Orientador : Graziela Maciel Barroso / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-17T14:11:34Z (GMT). No. of bitstreams: 1 Santos_JoaoUbiratanMoreirados_M.pdf: 15740551 bytes, checksum: 7c7e5402ff7e05eaafd51db776de6434 (MD5) Previous issue date: 1984 / Resumo: Expoe-se neste trabalho um estudo taxonomico de vinte e oito espécies brasileiras do gênero Aspilia Th. (Compositae), pertencente a tribo Heliantheae. Os taxa com suas respectivas distribuições geográficas, são descritos, discutidos e ilustrados. Elaborou-se uma chave dicomtômica com a finalidade de identificar as espécies estudadas, muitas das quais apresentam grandes afinidades entre si. Três espécies, a grazielae Santos, A hermogenesii Santos e A. vandenbergiana Santos e uma nova combinação, A. paraensis (Huber) Santos, foram descritas no decorrer deste estudo. Sinonimizou-se as espécies A. floribunda Gardn. à A. attenuata (Gardn.) Baker; A. glabra (Gardn.) Benth., A. elliptica (BC) Barker, A. foliacca var. angustifolia (Gardn.) Baker, A. foliacea var. hirsuta (Gardn.) Baker, A. foliacea var. oblonga Barker e A. pusilla Barker à A. foliacea (Spreng.) Barker; A. setosa Griseb. S. str. e A. montevidensis var. angustifólia (DC) Ktz. à A. monrevidensis (Spreng) Ktz. . O epíteto específico A. reflexa Baker, foi revalidado. A. camporum Chod., do Paraguai e A. pascalodes Griseb., da Argentina, têm sua ocorrência registrada pela primeira vez para o Brasil / Abstract: This paper is a taxonomic study of twenty ¿ eight Brazilian species of the genus Aspilia Th. (Compositae) of the tribe Heliantheae. The taxa are described, discussed and illustrated. A dichotomus key is given for the identification of the studied species, several of which are very similar to one another. Tree species, A. grazielae Santos, A. hermogenesii Santos, and A. vandenbergiana Santos, and a new combination, A. paraensis (Huber) Santos, were described during the course of this study. The following new synonumies are proposed: A. floibun da Gardn. Under A. attenuata (Gardn.) Baker; A. glabra (Gardn.) Benht., A. elliptica (DC) Barke, A. Foliacea var. angustifolia (Gard) Barker, A. foliaceae var. hirsute (Gardn) Baker, A. foliacea (Spreng.) Baker; A setosa Griseb. s.strs and A montevidensis var. angustifolia (DC)Ktz. Under aA. Montevidensis (Spreng.) Ktz. The species A. reflexa Baker is revalidated. A csmporum Chod. From Paraguay and A. pascaloides Griseb. from Argentina are reported for the first time for Brazil / Mestrado / Biologia Vegetal / Mestre em Ciências Biológicas

Orientador : Graziela Maciel Barroso / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-17T14:09:58Z (GMT). No. of bitstreams: 1 Tozzi_AnaMariaGoulartdeAzevedo_D.pdf: 29116244 bytes, checksum: 5e74052c64445e4b69c175c46774e334 (MD5) Previous issue date: 1989 / Resumo: O presente trabalha consiste de estudos taxonômicos dos gêneros Lonchocarpus Kunth e Deguelia Aubl. para o Brasil. O gênero Deguelia compreendendo as espécies americanas anteriormente incluídas em Derris Lour.. Millettia Wight & Arn. e Lonchocarpus subg. Phacelanthus Pittier. No conceito aqui adotado, estes gêneros mostram-se como grupos naturais, que são separados principalmente pela estrutura da inflorescência, permitindo uma classificação estável para o complexo Para o gênero Lonchocarpus foram reconhecidas 23 espécies, 3 das quais ainda não descritas na literatura (L. grandiflorus, L. bahianus e L. Montanus). Para o gênero Deguelia foram registradas 17 espécies, das quais 3 novas (D. glaucífolía, D. hatschbachii e D. duckeana), e 1 variedade ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: It is presented a taxonomic revision of the genera Lonchocarpus Kunth and Deguelia Aubl. in Brazil. The genus Desuelia comprising the american species formerly included in Derris Lour., Millettia Wight & Arn. and Lonchocarpus subg. Phacelanthus Pittier. These genera as here defined are considered as natural groups, distinguished from each other mainly by the characteristics of the infIorescence. For the genus Lonchocarpus 23 species are recognized, 3 of which are new (L. grandiflorus, L. bahianus and L. montanus). For the genus Deguelia 17 species are recognized, 3 of which are new (D. glaucifolia, D. hatschbachii and D. duckeana), and 1 variety ¿ Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Biologia Vegetal / Doutor em Ciências Biológicas

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

This case-control study involved a total of 29 autistic children (Au) aged 6 to 12 years, and 28 gender and age-matched typically developing children (TD). We evaluated a high number of peripheral oxidative stress parameters, erythrocyte and lymphocyte membrane functional features and membrane lipid composition of erythrocyte. Erythrocyte TBARS, Peroxiredoxin II, Protein Carbonyl Groups and urinary HEL and isoprostane levels were elevated in AU (confirming an imbalance of the redox status of Au); other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma Total antioxidant capacity and plasma carbonyl groups, erythrocyte SOD and catalase activities) were unchanged, whilst peroxiredoxin I showed a trend of elevated levels in red blood cells of Au children. A very significant reduction of both erythrocyte and lymphocyte Na+, K+-ATPase activity (NKA), a reduction of erythrocyte membrane fluidity, a reduction of phospatydyl serine exposition on erythrocyte membranes, an alteration in erythrocyte fatty acid membrane profile (increase in MUFA and in ω6/ω3 ratio due to decrease in EPA and DHA) and a reduction of cholesterol content of erythrocyte membrane were found in Au compared to TD, without change in erythrocyte membrane sialic acid content and in lymphocyte membrane fluidity. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity, and ADOS and CARS score are inversely related to peroxiredoxin II levels. Oxidative stress and erythrocyte structural and functional alterations may play a role in the pathogenesis of Autism Spectrum Disorders and could be potentially utilized as peripheral biomarkers.

Abstract The aim of this work was the development of a murine model of septic arthrosynovitis and osteomyelitis caused by Staphylococcus aureus, which could mimic the natural disease occurring in humans and which could be suitable for testing preventive and therapeutic interventions. This model could be particularly useful since S. aureus-mediated joints and bones infections are relevant in humans, both in terms of frequency and severity. Our attention focused in tracking bacterial infiltration in joints and bones over time using different microbiological and hystopathological tools, which allowed us to have a complete overview of the situation and to evaluate the immunological actions undertaken by the host to contain or eradicate the bacterial infection. Antibodies and cytokines profiles, as well as recruitment of host immune cells at joints of immunized and infected mice were therefore monitored for a time period that allowed us to study both the acute and the chronic phases of the disease in situ. Finally the Novartis vaccine formulation proposed against S. aureus infections was tested for its capacity to protect immunized mice from joints infections, and the preventive immunization was compared to a standard antibiotic prophylaxis. The availability of powerful tools to study specific bacterial-mediated diseases is nowadays an important requirement for the scientific community to shed light on the complex interactions between host and pathogens and to test treatments for preventing or contrasting infections. We believe that our work significantly contributes to the overall knowledge in the field of S. aureus-dependent pathologies, opening the possibility for further investigations in several fields of study.

CD99, glicoproteina di membrana codificata dal gene MIC2, è coinvolta in numerosi processi cellulari, inclusi adesione, migrazione, apoptosi, differenziamento e regolazione del trafficking intracellulare di proteine, in condizioni fisiologiche e patologiche. Nell’osteosarcoma risulta scarsamente espressa ed ha ruolo oncosoppressivo. L’isoforma completa (CD99wt) e l’isoforma tronca (CD99sh), deleta di una porzione del dominio intracellulare, influenzano in modo opposto la malignità tumorale. In questo studio, comparando cellule di osteosarcoma caratterizzate da differenti capacità metastatiche e diversa espressione di CD99, abbiamo valutato la modulazione dei contatti cellula-cellula, la riorganizzazione del citoscheletro di actina e la modulazione delle vie di segnalazione a valle del CD99, al fine di identificare i meccanismi molecolari regolati da questa molecola e responsabili del comportamento migratorio e invasivo delle cellule di osteosarcoma. L'espressione forzata di CD99wt induce il reclutamento di N-caderina e β-catenina a livello delle giunzioni aderenti ed inibisce l'espressione di molecole cruciali nel processo di rimodellamento del citoscheletro di actina, come ACTR2, ARPC1A, Rho-associated, coiled–coil-containing protein kinase 2 (ROCK2), nonché di ezrina, membro della famiglia ezrin/radixin/moesin e chiaramente associata con la progressione tumorale e la metastatizzazione dell’OS. Gli studi funzionali identificano ROCK2 come mediatore fondamentale nella regolazione della migrazione e della diffusione metastatica dell’osteosarcoma. Mantenendo cSRC in una conformazione inattiva, CD99wt inibisce la segnalazione mediata da ROCK2 inducendo una diminuzione dell’ezrina a livello della membrana accompagnata dalla traslocazione in membrana di N-caderina e β-catenina, principali ponti molecolari per il citoscheletro di actina. La ri-espressione di CD99wt, generalmente presente negli osteoblasti, ma perso nelle cellule di osteosarcoma, attraverso l'inibizione dell'attività di cSrc e ROCK2, aumenta la forza di contatto e riattiva i segnali anti-migratori ostacolando l’azione pro-migratoria, altrimenti dominante, dell’ezrina nell’osteosarcoma. Abbiamo infine valutato la funzione di ROCK2 nel sarcoma di Ewing: nonostante il ruolo oncogenico esercitato da CD99, ROCK2 guida la migrazione cellulare anche in questa neoplasia. / CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion, migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions and inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated coiled–coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration and metastatic spread. By maintaining cSrc in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. We propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of cSrc and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells. We also assessed ROCK2 function in Ewing sarcoma cells: despite the oncogenic role exerted by CD99 in these tumour cells, ROCK2 still drives cell migration.

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.

The oriental fruit moth, Grapholita molesta, is one of the main pests of peach trees worldwide. It is conThe oriental fruit moth, Grapholita molesta, is one of the main pests of peach trees worldwide. Plant volatiles are a promising technique to attract G. molesta under mating disruption conditions. In my thesis I have characterized the response of olfactory receptor neurons (ORN) to pheromone and plant odors by means of single-sensillum electrophisiology. I then used this information to determine that a previously reported pheromone-plant synergism does not result from pheromone-plant interactions at the ORN level. I have compared several plant volatile blends previously tested in Australia and China, and have found that none of them attracted moths in the field, but they synergized pheromone responses in the laboratory. Finally, I investigated the role of plant blends and alcohols on the response to unnatural pheromone blend ratios or overdosed pheromone concentrations. With these studies we hopefully advanced on basic and applied aspects of the olfactory neuroethology of this species. / Grapholita molesta es una de las principales plagas del melocotonero. Los volátiles de planta son una técnica prometedora para atraer G. molesta en condiciones de confusión sexual. En mi tesis he caracterizado la respuesta de las neuronas receptoras olfativas (ORN) a la feromona y volátiles de planta mediante registros de sensila única. Después determiné que el sinergismo entre volátiles de planta y feromona previamente publicado no ocurre a nivel de la ORN. Más adelante he comparado volátiles de planta que en estudios anteriores en China y en Australia habían dado buenos resultados pero en mi caso no hubo respuestas en campo, aunque en el túnel de vuelo sí que aumentaron la respuesta a la feromona. Finalmente he explorado el papel de volátiles de planta y alcoholes en la respuesta a mezclas subóptimas de feromona. Con estos resultados espero haber contribuido al conocimiento de la neuretología olfativa de esta plaga. / Grapholita molesta és una de les principals plagues del presseguer. Els volàtils de planta són una tècnica prometedora per atreure G. molesta en condicions de confusió sexual. En la meva tesi he caracteritzat la resposta de les neurones receptores olfactòries (ORN) a la feromona i volàtils de planta mitjançant registres de sensila única. Després vaig determinar que el sinergismo entre volàtils de planta i feromona prèviament publicat no ocorre a nivell de la ORN. Més endavant he comparat volàtils de planta que en estudis anteriors a Xina i a Austràlia havien dau bons resultats però en el meu cas no va haver-hi respostes en camp, encara que en el túnel de vol sí que van augmentar la resposta a la feromona. Finalment he explorat el paper de volàtils de planta i alcohols en la resposta a mescles subóptimas de feromona. Amb aquests resultats espero haver contribuït al coneixement de la neuretología olfactòria d'aquesta plaga.trolled with sex pheromones (mating disrupton) and insecticide applications. Under mating disruption conditions it is difficult to monitor the pest and to evaluate the control methods. Plant volatiles are a promising technique to attract G. molesta under mating disruption conditions. In addition plant volatiles could attract females, while the sex pheromone only attracts males.