Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Diploma thesis Author: Dana Kettnerová Supervisor: PharmDr. Jan Martin, Ph.D. Title of diploma thesis: Isolation of plant organelles and study of transport mechanisms Key words: isolation, chloroplast, protoplast, vacuole, cell wall Isolation of plant organelles and other cellular components is essential for the study of physiological and pathological processes within the plant cell. It is possible to analyze cell structures, detect accumulation of certain metabolites, ions, enzymes and other substances thanks to the isolation. The goal of this diploma thesis was to provide an overview of isolation methods used for the isolation of cell wall, protoplasts, chloroplasts and vacuoles of plant cells. Isolation processes used for individual types of cell structures, the pros and cons of the various isolation methods, components of used media and their functions, as well as the structure and function of individual plant structures were described.

Industrial machines during their operation generate vibration due to dynamic forces acting on the machines. This vibration may create noise, abrasion in the machine parts, mechanical fatigue, degrade performance, transfer to other machines via floor or walls and may cause complete shutdown of the machine. To limit the vibration pre-installation, vibration isolation measures are usually employed in workshops and industrial units. However, such vibration isolation may not be sufficient due to varying operating and physical conditions, such as machine ageing, structural changes and new installations etc. Therefore, it is important to assess the quantity of vibration generated and transmitted during true operating conditions. The thesis work is aimed at the estimation of vibrational transmissibility or transfer from industrial machines to floor and to other adjacent installed machines. This study of transmissibility is based on the measurement and analysis of various spectral estimation tools such as Power Spectral Density (PSD), Frequency Response Function (FRF) and Coherence Function. The overall study is divided into three major steps. Firstly, the initial measurements are carried in BTH on simple Single Degree of Freedom (SDOF) systems to gain confidence in measurement and analysis. Then the measurements are performed on a Lathe machine “Quick Turn Nexus 300-II” in a laboratory at BTH. Finally, the measurements are taken on the machines of an Industrial workshop (KOSAB). The analysis results revealed that vibration measurements in industry are challenging and not easy as measurement in labs. Measurements are contaminated by noise from other machines, which degrade the coherence function. However, vibration transferred from one machine to the floor or other machines may be studied using FRF and PSD. Appropriate further isolations may be employed based on the spectral analysis.

Background: Accurate diagnostic methods for Clostridium difficile infection (CDI) are required for optimal patient management, appropriate implementation of infection control measures and surveillance. An assay that also provides rapid results, is easy to implement in a routine diagnostic lab and is cost-effective would be ideal. Laboratory testing for Clostridium difficile infection is rapidly evolving. Recently published literature has shown that immunoassays for toxin detection, whilst being cheap and easy to implement, lack sensitivity. Molecular diagnostics that are sensitive and provide rapid results are now available. However, the high cost of these assays is of concern. As reflected in the literature the optimal test or testing algorithm for Clostridium difficile infection diagnosis is not clear. Objectives: This study aimed to compare the performance of a real-time PCR assay and two immunoassays, and to establish the optimal testing strategy for Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). Methods: Using toxigenic culture as the gold standard, the Roche PCR assay for the detection of the tcdC gene, the Immuno Card Toxins A & B immunoassay and the C. Diff Quik Chek Complete immunoassay were evaluated as stand alone assays and as part of testing algorithms. Results: The sensitivity, specificity, positive predictive value and negative predictive value of the various assays and algorithms ranged from 38% to 81%, 98% to 100%, 92% to 100% and 85% to 95%, respectively. The charge per sample tested varied widely depending on the assay and algorithm used. The maximum turnaround time ranged between four and twenty four hours. Conclusion: The algorithm combining glutamate dehydrogenase and toxin immunoassay testing of all samples followed by PCR testing of only a subset of samples, performed the best, providing accurate results rapidly and cost-effectively.

O coronavírus canino (CCoV) causa gastroenterite em cães jovens, podendo ser letal, sobretudo quando há coinfecção com parvovírus canino (CPV). Os objetivos do presente projeto foram investigar a presença de CCoV e CPV em amostras fecais de cães jovens; estudar a diversidade molecular das amostras de CCoV com base em sequenciamento parcial dos genes M, S, 3b e N, incluindo amostras vacinais, e estudar a evolução in vitro de CCoV em células A72 de fibroma canino. Foram detectados 40,17% (47/117) animais positivos para CCoV e 13,68% (16/117) para CPV. Estudos filogenéticos demonstraram que oito amostras foram classificadas como CCoV-II, vinte e cinco como CCoV-I. Análises para o gene M destacaram alta identidade de CCoV-I com amostras de coronavírus da peritonite infecciosa felina (PIF) e uma possível amostra pantrópica foi demonstrada pela análise do gene S. O gene do nucleocapsídeo de CCoV é altamente conservado entre os tipos I e tipo II, com uma resolução mais baixa em relação a árvores para os genes M e S. Amostras dos tipos I e II apresentam um polimorfismo baixo para o gene 3b, sem marcadores estáveis para diferenciação dos tipos de CCoV. Uma amostra de CCoV-II putativamente pantrópica foi isolada em células A72, resultando em efeito citopático no 5o dia da 5a passagem. No estudo evolutivo, a amostra vacinal CCV 1-71 e nove passagens desta em células A72 foram submetidas a amplificação parcial e clonagem molecular do gene S seguida de sequenciamento de DNA. Os resultados mostraram mutações não silenciosas, silenciosas e três deleções de aminoácidos, mas nenhuma mutação compartilhada entre as diversas passagens. Amostras vacinais de CCoV-II adaptadas em células podem ser altamente geneticamente estáveis após passagens em série em uma mesma linhagem celular, acumulando substituições de nucleotídeos principalmente sinônimas no gene S devido a relação célula - hospedeiro estável. Estes resultados de epidemiologia molecular e processos evolutivos de CCoV podem servir para uma melhor compreensão da virologia básica e ser base de dados para estudos em outros coronavírus / Canine coronavirus (CCoV) causes gastroenteritis in young dogs and can be lethal, especially when there is co-infection with canine parvovirus (CPV). The objectives of this project were to investigate the presence of CCoV and CPV in stool samples from young dogs, the molecular diversity of CCoV strains based on partial sequencing of genes M, S, N and 3b, and the in vitro evolution of CCoV in A72 canine fibroma. Of the fecal samples studied, 40.17% animals (47/117) were positive for CCoV and 13.68% (16/117) for CPV. Phylogenetic studies have shown that eight strains were CCoV-II and twenty five CCoV-I. Phylogenetic analysis for the M gene highlighted the high identity of CCoV-I strains with a feline coronavirus strain (FCoV) that causes feline infectious peritonitis (FIP). Further analysis based on the spike gene showed a putative pantropic CCoV strain (CCoV-II/dog50). CCoV nucleocapsid gene is highly conserved among type I and type II, with a lower resolution relative to trees based on M and S genes. CCoV Types I and II had a low polymorphism for 3b gene, without any stable markers to differentiate thee types. Regarding the virus isolation trial, a putative pantropic CCoV-II strain was successfully isolated in A72 cells from, resulting in cytopathic effect on the 5th day of the 5th passage. In the evolutionary study, the vaccine strain CCV 1-71 and nine passages of this strain in A72 were submitted to partial S gene amplification and molecular cloning followed by DNA sequencing. Missense, silent, and three amino acids deletions were found amongst diverse clones of each passage, but no mutation was repeatedly found among passages. Cell culture-adapted CCoV-II vaccine strains can be highly genetically stable upon serial passage in a same cell line, accumulating primarily synonymous nucleotide substitutions in the S gene due to a stable cell-host relationship. In short, all data gathering herein on molecular epidemiology and evolutionary processes of CCoV can serve for a better understanding of basic virology and as a basis for studies on other coronaviruses

Thesis advisor: Steven D. Bruner / Thesis advisor: Mary F. Roberts / The fuscachelins are a group of novel small molecule secondary metabolites produced by the thermophilic actinomycete <italic>Thermobifida fusca</italic>. A genome mining approach was employed to identify the fuscachelin nonribosomal peptide synthetase biosynthetic gene cluster in <italic>T. fusca</italic>. The peptide natural products were predicted to be siderophores, iron-scavenging small molecules. An assay guided fractionation approach was utilized to isolate the fuscachelins. Structure elucidation efforts employed nuclear magnetic resonance, mass spectrometric and chemical degradation techniques to determine the structure of the isolated compounds. Once the structure was known, a total synthesis was undertaken. The established synthetic route to the fuscachelins will allow for the facile development of custom-designed chemical tools for the further study of the fuscachelin biosynthetic enzymes and utilization proteins. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A aquicultura é apontada como um mercado estratégico para o desenvolvimento sustentável, produção de alimentos e ampliação de fronteiras inexploradas no Brasil. No entanto, como outros sistemas de produção animal, este setor enfrenta problemas com doenças resultantes de sua intensificação, como os aspectos sanitários da produção e a falta de estrutura para o diagnóstico das principais enfermidades infecciosas. Durante os últimos 20 anos, os vírus da família Iridoviridae, em especial membros do gênero Ranavirus, têm sido responsáveis por epizootias de grande impacto ecológico e econômico, envolvendo um grande número de espécies de peixes, anfíbios e répteis de importância na aquicultura de várias partes do mundo. No entanto, as informações sobre a ocorrência de infecções de peixes e anfíbios causadas por ranavírus no Brasil são limitadas. O presente projeto compreendeu o isolamento em cultivo celular de estirpe de Frog Virus 3-símile, no Estado de São Paulo, confirmada por sequenciamento nucleotídico do gene MCP e subsequente RFLP, assim como caracterização fenotípica e de cinética de replicação do isolado. Amostras de fígado, baço e rins de rãs-touro gigante provenientes de ranário comercial, positivas ao diagnóstico molecular para Ranavirus, foram utilizadas para isolamento viral em cultivo celular. Efeito citopático foi detectado na segunda passagem em células BF-2 e posterior confirmação do isolamento foi feita por PCR e RFLP. A caracterização fenotípica viral foi feita com microscopia eletrônica de transmissão, a qual permitiu a confirmação de morfologia semelhante às partículas de Ranavirus, além da realização de curva de replicação, indicando maiores títulos virais no quarto dia após inoculação. Também foi feito teste de susceptibilidade a solventes, que confirmou a presença de partículas envelopadas. Os resultados obtidos nesse estudo poderão contribuir para a futura compreensão da biologia dos iridovírus circulantes como agentes etiológicos de ranaviroses em anfíbios no Estado de São Paulo. / Aquaculture is appointed as a strategic market for sustainable development, food production and expansion of unexplored frontiers in Brazil. However, just like other animal production systems, this area faces problems due its intensification, like the production sanitation aspects and the lack of structure for the diagnosis of major infectious diseases. During the last 20 years, viruses from Iridoviridae family, especially Ranavirus genera, have been responsible for major economic and ecological impactant epizootic diseases in a great number of fish, amphibian and reptile species in many parts of the world. However, information on the occurrence of infections in fishes and amphibians caused by Ranavirus in Brazil are limited. In this context, this project aimed to isolate a Frog Virus 3-like strain detected in São Paulo state in cell culture, which was later confirmed by nucleotide sequencing of MCP gene and further RLFP technique and also phenotipical characterization and replication kinetics of the obtained strain. Liver, spleen and kidney samples from american bullfrogs from commercial frog ponds, positive by molecular diagnostic for Ranavirus, were used for viral isolation in cell culture. Citopathic effect was detected during the second passage in cells and later confirmed by PCR and RFLP. The viral characterization was carried out with transmission electronic microscopy, which confirmed similar morphology of viral particles to those of Ranavirus, besides the construction of a growth curve which indicated larger titres on the fourth day post inoculation. A test for solvent sensitivity was also performed and confirmed the presence of enveloped particles. The results obtained in this study will contribute to the understanding of the biology of the circulating iridovirus in São Paulo state as an etiological agent of Ranavirus infection in amphibians.

Examining the ecological processes generating evolutionary patterns is crucial to understanding how biodiversity arises and evolves. One of the most striking examples of evolutionary diversification is provided by the flowering plants (angiosperms) and their flowers. Pollinators are traditionally considered to be the most important selective agents and drivers of floral diversity. However, many angiosperms have a generalized floral morphology and are visited by a diverse and overlapping suite of pollinators, making it unclear how pollinators could have driven diversification in these taxa. In addition, flowers and plant reproductive success are likely to be influenced by factors other than pollinators, such as herbivores, precipitation, and temperature. These factors need to be considered along with pollinators in order to improve our understanding of angiosperm evolution and diversification. In my thesis, I focussed on the processes influencing adaptation and diversification in flowering plants in the genus Mertensia (Boraginaceae), which have relatively unspecialized flowers that attract a variety of nectar- and pollen-feeding insects. In Chapter One, I explored correlations among floral traits, vegetative traits, and flowering phenology across 12 Mertensia species. In Chapter Two, I assessed reproductive isolating barriers between related Mertensia species occurring in sympatry. In Chapter Three, I examined the ecological function of floral orientation in two Mertensia species with respect to pollinators and precipitation. First, across Mertensia species, I found that early-flowering species were shorter, produced fewer flowers, and occurred at higher altitudes than late-flowering species—suggesting a life-history trade-off between plant size and flowering phenology, as well as an altitudinal effect on both traits. Interspecific variation in floral traits was not strongly associated with variation in flowering phenology or plant size. Second, between sympatric M. brevistyla and M. fusiformis populations, I found weak reproductive isolating barriers and possible hybridization. Most pre-pollination barriers were weak, as the two Mertensia species shared similar habitats, flowering phenology, and pollinator assemblages. The two relatively strong barriers were floral (ethological and mechanical) isolation and post-pollination isolation: Pollinators transferred significantly more of a pollen analogue among conspecific than heterospecific plants in mixed-species arrays, and flowers yielded higher seed set when receiving conspecific rather than heterospecific pollen in hand-pollination experiments. Lastly, I found that floral orientation was more likely to be under selection by precipitation than by pollinators, in that paternal fitness (i.e., pollen germination) was reduced by contact with water and that pollinator-mediated selection via maternal fitness (i.e., seed set) was not detected. A more pendant floral orientation likely protects the relatively long and exposed anthers of M. fusiformis from rain, while the less pendant M. brevistyla does not require this protection because of its shorter, more concealed reproductive structures. Although I detected an effect of floral orientation on seed set, I was not able to identify the selective agents driving this effect. In summary, my results suggest that pollinators play a minor role in influencing floral adaptation and diversification in Mertensia. Instead, the dominant influences on the traits I examined appear to be life-history trade-offs, environmental conditions that vary along altitudinal gradients, and abiotic variables (e.g., precipitation). It is important to consider these factors and their influences on paternal and maternal fitness in order to gain a broader perspective on floral evolution in plants with generalized pollination systems.

A xilanase e a &alpha;-amilase são enzimas responsáveis por degradar parte da matéria orgânica nos solos, atuando na mineralização da hemicelulose e do amido, respectivamente. Do ponto de vista biotecnológico, estas enzimas, assim como os subprodutos gerados pela sua ação enzimática, podem ser utilizados em diversos setores industriais. Os manguezais podem representar uma fonte promissora e inexplorada para a busca de enzimas microbianas em função das altas quantidades de matéria vegetal em decomposição presente neste ecossistema, onde ocorrem condições ambientais únicas, representadas pela combinação da anaerobiose e salinidade. No presente trabalho, métodos independentes e dependentes de cultivo microbiano foram utilizados para a bioprospecção de enzimas xilanolíticas e amilolíticas em um ambiente de manguezal. A partir da triagem funcional de uma biblioteca metagenômica foram obtidos 3 clones xilanolíticos. Estes foram sequenciados e os reads utilizados para montagem dos contigs e posterior anotação dos genes. Uma das ORFs geradas da anotação do contig MgrBr135 foi utilizada para subclonagem em vetor de expressão demonstrando atividade amilolítica. A análise filogenética do contig MgrBr135 mostrou afiliação da mesma ao filo Planctomycetes. Por meio do método dependente de cultivo, foram isolados 44 bactérias xilanolíticas do solo de manguezal. Destas, 73% apresentaram índices enzimáticos (IE) elevados quando submetidos ao teste qualitativo (cup plate). A estirpe bacteriana 11 foi a que se destacou das demais por ter apresentado IE de 10,9. Quando submetidas ao teste quantitativo, o isolado 39 se destacou produzindo uma atividade enzimática de 0,43 U/mL. O sequenciamento parcial do gene 16S rRNA das estirpes permitiu a identificação dos isolados como pertencentes a dois gêneros: Bacillus e Paenibacillus. Estes resultados destacam a importância de estudos sobre as bactérias encontradas nos manguezais, e indicam os grupos bacterianos que hospedam estas características, o que deve dar maior suporte a exploração deste recurso como ferramentas biotecnológicas, a serem utilizadas na degradação da hemicelulose e amido. / Xylanase and &alpha;-amylase are enzymes that degrade organic matter, acting in the mineralization of hemicellulose and starch, respectively. These enzymes, as well as the byproducts generated by them, can be used in various industrial sectors. Mangroves represent a promising and untapped source of microbial enzymes due to the high content of decomposing organic matter present in this ecosystem, where unique environmental conditions prevail, represented by the combination of anaerobiose and salinity. In this study, independent and dependent microbial cultivation methods were used for bioprospecting xylanolytic and amylolytic enzymes in a mangrove environment. Through the functional screening of a metagenomic library, 3 xylanolytic clones were obtained. These clones were sequenced, and the reads were used for contig assembly followed by gene annotation. One of the ORFs generated by the annotation of contig MgrBr135 was used for subcloning into the expression vector, showing later amylase activity. Phylogenetic analysis of contigs MgrBr135 indicated its affiliation to the phylum Planctomycetes. Through the cultivation dependent method, 44 xylanolytic bacteria were isolated from mangrove soil. From these, 73% had considerable enzymatic index (EI) when subjected to the qualitative test (cup plate). The bacterial strain 11 was the one that stood out from the others because it presented the highest EI, 10.9. When subjected to quantitative test, the isolated 39 stood out producing an enzyme activity of 0.43 U/mL. The partial 16S rRNA gene sequencing of the strains allowed the characterization of two genera: Bacillus and Paenibacillus. These results highlight the importance of studying the bacterial community found in mangroves, and indicate the bacterial groups hosting these features, supporting further exploitation of this resource as biotechnological tools that can be used in the degradation of hemicellulose and starch.

Hantavirus é um gênero na família Hantaviradae, incluindo agentes polimórficos, envelopados e com genoma de RNA fita simples, polaridade negativa e trissegmentado. Os Hantavírus são zoonóticos e mantidos na natureza em reservatórios das ordens Rodentia, Soricomorpha e Chiroptera. No Brasil, foram descritos 8 Hantavírus e destes, 6 causam doença humana grave e de alta letalidade, a Síndrome Pulmonar e Cardiovascular, sendo dentre eles o Araraquara (ARQV) o vírus de ocorrência nas regiões de Cerrado do Sudeste (incluindo Ribeirão Preto) e Planalto Central. ARQV destaca-se por produzir a maior letalidade dentre todos os Hantavírus existentes e possuir como reservatório o roedor silvestre Necromys lasiurus que o transmite ao homem por inalação dos aerossóis contaminados de suas excretas. Neste trabalho, detectamos genoma de Hantavírus em tecidos de Necromys lasiurus e em tecidos de morcego Desmodus rotundus, ambos capturados no Nordeste do Estado de São Paulo. Destes animais, foram feitas tentativas de isolamento de Hantavírus a partir de amostras de tecido de Necromys lasiurus e Desmodus rotundus. Os procedimentos foram realizados em laboratório de nivel de biossegurança 3, onde fizeram-se as inoculações em cultura de células VERO E6, com detecção viral após uma única passagem de 4 dias. Desta forma, foi possível isolar um Hantavírus a partir de sobrenadante do lisado de coração do roedor, o que foi confirmado pela presença do genoma viral do isolado por RT-PCR do sobrenadante de cultura celular e dos antígenos virais nas células Vero E6, por imunofluorescência indireta e western blot. O processo de isolamento mostrou-se reprodutível, por 3 vezes, para o mesmo tecido do roedor. Tais resultados encorajam que esta metodologia para isolamento de Hantavírus deva ser experimentada por mais vezes. O Hantavírus isolado (provavelmente ARQV) deverá fornecer importantes informações sobre seu genoma completo, além de propiciar vários estudos futuros. / Hantavirus is a genus in the Hantaviradae family, including polymorphic agents, it is enveloped and with a single stranded RNA genome, negative and tri-polarity polarity. Hantaviruses are zoonotic and kept in nature in reservoirs of the orders Rodentia, Soricomorpha and Chiroptera. There are 8 hantaviruses recorded in Brazil, 6 of them cause severe human disease and high lethality, a Pulmonary and Cardiovascular Syndrome, among which Araraquara (ARQV) is the virus that occurs in the Southeastern Cerrado (including Ribeirão Preto) and Central Plateau. ARQV stands out for producing higher lethality among all existing hantaviruses and has as reservoir the wild rodent Necromys lasiurus, which transmits to humans by inhalation of the contaminated aerosols of their excreta. In this work, we detected the genome of hantavirus in Necromys lasiurus and Desmodus rotundus, bat tissues, both captured in the Northeast of the State of São Paulo. Of these animals, attempts were made to isolate hantavirus from tissue samples of Necromys lasiurus and Desmodus rotundus. The laboratory tests of biosafety level 3, where inoculations in culture of VERO cells E6 were done, with viral detection after a single passage of 4 days. Thus, it was possible to isolate hantavirus from the rodent heart lysate supernatant, which was confirmed by the presence of the viral genome of the isolate by RT-PCR of the cell culture supernatant and the viral antigens in the Vero E6 cells by Indirect Immunofluorescence and Western Blot. The isolation process was reproducible, 3 times, for the same tissue of the rodent. These results indicate that this methodology for hantavirus isolation should be tested more often. The isolated Hantavirus (ARQV), should provide important information about its complete genome, as well as providing several future studies.

Coronavirus bovino (BCoV), um membro da família i>Coronaviridae, causa severa diarréia em bezerros neonatos e tem sido associado a diarréias de inverno em vacas leiteiras em vários paises, incluindo o Brasil. A morbidade da disenteria de inverno e alta chegando ate 100% , sendo um fator importante para economia já que causa queda da produção leiteira, levando a grandes perdas as criações de vacas leiteiras. O objetivo deste trabalho foi pesquisar a ocorrência de BCoV em vacas, diagnosticando amostras positivas por RT-PCR gene Rp Rd e isolando estas amostras positivas em células da linhagem HRT-18G. As amostras de fecais foram obtidas de 43 vacas leiteiras com disenteria de 8 propriedades dos Estados de São Paulo e Minas Gerais, Brasil. Das dez (10/43=23%) amostras positivas para esta técnica, 7 foram inoculadas em células da linhagem HRT-18G, sendo que o isolamento foi comprovado pela mesma técnica após seis passagens seriadas em 4 inoculações. Com isso, mostra-se que o BCoV também esta envolvido em disenterias de inverno em vacas leiteiras no Brasil. E através de isolamentos deste vírus, podemos contribuir para estudos continuados ajudar no esclarecimento de sua epidemiologia e possibilitar com um banco de vírus a prevenção de ordem também especifica da enfermidade. / Bovine coronavirus (BCoV), a member of Coronaviridae family, causes severe diarrhea in newborn calves and has been associated with outbreaks of winter dysentery (WD) in adult cattle in several countries, including Brazil. The morbidity rate of WD is very high (50-100%) and the disease causes severe economic losses once it decreases milk production. The aim of the present study was to survey for the occurrence of BCoV in cows using a RT-PCR targeted to the replicase gene and to isolate positive samples in HRT-18G cells. The fecal samples were obtained from 43 adult dairy cows with dysentery from São Paulo and Minas Gerais States, Brazil. Ten (23%) of the 43 fecal samples were positive for BCoV and 7 of these were inoculated in HRT-18G cells, when the isolation of 4 samples was proved by RT-PCR after sex passages. These findings indicate that BCoV is also involved in outbreaks of dysentery in adult cattle in Brazil. This shows the importance of more comprehensive studies on coronavirus in dairy cattle in the surveyed area and, with the isolation of the virus strains studied herein, one may contribute to other studies to enlighten the epidemiology and prevention of the disease.