L’infection par le virus de la maladie de Marek (MDV) induit chez les poussins une immunosuppression précoce et transitoire associée à une atrophie du thymus (TA) et de la bourse de Fabricius (BA). L’objectif de ce travail était de mieux comprendre les mécanismes cellulaires responsables de cette atrophie. La 1ère étape de mon travail a été d'établir un modèle d'infection in vivo menant à une BA et TA suffisante et reproductible. Une fois établi, ce modèle nous a permis d’étudier l’effet du MDV au sein de ces organes à 6, 10 et 14 jours post-infection. Les résultats indiquent que l'apoptose et l'inhibition de prolifération peuvent contribuer à la BA, tandis que seule l'augmentation de l'apoptose conduit à la TA. La numération des lymphocytes sanguins, a mis en évidence une diminution des lymphocytes B durant les 2 premières semaines post-infection concomitamment à la BA. Cette technique semble donc un outil prometteur non invasif pour diagnostiquer la BA induite par le MDV. Une différence de sensibilité à la TA et BA induite par le MDV a été mise en évidence dans deux lignées de poule White Leghorn d’haplotype B différents, toutes deux très sensibles au développement des tumeurs. / Marek's disease virus (MDV) infection induces early and transient immunosuppression associated with atrophy of the thymus (TA) and the bursa of Fabricius (BA) in chicks. The aim of this work was to better understand the cellular mechanisms responsible for this atrophy. The first step in my work was to establish an in vivo infection model leading to a sufficient and reproducible BA and TA. Once established, this model allowed us to study the effect of MDV in these organs at 6, 10 and 14 days post-infection. The results indicate that apoptosis and inhibition of proliferation can contribute to BA, whereas only the increase in apoptosis leads to TA. The lymphocyte blood count showed a decrease in B-lymphocytes during the first 2 weeks of infection that seems related to BA. This technique therefore seems a promising non-invasive tool to diagnose BA at early times of MDV infection. A difference in sensitivity to MDV-induced TA and BA was found in two lines White Leghorn of different B haplotype, both of which very sensitive to tumor development.

Orientador : Profª. Drª Daniela Parada Pavoni / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 31/08/2016 / Inclui referências : f. 106-114 / Resumo: A doença infecciosa da bursa (IBD) ou doença de Gumboro é uma doença viral de galinhas (Gallus gallus) relevante para o comércio internacional de animais e de seus produtos. Está associada à mortalidade e imunossupressão dos animais. Considerando a importância econômica da avicultura para o Brasil e o estado do Paraná, é obrigatório a tomada de medidas de controle em granjas comerciais, que visem a prevenção de surtos e a vigilância sobre o aparecimento de novas variantes, mais patogênicas, e que não respondem às vacinas. Testes diagnósticos confiáveis são ferramentas importantes no controle dessa infecção. A utilização de proteínas recombinantes como substrato de testes sorológicos é uma atraente alternativa para a produção nacional de reagentes, uma vez que os testes atualmente utilizados são importados. Sequências das proteínas antigênicas VP2 e VP3 de cepas brasileiras e estrangeiras, selvagens e vacinais foram buscadas no banco de dados do NCBI e alinhadas utilizando o programa ClustalW. Foram selecionadas 10 sequências nucleotídicas para síntese comercial clonadas no pUC57, das quais 9 da VP2 e uma da VP3. Os genes sintéticos foram clonados no vetor pET-28a (Novagen) para expressão em Escherichia coli e pMIB/V5-His (Invitrogen) para expressão em células de insetos. Fragmentos da VP3 foram obtidos por PCR fusionados à GFP para aumentar a massa molecular dos peptídeos. A expressão da VP2 em células de insetos e em E. coli não foi obtida. A VP3 e os fragmentos foram expressos na cepa BL 21 Star. A VP3 foi expressa em corpos de inclusão e solubilizada com 2 M de ureia. Os fragmentos foram obtidos na fração solúvel. A purificação das proteínas foi realizada por afinidade em resina de níquel. A reatividade da VP3 e fragmentos foi testada no teste de ELISA indireto. O teste de ELISA da VP3 com soros de animais vacinados e não vacinados apresentou 93,75% de sensibilidade e 85,19% de especificidade. Utilizando somente soros de animais imunizados o teste de ELISA apresentou 100% de sensibilidade e especificidade. O mapeamento das regiões antigênicas da VP3 mostrou duas regiões, fragmentos 2 e 7, que concentraram maior número de epitopos. Os fragmentos 5, 6, e 7 foram as regiões da VP3 mais reativas. Este estudo mostrou que a proteína recombinante VP3 pode ser utilizada para avaliar o status imunológico pós-vacinal das aves. A utilização da VP3 recombinante para diagnosticar a presença/ausência da doença em animais não vacinados deve ser melhor avaliada utilizando uma amostragem maior desses animais no teste de ELISA. Através dos resultados do mapeamento foi possível estabelecer regiões da proteína VP3 mais antigênicas, entretanto mais testes devem ser conduzidos antes de considerar a utilização dos fragmentos no diagnóstico do vírus da doença infecciosa da bursa (IBDV) em galinhas. Palavras-chave: doença infecciosa da bursa, doença de Gumboro, proteína recombinante, vírus da doença infecciosa da bursa, proteína VP2, proteína VP3, mapeamento de regiões antigênicas, teste de ELISA, diagnóstico da doença infecciosa da bursa. / Abstract: Infectious bursal disease (IBD) or Gumboro disease is a viral disease of chickens (Gallus gallus) relevant to international trade in animals and their products. It is associated with mortality and immunosuppression in animals. Considering the economic importance of poultry production to Brazil and to the state of Paraná, taking control measures on commercial farms is mandatory for the prevention of outbreaks and surveillance of the appearance of new more pathogenic variants that do not respond to vaccines. Reliable diagnostic tests are important tools in the control of this infection. The use of recombinant proteins as serological tests substrates is an attractive alternative to the domestic production of reagents, since the currently used tests are imported. Sequences of antigenic proteins VP2 and VP3 of Brazilian and foreign strains, wild and vaccine were sought in the NCBI database and aligned using the ClustalW program. Ten nucleotide sequences were selected, 9 sequences of VP2 and one of VP3. These sequences were commercially synthesized and cloned into pUC57. The synthetic genes were cloned into pET-28a vector (Novagen) for expression in Escherichia coli and into pMiB/V5-His (Invitrogen) for expression in insect cells. Fragments obtained by PCR using VP3 as template were used fused to GFP to increase the molecular weight of the peptides. Expression of VP2 in insect cells and E. coli was not obtained. The VP3 and the fragments were expressed in BL 21 Star strain. The VP3 was expressed in inclusion bodies and was solubilized with 2 M urea. The VP3 fragments were obtained in the soluble fraction. Purification of the protein was accomplished by nickel affinity resin. The reactivity of VP3 and fragments was tested by ELISA. The ELISA test with VP3 sera of animals vaccinated and non-vaccinated showed sensitivity of 93.75% and specificity of 85.19%. Using only the sera of immunized animals ELISA showed sensitivity and specificity of 100%. Mapping the antigenic regions of VP3 showed that two regions, fragments 2 and 7, present the highest proportion of epitopes. Fragments 5, 6, and 7 were the most reactive VP3 regions. This study showed that the recombinant protein VP3 can be used to assess post-vaccination immune status of the birds. The use of recombinant VP3 for diagnosing the presence / absence of the disease in non-vaccinated animals should be further evaluated using a larger sample of these animals in the ELISA test. Through the results of the mapping it was possible to establish regions of more antigenic protein VP3, though more tests should be conducted before considering the use of fragments in the diagnosis of IBDV in chickens. Keywords: infectious bursal disease, Gumboro disease, recombinant protein, infectious bursal disease vírus, VP2 protein, VP3 protein, mapping of antigenic regions ELISA test, diagnosis of infectious bursal disease.

B cell development in chicken takes place in a specific primary lymphoid organ, the bursa of Fabricius. The bursa is considered to provide a microenvironment that promotes B lymphocyte survival and maturation. The most important maturation step in the bursa is the immunoglobulin (Ig) gene conversion, a process that is responsible for immunoglobulin repertoire in avian species. The Ig-gene conversion is strictly regulated, and only progenitors that are able to initiate the process will develop into fully functional B lymphocytes. In this study the late embryonic B lymphocyte stages are investigated, the bursal stem cell stage and the onset of Ig-gene conversion stage. Previous studies identified functional and phenotypic differences between the two stages, showing high rate of proliferation at both, but a significant increase in apoptotic activity at the onset of gene conversion stage. The molecular basis behind the initiation of Ig-gene conversion is not well understood. Here two approaches are presented to provide information on the B lymphocyte developmental process. In chapter II proteomic analysis of the two cell stages was performed. The proteins were sorted into functional groups and signal transductions pathways were identified that are associated with proliferation, differentiation, cell adhesion and apoptosis. The project identified differences in protein profiles that might explain the changes in B lymphocyte physiology and bursal microenvironment at the initiation of Ig-gene conversion. In chapter III the antigen recognized by a bursal secretory dendritic cell specific monoclonal antibody, GIIF3 was identified and cloned. The antigen was shown to be expressed by bursal secretory dendritic cells only during the late embryonic period. The antigen was identified as smooth muscle gamma actin. Futher work will investigate what role the gene plays in dendritic cell funtion.

Yes / Capsella bursa-pastoris (L.) Medik. is a species with a cosmopolitan distribution which shows considerable morphological variation. Numerous authors have recognised widely differing numbers of varieties, microspecies or other infraspecific subdivisions (segregates) of this species. In an attempt to clarify this situation, we grew British material of the species under controlled conditions through to the F) generation to remove environmental variation, and assessed the plants on the basis of a range of morphological criteria, namely leaf shape, capsule size and also length of time taken to flower. Analysis of these characteristics consistently produced four basic groups, which had been previously described. Herbarium specimens could also nearly always be assigned to one of these groups. Limited chromosome counts suggest that two of these groups are diploid and two are tetraploid. We suggest this fourfold division into broad groups reflects the major genetic separations within the species, but that there is also considerable phenotypic plasticity shown by C. bursapastoris in response to factors such as shade or trampling. These four groups appear to differ in their geographic.al distribution in Britain. KEYWORDS: Shepherd's Purse, morphological variation, leaf characters, capsule characters, chromosome

No

Historic commercial centres are subject to continuous change and transformation due to the changing socio-economic and cultural structure and relations of production and consumption. Organized within a traditional structure, these spaces have survived until today with their altering meanings and different identities as well as the restructuration they went through because of the socio-economic transformation processes. Besides, they are not only shopping areas or consumption spaces but also urban areas with a traditional style of manufacture and crafts. Theoretical discussions on the conservation of historic city centres focus on issues such as integration of city centres into contemporary life with functions that do not have adverse effects on the authentic characteristics of historic streets and urban fabrics, the role of new functions, activities and structures in the historic fabric, and participation in the conservation process. Historic buildings adopt functions that are different from their original functions due to changing life styles. Having functioned in a well-organized order and shaped by the socio-cultural and economic structure of their eras, they are subject to interventions due to the functions required by the present conditions, such as the addition of a mezzanine or the establishment of a connection between spaces. As a result of these interventions, the original plan schemes and authentic characteristics of historic buildings have changed. This thesis aims at identifying the types of commercial functions that intervene into the authentic design characteristics of commercial buildings located in the historic commercial centre. To this end, the Commercial Centre of Bursa was chosen as the case and the change in the functions of the hans located in this area was examined in detail.

Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

<p>Инфективна болест бурзе (Гамборо болест) је контагиозна вирусна болест младих пилића. Узрочник је РНК вирус који припада фамилији Birnaviridae. Испољава тропизам према Фабрицијусовој бурзи доводећи до оштећења лимфоцита и атрофије. Болест се контролише вакцинацијом родитељских јединки и пријемчивих пилића. Употреба атенуираних вакцина може довести до смањења неких технолошких параметара. Циљ овог рада је био да се докаже које од примењених атенуираних вакцина, интермедијарних Гумбокал, Гумбокал D78 и интермедијарне-плус Гумбокал 228Е, индукује најбољи развитак имнунолошког одговора и доводе до најмањих оштећења у ткиву Фабрицијусове бурзе и слезене. Пратили смо да ли делују имуносупресивно на вакцину против Њукастл болести и да ли утичу на смањење телесне масе бројлерских пилића. Основне огледне групе су вакцинисане четрнаестог дана старости против Гамборо болести, а затим су од основних огледних група оформљене подгрупе које су вакцинисане против Њукастл болести у различитим временским периодима: О1-1, О2-2, О3-1 после седам дана, О1-2, О2-2, О3-2 после четрнаест дана, О1-3, О2-3, О3-3 после двадесетједан дан од вакцинације против Гамборo болести .Од првог до четрдесетдругог дана експеримента сваких седам дана је вађена крв пилићима, мерена њихова телесна маса и узорковане бурзе и слезене. Крвни серуми су испитивани на висину титра антитела ЕЛИСА тестом за Гамборо болест, а методом инхибиције хемагутинације за Њукастл болест. Највишу висину титра на Гамборо болест је показала огледна група О1, затим група О2 и најнижи О3. У огледним подгрупама О3-1, О3-2 и О3-3 се испољио имуносупресивни ефекат на имунолошки одговор према Њукастл болести. Фабрицијусовим бурзама је одређивана релативна маса и бурзални индекс да би се установило да ли је дошло до атрофије након примене вакцина. Најнижи бурзални индекс је установљен у огледној групи О3, затим у О2 и у О1. Таквим редоследом су устновљене и патохистолошке промене у бурзи. Добијени резултати указују да употреба интермедијане-плус вакцине индукује најбољи имунолошки одговор, али и најнижу релативну масу и вредност бурзалног индекса, као и нижу телесну масу у односу на инермедијарне вакцине. Примењене вакцине нису утицале на релативну масу слезене и нису довеле до појаве патохистолошких промена у њој.</p> / <p>Infektivna bolest burze (Gamboro bolest) je kontagiozna virusna bolest mladih pilića. Uzročnik je RNK virus koji pripada familiji Birnaviridae. Ispoljava tropizam prema Fabricijusovoj burzi dovodeći do oštećenja limfocita i atrofije. Bolest se kontroliše vakcinacijom roditeljskih jedinki i prijemčivih pilića. Upotreba atenuiranih vakcina može dovesti do smanjenja nekih tehnoloških parametara. Cilj ovog rada je bio da se dokaže koje od primenjenih atenuiranih vakcina, intermedijarnih Gumbokal, Gumbokal D78 i intermedijarne-plus Gumbokal 228E, indukuje najbolji razvitak imnunološkog odgovora i dovode do najmanjih oštećenja u tkivu Fabricijusove burze i slezene. Pratili smo da li deluju imunosupresivno na vakcinu protiv NJukastl bolesti i da li utiču na smanjenje telesne mase brojlerskih pilića. Osnovne ogledne grupe su vakcinisane četrnaestog dana starosti protiv Gamboro bolesti, a zatim su od osnovnih oglednih grupa oformljene podgrupe koje su vakcinisane protiv NJukastl bolesti u različitim vremenskim periodima: O1-1, O2-2, O3-1 posle sedam dana, O1-2, O2-2, O3-2 posle četrnaest dana, O1-3, O2-3, O3-3 posle dvadesetjedan dan od vakcinacije protiv Gamboro bolesti .Od prvog do četrdesetdrugog dana eksperimenta svakih sedam dana je vađena krv pilićima, merena njihova telesna masa i uzorkovane burze i slezene. Krvni serumi su ispitivani na visinu titra antitela ELISA testom za Gamboro bolest, a metodom inhibicije hemagutinacije za NJukastl bolest. Najvišu visinu titra na Gamboro bolest je pokazala ogledna grupa O1, zatim grupa O2 i najniži O3. U oglednim podgrupama O3-1, O3-2 i O3-3 se ispoljio imunosupresivni efekat na imunološki odgovor prema NJukastl bolesti. Fabricijusovim burzama je određivana relativna masa i burzalni indeks da bi se ustanovilo da li je došlo do atrofije nakon primene vakcina. Najniži burzalni indeks je ustanovljen u oglednoj grupi O3, zatim u O2 i u O1. Takvim redosledom su ustnovljene i patohistološke promene u burzi. Dobijeni rezultati ukazuju da upotreba intermedijane-plus vakcine indukuje najbolji imunološki odgovor, ali i najnižu relativnu masu i vrednost burzalnog indeksa, kao i nižu telesnu masu u odnosu na inermedijarne vakcine. Primenjene vakcine nisu uticale na relativnu masu slezene i nisu dovele do pojave patohistoloških promena u njoj.</p> / <p>Infectious bursal disease (Gumboro disease) is a contagious viral disease of young chickens. The causative agent is an RNA virus that belongs to the family Birnaviridae. It exhibits tropism toward bursa of Fabricius causing damage to cells and atrophy. The disease is controlled by vaccination of susceptible broiler breeders and chickens. The use of attenuated vaccines may lead to the reduction of certain production parameters. The aim of this study was to prove which of the applied attenuated vaccines, intermediate Gumbokal, Gumbokal D78 and intermediate-plus Gumbokal 228E, induces the best immunological response and leads to fewest damages to burza of Fabricijus tissue and spleen. We tracked whether it influences on immunosuppressive vaccine against Newcastle disease and if it leads to reduce of body mass of broiler chickens. Basic experimental groups were vaccinated on the fourteenth day of age against Gamboro diseases, and sub-groups were formed of the basic experimental groups which have been vaccinated against Newcastle disease in different time periods: O1-1, O2-2, O3-1 after seven days, O1-2 , O2-2, O3-2 after fourteen days, O1-3, O2-3, O3-3 after twenty-one days of vaccination against Gumboro disease. From the first to the fourty second day of the experiment, blood was extracted form the chicks every seven days, their body weight was measured and burza and spleen were sampled. Blood serum were assayed for the amount of the antibody titer by enzyme-linked immunosorbent assay test (ELISA) for Gumboro disease, and with Hemagglutination-inhibition test (HI) for Newcastle disease. The biggest titer for Gamboro disease were showed by experimental group O1, then O2 and group O3 the lowest. In the experimental subgroups O3-1, O3-2 and O3-3 immunosuppressive effect was exhibited on the immune response to Newcastle disease. Relative weight and bursal index were determined for burza of Fabricius to determine whether there was atrophy after administration of the vaccine. The lowest bursal index was established in the experimental group O3, followed by O2 and O1. Such sequence was establilshed also for histopathological changes in the burza. The obtained results indicate that the use of the intermediate-plus vaccine induces the best immune response, but also the lowest relative value of the mass and burzal index, as well as a lower body weight compared to the inermediate vaccine. Applied vaccines did not affect the relative weight of the spleen and have not led to the appearance of histopathological changes in it.</p>

This thesis deals with the theme of folk baroque. The aim of this thesis is to introduce this theme to pupils of secondary school in vivid and entertaining form and thus achieve bigger efficiency of educational process and acquisition of newly acquired information. As the best form of teaching i have considered watching a documentary video. The theoretical part of this thesis describes the history and formation of folk baroque, the most important areas in South Bohemia and also the life and work of Master bricklayer Jakub Bursa. This thesis also summarizes the history of documentary videos, its individual categories, the basics of film language, but also the essential aspects of the usage of video as a methodological aid for teaching. The practical part of this thesis deals with the implementation of an educational documentary video with the theme of folk baroque. The thesis also describes the creation of a supplementary animation program, which is completed by an artistic output in the form of artistic techniques of frottage.

My diploma work purpose is to frame and describe the development of some monuments of rural architecture in region {\clqq}Podšumaví``. In the first chapter I briefly open the main topic of the work. More detailed description of typical features of rural buildings is presented in next part, concentrated on specific area - the South Bohemia. In this range I aimed the focus especially on mikroregion {\clqq}Volyňsko`` and {\clqq}Vlachobřezsko``. Jakub Bursa, the bricklayer´s master, worked in this area in the second third of the 19th century and influenced on the final figure of local architecture. The principles of the bricklayer´s guild {--} J. Bursa was a member of it {--} are mentioned in the third chapter. Tha last and main part of this diploma work is presented by the state protected collection of rural architecture in the village {\clqq}Zechovice``. In this place we can understand the development of particular buildings in various moments of its history up to now (it depends on the fact, if the period documents are reachable). I also think about the possible, but not proved authorship of J. Bursa´s work in the case of some mentioned buildings. At the end of this part I remind the consequences of the state protection of buildings in the village.