. chrysogenum was reported to produce the mycotoxins cyclopiazonic acid (CPA) and roquefortine-C (ROQ-C) [4]. These results will be presented and discussed. [1] A. Biancardi, R. Piro, G. Galaverna, C. Dall’Asta, "A simple and reliable liquid chromatography–tandem mass spectrometry method for determination of ochratoxin A in hard cheese" International Journal of Food Sciences and Nutrition 64 (5), 2013, 632 – 640. [2] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of Carcinogenic Risks to Humans” 31, 1983, 191 – 199. [3] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of carcinogenic Risks to Humans”, suppl. 7, 1987, 72. [4] J. I. Pitt, D. A. Hocking, “Fungi and Food Spoilage” 1997, 291.

Avaliação da predação de Podocnemis expansa e Podocnemis unifilis (Testudines, Podocnemididae) no rio Javaés, Tocantins

Podocnemis expansa e P. unifilis são animais de vida longa, com uma demorada maturação sexual, o que influencia uma baixa taxa de substituição de indivíduos. Suas populações são caracterizadas por uma pequena mortalidade dos animais adultos, mas alta taxa de mortalidade de filhotes e embriões. Sendo a predação natural de ninhos e filhotes um dos fatores mais importantes do baixo sucesso de eclosão dessas espécies. No rio Javaés, os ovos e recém-eclodidos podem ser predados por uma grande diversidade de animais: dentre as aves, urubus (Coragyps atratus e Cathartes aura), carcará (Polyborus plancus), jaburu (Jabiru mycteria); lagartos (Tupinambis teguixin) e mamíferos de pequeno porte, coati (Nasua nasua) e cachorro-do-mato (Cerdocyon thous). Do total anual de desovas de P. unifilis em média 65,98% são predadas, sendo 41,68% de forma total e 24,30% parcialmente. Enquanto que apenas 5,31% das ninhadas de P. expansa são sempre parcialmente predadas. Dentre os predadores aquáticos existem diversos peixes, principalmente piranhas (Serrasalmus nattereri) e jacarés (Melanosuchus niger e Caimam crocodilus). Os predadores das fêmeas de P. unifilis são: jacaré-açu (Melanosuchus niger), onça-pintada (Panthera onca) e onça-parda (Puma concolor). Enquanto que as fêmeas de P. expansa em postura, somente são predadas por P. onca. As fêmeas de P. unifilis em postura são predadas num total médio de 3,93% anualmente, enquanto que para P. expansa a média anual é 5,66% das fêmeas.

Superação de dormência na qualidade de sementes e mudas: influência na produção de Senna multijuga (L. C. Rich.) Irwin & Barneby

O objetivo deste trabalho foi determinar a qualidade fisiológica e sanitária de sementes de Senna multijuga(L. C. Rich.) Irwin & Barneby relacionada aos métodos de superação de dormência e à interferência na produção de mudas. As sementes foram submetidas aos seguintes métodos: imersão em água fervente, as sementes foram imersas em água, com temperatura de 100°C, até esfriar, por 24 horas; escarificação ácida, onde as sementes foram imersas em ácido sulfúrico (H2SO4) a 90%, por 10 e 20 minutos, e testemunha (sem tratamento). Foram realizados os testes de sanidade, germinação, tetrazólio e avaliação da qualidade das mudas. O delineamento experimental foi inteiramente casualizado. Para a avaliação da germinação foi utilizado um esquema fatorial (4 X 2), com quatro métodos de superação de dormência X dois fotoperíodos, para os substratos rolo-de-papel e vermiculita. A escarificação ácida constituiu-se no método mais eficiente para a superação da dormência das sementes de Senna multijuga. Penicillium sp. e Aspergillus sp. tiveram sua incidência aumentada quando o tegumento foi danificado pela escarificação ácida por 20 minutos. O controle de Fusarium spp. aumentou gradativamente com o aumento do tempo de exposição ao ácido sulfúrico.