Orientadores: Sandra Maria Carmello-Guerreiro, Marilia de Moraes Castro, Lia Ascensão / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T00:05:44Z (GMT). No. of bitstreams: 1 Lacchia_AnaPaulaStechhahn_D.pdf: 8477823 bytes, checksum: a1685fc288b20acce389a79bd6302d17 (MD5) Previous issue date: 2006 / Resumo: Anacardiaceae é composta por 70 gêneros com aproximadamente 600 espécies de distribuição principalmente pantropical, com poucas de regiões temperadas. A classificação infrafamiliar mais amplamente utilizada, proposta por Engler (1892), divide a família em cinco tribos: Anacardieae, Rhoeae, Semecarpeae, Spondiadeae e Dobineeae. No presente trabalho, foram estudadas uma espécie da tribo Anacardieae (Anacardium humile St.Hil), uma espécie da tribo Rhoeae (Lithraea molleoides (Vell.) Engl. e duas espécies da tribo Spondiadeae (Spondias dulcis G.Forst e Tapirira guidnensis Aubl.). As estruturas secretoras dos órgãos vegetativos e reprodutivos destas espécies foram caracterizadas anatômica e histoquimicamente e apenas os canais secretores foram caracterizados ultra-estruturalmente. Os canais têm epitélio unisseriado; os associados ao floema são resiníferos (lato sensu) e os medulares de A. humile e S. dulcis, são mucilaginosos. A presença de canais produzindo secreções quimicamente distintas em um mesmo órgão, de uma mesma espécie, está sendo descrita pela primeira vez neste tranalho. Em A. humile, L. molleoides e T.guianensis os canais estão associados ao floema da pétala, sépala, receptáculo, pedicelo florais de todas as flores e ao floema do mesofilo ovariano das flores pistiladas e do pistilódio das flores estaminadas de L. molleoides e, T. guianensis. Canais também são encontrados no fruto e pedúnculo de A. humile e no fruto de T. guianensis. Os canais têm epitélio unisseriado; os florais de A. humile, L. molleoides e T. guianensis são resiníferos (lato sensu), os do fruto e pedúnculo de A. humile são resiníferos (stricto sensu) e os do fruto de T. guianensis são resiníferos (lato sensu). Em uma mesma espécie os canais secretores presentes em diferentes órgãos podem produzir substâncias distintas¿Observação: O resumo, na íntegra poderá ser visualizado no texto completo da tese digital / Abstract: Anacardiaceae is comprised of 70 genera with approximately 600 species of pantropical distribution, mainly, with few species from temperate regions. The most used infrafamilial classification, proposed by Engler (1892), divides this family in five tribes: Anacardieae, Rhoeae, Semecarpeae, Spondiadeae, and Dobineeae. In the present work, there were studied a species from Anacardieae tribe (Anacardium humile St.Hil), a species from Rhoeae tribe (Lithraea molleoides (Vell.) Engl., and two species from Spondiadeae tribe (Spondias dulcis G.Forst and Tapirira guianensis Aubl.). The secretory structures of the vegetative and reproductive organs of these species were characterized anatomically and histochemically, and the secretory canals present in them were ultrastructurally characterized. The canals present uniseriate epithelium; the ones associated to the phloem are resiniferous (lato sensu) and the medullar ones of A. humile and S. dulcis are mucilaginous. The presence of canals that produce chemically distinct secretions in the same organ of a species is being described for the first time. In A. humile, L. molleoides, and T. guianensis, the canals are associated to the phloem of petal, sepal, receptacles, and ovarian mesophyll; only in the pistil-like structure of the male flowers of L. molleoides and T. guianensis, there are found some canaIs. The canals are also found in the fruit and peduncle of A. humile and in the fruit of T. guianensis. The canaIs have uniseriate epithelium; the floral canaIs of A. humile, L. molleoides, and T. guianensis are resiniferous (lato sensu), the ones from fruit and peduncle of A. humile are resiniferous (stricto sensu), and the ones from the fruit of T. guianensis are resiniferous (lato sensu). In the same species, the secretory canaIs present in different organs may produce distinct substances...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Doutor em Biologia Vegetal

Tricomas secretores são apêndices epidérmicos presentes nas maiorias das plantas desempenhando funções diversas, dentre elas a defesa contra herbivoria. Dentre as famílias que apresentam tricomas secretores destaca-se Cannabaceae, que é amplamente estudada por produzir compostos psicoativos e outros utilizados como aromatizantes de cerveja. Recentemente alguns gêneros pertencentes à Ulmaceae foram inseridos em Cannabaceae, como Celtis e Trema. Tais gêneros são os mais numerosos em espécies da família. Considerando que tricomas secretores são os principais sítios de produção de substâncias de interesse econômico, e que são amplamente utilizados como caráter de valor taxonômico, o presente trabalho comparou a distribuição e morfologia de tricomas secretores de duas espécies de Cannabaceae (Celtis pubescens e Trema micrantha) e três espécies afins de Ulmaceae (Ampelocera glabra, Phyllostylon rhamnoides e Ulmus parvifolia). Os dados foram comparados aos existentes na literatura e discutidos em um contexto sistemático. Os tricomas secretores de Trema micranta foram, ainda, analisados em detalhe quanto à ontogenia, análise química do exsudato (in situ e direto) e ultraestrutura. Para tal, amostras de folhas e flores (pistiladas e estaminadas) foram coletadas, fixadas e processadas para análise de microscopia de luz, eletrônica de varredura e de transmissão. Foram encontrados cinco morfotipos diferentes nas espécies estudadas. C. pubescens e T. micrantha apresentaram tricomas secretores capitados e filiformes, que diferiram quanto ao pedúnculo, unisseriado em C. pubescens e bisseriado em T. micrantha. As três espécies de Ulmaceae apresentaram um único morfotipo (capitado com pedúnculo unicelular). Os tricomas de T. micranta iniciam o desenvolvimento com uma divisão anticlinal, semelhante ao que ocorre em Cannabis sativa. Seus tricomas secretam terpenos, alcaloides e compostos fenólicos. Testes químicos comprovaram a presença de alcaloides e os indicadores de canabinoides sugerem que T. micrantha apresenta potencial para produção desses compostos. A morfologia dos tricomas encontrados nas espécies de Cannabaceae é diferente da observada nas espécies de Ulmaceae, o que apoia a atual circunscrição destas famílias. A diversidade morfológica e de termos utilizados torna difíceis a classificação e comparação entre os tipos de tricomas secretores, sendo necessários esforços no sentido de padronizar a caracterização destas estruturas secretoras, em especial nas famílias que compõem o clado urticoide. / Secretory trichomes are epidermal appendages that are present in most plants performing various functions, among them the defense against herbivory. Cannabaceae comprises plants covered with secretory trichomes, and is a well-known family for producing psychoactive compounds and others used as beer flavorings. Recently some genera belonging to Ulmaceae were inserted into Cannabaceae, such as Celtis and Trema, the species-richest genera of the family. As the secretory trichomes are the main plant source of economically important substances and are widely used as taxonomic markers, the present study compared the distribution and morphology of secretory trichomes of two species of Cannabaceae (Celtis pubescens and Trema micrantha) and three related species of Ulmaceae (Ampelocera glabra, Phyllostylon rhamnoides and Ulmus parvifolia). The data were compared to those in the literature and discussed in a systematic context. The secretory trichomes of Trema micranta were also analyzed in detail regarding ontogeny, histolocalization of substances and ultrastructure. For this, samples of leaf and flower (pistillate and staminate) were collected, fixed and processed for analyses of light microscopy, scanning electron microscopy and transmission electron microscopy. For T. micranta chemical tests in situ and direct were carried out; in addition, samples were prepared for ultrastructural study in transmission electron microscopy. Five different morphotypes of secretory trichomes were found in the species studied. C. pubescens and T. micrantha exhibited capitate and filiform secretory trichomes that differed in the stalk, uniseriate in C. pubescens and biseriate in T. micrantha. The three species of Ulmaceae had a single morphotype (capitate with a unicellular stalk). The trichomes of T. micranta originate of a first anticlinal cell division, similar to what occurs in Cannabis sativa. They secrete terpenes, alkaloids and phenolic compounds. Chemical tests have confirmed the presence of alkaloids and cannabinoid indicators suggest that T. micrantha is a putative species for the production of these compounds. The morphology of the trichomes found in the species of Cannabaceae is different from that observed in the species of Ulmaceae, which supports the current circumscription of these families. The diversity of morphology and terms employed makes it difficult to classify and compare the types of secretory trichomes. In this sense, efforts are needed to standardize the characterization of these secretory structures, especially in the families that comprise the urticalean rosids.

A circunscrição de Apocynaceae gerou diversas discussões ao longo da história devido às semelhanças morfológicas de suas flores com as dos representantes de Asclepiadaceae e, atualmente, essas duas famílias foram unidas, sendo subdivididas em cinco subfamílias. Nesse novo conceito, Apocynaceae apresenta uma sinorganização não usual de estruturas florais e somente através de uma análise comparativa das diferentes subfamílias é possível compreender como esse grupo atingiu esse alto grau de elaboração floral. Neste contexto, o presente estudo teve o propósito de caracterizar anatomicamente as estruturas secretoras florais de três representantes de diferentes subfamílias: Tabernaemontana, Mandevilla e Ditassa. Essa análise comparativa demonstrou que os gêneros analisados apresentam diferentes estratégias para atração do polinizador e que houve uma alteração de estrutura e posição do nectário em Apocynoideae em relação à Asclepiadoideae. Enquanto o nectário de Mandevilla é uma projeção glandular ao redor do ovário, em Ditassa apenas a epiderme do tubo dos filetes é nectarífera. Essa alteração de posição está relacionada a diferentes estratégias de dispersão do pólen: mônades em Mandevilla e polínias em Ditassa. A dispersão do pólen em mônades ou em polínias depende diretamente da secreção produzida pela cabeça do estilete que apresenta forma e função distintas nas diferentes subfamílias. O estudo de Tabernaemontana e Mandevilla evidenciou que a cabeça do estilete produz uma secreção viscosa e fluida que auxilia na adesão do pólen ao polinizador durante a coleta do néctar no fundo do tubo floral, além de reter os grãos no nível do estigma, após a visitação de uma outra flor. Por outro lado em Ditassa, a cabeça do estilete produz uma secreção rígida que se adere às polínias e ao polinizador sendo transferida em conjunto durante a coleta do néctar que se encontra no androceu. Além dessas glândulas, coléteres e osmóforos foram encontrados em Ditassa, demonstrando uma maior diversidade de estruturas secretoras associada a uma maior elaboração da morfologia floral dentro do grupo. / Apocynaceae circumscription led to several discussions throughout its history because of the morphological similarities of their flowers with those of Asclepiadaceae and currently these two families are united, being subdivided into five subfamilies. In this new concept, Apocynaceae presents an unusual synorganization of floral structures and, only through a comparative analysis of different subfamilies, is possible to understand how this group reached this high degree of floral complexity. In this context, this study aimed to characterize anatomically the floral secretory structures of three representatives of different subfamilies: Tabernaemontana, Mandevilla and Ditassa. This comparative analysis showed that the analyzed genera have different strategies to attract the pollinator and that there was structural and position shifts in Apocynoideae in relation to Asclepiadoideae. While the Mandevilla nectary is a glandular projection around the ovary, in Ditassa only the epidermis on the filaments tube is nectariferous. This change of position is related to different pollen dispersal strategies: monads in Mandevilla and pollinia in Ditassa. The pollen dispersal in monads or pollinia depends directly on the secretion produced by the style head which has distinct shape and function in the different subfamilies. The study of Tabernaemontana and Mandevilla showed that the style head produces a viscous and fluid secretion that aids in the adhesion of pollen to the pollinator, while collecting nectar at the bottom of the floral tube, and retaining the grains in the stigma level after the visitation of another flower. On the other hand, the style head of Ditassa produces a rigid secretion that adheres to pollinia and to pollinator, being moved together while collecting nectar which is in androecium. In addition to these glands, colleters osmophores were found in Ditassa, demonstrating a greater diversity of secretory structures associated with a further elaboration of floral morphology within the group.

abstract: There is increasing interest and demand in biology studies for identifying and characterizing rare cells or bioparticle subtypes. These subpopulations demonstrate special function, as examples, in multipotent proliferation, immune system response, and cancer diagnosis. Current techniques for separation and identification of these targets lack the accuracy and sensitivity needed to interrogate the complex and diverse bioparticle mixtures. High resolution separations of unlabeled and unaltered cells is an emerging capability. In particular, electric field-driven punctuated microgradient separations have shown high resolution separations of bioparticles. These separations are based on biophysical properties of the un-altered bioparticles. Here, the properties of the bioparticles were identified by ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. As part of this dissertation, high-resolution separations have been applied to neural stem and progenitor cells (NSPCs). The abundance of NSPCs captured with different range of ratio of EK to DEP mobilities are consistent with the final fate trends of the populations. This supports the idea of unbiased and unlabeled high-resolution separation of NSPCs to specific fates is possible. In addition, a new strategy to generate reproducible subpopulations using varied applied potential were employed for studying insulin vesicles from beta cells. The isolated subpopulations demonstrated that the insulin vesicles are heterogenous and showed different distribution of mobility ratios when compared with glucose treated insulin vesicles. This is consistent with existing vesicle density and local concentration data. Furthermore, proteins, which are accepted as challenging small bioparticles to be captured by electrophysical method, were concentrated by this technique. Proteins including IgG, lysozyme, alpha-chymotrypsinogen A were differentiated and characterized with the ratio factor. An extremely narrow bandwidth and high resolution characterization technique, which is experimentally simple and fast, has been developed for proteins. Finally, the native whole cell separation technique has also been applied for Salmonella serotype identification and differentiation for the first time. The technique generated full differentiation of four serotypes of Salmonella. These works may lead to a less expensive and more decentralized new tool and method for transplantation, proteomics, basic research, and microbiologists, working in parallel with other characterization methods. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

Orientador: Ana Paula Fortuna Perez / Resumo: Poiretia Vent. (Leguminosae, Papilionoideae, Dalbergieae, Clado Adesmia) é caracterizado pela presença de glândulas punctiformes em todo corpo vegetal, hábito arbustivo, subarbustivo ou trepador, estames monadelfos, anteras dimórficas e frutos do tipo lomento. A última revisão taxonômica do gênero data da década de 80 e muitas de suas espécies ainda apresentam uma delimitação difícil com sobreposição de caracteres, dificultando uma precisa identificação dos táxons. Como mencionado, uma das características morfológicas mais marcantes do gênero é a presença de glândulas orbiculares com forte odor por toda a planta e apesar disto, estudos investigativos morfológicos e anatômicos, especialmente destas glândulas, objetivando a busca de potenciais caracteres com valor taxonômico são inexistentes no gênero. Por isso, um estudo taxonômico e anatômico, com ênfase nas estruturas secretoras de folíolos, visando a precisa delimitação dos táxons deste gênero, a partir de técnicas usuais em taxonomia e anatomia, foi realizado. Como resultado, são apresentados dois capítulos nesta tese. O primeiro capítulo apresenta a revisão taxonômica, no qual foram reconhecidas 12 espécies exclusivamente Americanas e predominantemente Tropicais. Todas ocorrem no Brasil, das quais nove são endêmicas. Foram realizadas duas lectotipificações e duas neotipificações. Além disso, é fornecida uma chave para identificação, descrições, comentários quanto às suas distribuições geográficas, incluindo mapas, status ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Poiretia Vent. (Leguminosae, Papilionoideae, Dalbergieae, Adesmia clade) is characterized by presence of punctiform glands throughout the entire plant body, shrubby, subshrubby and climbing plants, monadelfhous stamens, dimorphic anthers and fruit of the lomento type. The last taxonomic revision of the genus dates back to the 1980s and many of its species still have a difficult delimitation with overlapping characters, making it difficult to accurately identify taxa. As mentioned, one of the most striking morphological characteristics of the genus is the presence of orbicular glands with a strong odor throughout the plant and despite this, morphological and anatomical investigative studies, especially of these glands, aiming at the search for potential characters with taxonomic value are nonexistent in the genus. For this reason, a taxonomic and anatomical study, with emphasis on the secretory structures of leaflets, aiming at the precise delimitation of the taxa of this genus, using techniques usual in taxonomy and anatomy, was carried out. As a result, two chapters are presented in this thesis. The first chapter presents the taxonomic revision, which recognized 12 exclusively American and predominantly Tropical species. All of them occur in Brazil, of which nine are endemic to the country. Two lectotypifications and two neotypifications were performed. In addition, it is provided an identification key, descriptions, comments about it’s geographical distributions, including ... (Complete abstract click electronic access below) / Doutor

Stomata are structures in plant epidermis which regulate contact between inner and outer environment of the plant by mediating their stomatal aperture. Many inner and outer signals contribute to the ontogenesis of the stomatal pattern. Guard cells undergo significant change of volume and surface during stomatal movement. This change of surface must be compensated by intracellular trafficking of membrane material because biological membrane has limited elasticity. Most of this trafficking takes place between plasma membrane and endosomal compartments. Complex exocyst is protein complex that ensures proper targeting of secretory vesicles to their destination on the plasma membrane. Function of this complex is essential for many cellular processes that require precise targeting of secretion. Mutation in gene Exo70B1 causes different development of the stomatal pattern. Plants with mutated Exo70B1 differ in stomatal size depending on the cultivation conditions more than wild type plant. Protein EXO70B1 is also directly involved in stomatal dynamics because mutants exo70B1 have retarded stomatal opening in response to light. This direct connection can be observed on the fluorescently labeled protein EXOB1 which significantly changes its localization during stomatal movements. None of these observed phenotypes is...

(English): Trichobilharzia regenti is a neurotropic parasite of birds from the family Schistosomatidae. Cercariae, the invasive stages of these trematodes actively penetrate the host skin employing excretory- secretory products (ESPs), which contain proteolytic enzymes able to disrupt host tissues and thus reach the successful transmission. The most abundant secreted enzyme responsible for cercarial penetration of the human schistosome S. mansoni is a cercarial elastase. This serine peptidase is well known for the degradation of skin proteins such as elastin, keratin, collagen or laminin. However, the active expression of the orthologue of this enzyme has never been found in the genus Trichobilharzia. For this reason, it was firmly believed, that cercaria of T. regenti uses mainly cysteine peptidases for the invasion of the host, particularly cathepsins, which were repeatedly identified in this life stage. To strengthen this hypothesis, we incubated T. regenti cercariae in the apparatus with the excised duck skin stimulating the release of their glands' content. The collected ESPs were further analysed by shotgun mass-spectrometry and for the first time, the protein form of cercarial elastase was identified. Unfortunately, we failed to produce its active recombinant protein in yeast and bacterial...

Monoclonal antibodies are well-established as a therapeutic in the biopharmaceutical market, targeting a variety of diseases and with 79 approved products by the United States Food and Drug Administration in December 2019. Therapeutic monoclonal antibodies are commonly produced as recombinant proteins in mammalian cell lines, due to their capacity of post-translational modifications, most notably glycosylation. Furthermore, an identified bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the endoplasmic reticulum. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion. Several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems. The aim of this study was to evaluate the expression of three human recombinant antibodies in Human Embryonic Kidney 293 (HEK293) cells by evaluating 16 different signal peptide combinations, consisting of eight heavy chain (HC) and two light chain (LC) signal peptides. The impact goal was an efficient secretion of recombinant antibodies, and thus lower production cost of recombinant antibodies in HEK293 cells. First, 16 HC and LC signal peptide plasmid constructs were generated for each of the three recombinant antibodies. Thereafter, transient gene expression in HEK293 cells were performed at three independent experiments. Finally, the antibody titers were quantified using Biacore concentration analysis. The produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, the evaluated HC and LC signal peptide combinations resulted in 3 times higher and 2 times higher antibody titers compared to the original signal peptides used by the Drug Discovery and Development platform at Science for Life Laboratory, for two of the studied antibodies respectively. The results presented in this report further demonstrates the necessity to evaluate signal peptides in order to achieve increased expression of recombinant antibodies in mammalian systems.

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [ H]NE uptake capacity, an effect characterized by a robust decrease in the V of the transport of [ H]NE. As expected, reserpine did not displace the binding of [ H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [ H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [ H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca /Ca -calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [ H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [ H]NE uptake and eliminated the inhibitory effects of reserpine on [ H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca -independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

The response of the atrial natriuretic peptide (ANP) secreting cells from both rat atria and the apex of the ventricles to dexamethasone (DEX) was analyzed by the plaque assay. In right atrial cardiocytes, 25% of the cells secreted ANP basally; DEX treatment did not alter this percentage. However, in the left atrial secretory population, a discordance between the basal (15%) and DEX stimulated (25%) percent plaque formation was found. ANP secreting cells from the ventricular apex responded similarly to DEX exposure (26%), with 8% of the cells basally releasing the hormone. These data suggest that in both the left atria and apex of the rat ventricles, exposure to DEX recruits ANP secretory cells from a non-secreting population. Consequently, the release of ANP from these tissues would increase after glucocorticoid stimulation.