Atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure. The early gut microbiota appears to be important for immune maturation. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. The aim of this thesis was to investigate whether early colonization with certain gut microbiota species influences childhood immune responses and allergy development up to age five. Also, as human milk oligosaccharides (HMOs) might stimulate the growth of certain gut microbiota species, the consumption of neutral colostrum HMOs was investigated for their role in allergy development up to 18 months. The concentrations of neutral colostrum HMOs varied considerably between women; however this variation could not be explained by their allergic status. Neither was the consumption of neutral colostrum HMOs related to allergy development in their children up to 18 months. Infants who harboured lactobacilli group I and Bifidobacterium adolescentis one week after birth developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time. Also, colonization with several Bifidobacterium species was associated with higher levels of house dust endotoxin and larger family size. The early Bifidobacterium flora influenced levels of salivary secretory IgA at six and 12 months but not during later childhood. Moreover, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous Toll-like receptor 4 mRNA expression in peripheral blood cells collected 12 months after birth. In conclusion, these results indicate that the early infant gut microbiota influences systemic and mucosal immune maturation during infancy, and that it might be altered in infants developing allergic disease.

The central role played by the Endoplasmic Reticulum (ER) in the correct folding and assembly of secretary and membrane proteins cannot be overstated. As the first compartment in the secretary pathway, it is responsible for the synthesis, modification and targeting of proteins to their proper destinations within the secretary pathway and the extracellular space. Protein folding in this specialized compartment is dynamic and involves a host of molecular chaperones and folding catalysts. Once inside the ER lumen, proteins fold into their native conformation and undergo a multitude of post-translational modifications, including N-linked glycosylation and disulfide oxidation. The proper conformational maturation of nascent proteins that traverse the secretary pathway is both aided and monitored by a complex process termed ER quality control. A variety of quality control mechanisms that rely on the chaperone systems operate in the ER. These act in close concert with the molecular machinery involved in degradation of non-native proteins to maintain homeostasis. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. As a general rule, only those proteins that have successfully completed their folding and passed a stringent selection process are allowed to exit the ER on their way to their final destinations. The importance of the normal functioning of the ER is underlined by the fact that disruption in protein folding, resulting in ER stress, has now been identified as the biochemical basis of many ER storage diseases including Diabetes mellitus, Endocrinopathies and Hemophilia A. Processing events occurring inside the ER lumen are known to influence the efficiency of protein secretion. Vastly different rates of exocytose observed among secretary proteins have been found to correlate with the rate of exit from the ER. One such example is the interesting secretion property exhibited by Retinol Binding Protein (RBP) The principal carrier of retinol (Vitamin A) in plasma. RBP is a single domain protein consisting of three intramolecular disulfide bonds and helps transport retinol from the liver stores to the various target tissues in the body. Availability of its ligand, retinol, while not affecting its synthesis, is known to be the major factor in regulating RBP secretion from the liver. In the absence of retinol, apo-RBP has been shown to be retained in the ER by a hitherto unclear mechanism. Like most other secretary proteins, RBP is co-translationally targeted to the ER lumen, where it undergoes disulfide oxidation as the only modification. It has been shown to form a complex with another secretary protein, Transthyretin (TTR) in the ER and this complex formation is thought to prevent premature glomerular filtration of the otherwise small RBP with its bound retinol. Despite attaining a mature conformation, apo-RBP is not secreted and awaits conversion to its ligand-bound, holo form in order to exit the ER. It is widely believed that ligand binding may relieve this retention of RBP from the ER quality control machinery. However the precise mechanisms that mediate and regulate RBP folding, ligand binding, TTR assembly and secretion are not clearly understood. Though the folding and secretion properties of RBP have been described in HepG2 cells, its interactions with the ER resident chaperones have not been addressed. Apart from being an important cell biological question, the study of RBP assumes a lot of significance with its recent emergence as a key player in the pathogenesis of type 2 diabetes mellitus. It has been proposed that lowering of serum RBP levels could be a new strategy for treating type 2 diabetes mellitus. The present study was undertaken with the intention of analyzing the oxidative folding of RBP in the ER more closely. A systematic approach aimed at understanding the early events associated with folding and maturation of RBP, with particular emphasis on the role of ER-resident chaperones and the quality control machinery, is likely to provide interesting insights into the mechanisms involved in its ligand dependent secretion. Reconstitution of RBP biogenesis in a cell free system. The folding of RBP in cells is extremely quick with rapid oxidation kinetics. This makes it difficult to systematically analyze the early folding events in cultured cells. It was necessary to make use of a simplified system that would faithfully recapitulate the folding process in the ER. Therefore, a cell free translation system consisting of rabbit reticulocyte lysate and canine pancreatic microcosms as a source of ER-derived membranes was developed. This system affords the advantage of easy manipulation while still preserving the overall environment that prevails in the ER of intact cells. Extensive biochemical and functional characterization of the isolated microcosms was carried out and in vitro translation and microsomal translocation of RBP was established. Though initially confined to studies on membrane insertion and core glycosylate, the cell free system supplemented with microcosms has subsequently been used to analyze folding and assembly of a number of secretary and membrane proteins. A similar strategy has been adopted in the present study of RBP folding and maturation. Oxidative folding of RBP in isolated microcosms: Delineation of its disulfide oxidation pathway Using glutathione (GSSG) as the oxidant, co- and posttranslational disulfide oxidation of RBP was carried out in isolated microcosms. The ability to manipulate the redox status of this cell free system has helped to considerably slow down the oxidative folding of RBP so that a more careful analysis of the folding process could be performed. RBP was found to undergo oxidative folding with a t1/2 of 30 minutes and folding proceeded through at least one disulfide-bonded intermediate. Non-reducing SDS PAGE was used to resolve the folding intermediates. The pattern of oxidation was in good agreement with that reported earlier in HepG2 cells. No significant effect of retinol was observed on either the folding kinetics or the pattern of disulfide oxidation of RBP in isolated microsomes.A DTT sensitivity assay, used to probe the conformational maturity of folding RBP, revealed that RBP was capable of maturing into a DTT-resistant conformation in isolated microsomes. With the aid of disulfide mutants, the probable disulfide oxidation pathway of RBP in the ER has been determined. Single and double disulfide mutants of RBP were generated by site-directed mutagenesis and their posttranslational oxidation patterns were analyzed and compared with that of the wild type protein. Based on the results obtained, it was clear that the folding intermediate was made up of one of the two big disulfide loops and that the presence of both these loops was essential for RBP to fold into a fully oxidized, compact form. It has not been possible to determine the contribution of the third, smallest disulfide loop to the oxidative folding of RBP. Molecular events associated with the early oxidative folding of RBP To gain insights into the possible role of ER chaperones in the oxidative folding of RBP, the oligomeric state of folding RBP was analyzed by velocity sedimentation and chemical crosslinking assays. Velocity sedimentation analysis revealed that the reduced form of RBP was present in a large complex of size >100 S20,W. Upon disulfide oxidation, it readily dissociated from the complex and assumed a monomeric state. This was evident even during co-translational oxidation which suggested that RBP transiently associated with the large complex during its oxidative folding. Dynamic nature of this complex indicated that this could be a folding complex containing the chaperone machinery of the ER. These results were also supported by crosslinking analysis performed in unbroken microsomes using the homo-bifunctional crosslinker, DSP. The early folding forms of RBP could be crosslinked to a large complex while upon disulfide oxidation, RBP matured to its monomeric form and was no longer crosslinkable. Sedimentation and crosslinking analyses of the RBP disulfide mutants revealed that while the double disulfide mutant remained irreversibly associated with the large complex, the single mutants were released upon acquiring one of the two big disulfide loops. This suggested that despite the lack of one of the two major disulfides, these mutants were considered ‘folded’ by the quality control machinery in the ER while the double mutant probably resembled a molten globule state and was therefore considered ‘unfolded’ and irreversibly retained. Results from crosslinking analysis in microsomes not engaged in active translation suggested that chaperones of the ER were organized in a complex constitutively thereby lending support to the concept of ER-matrix, a large network of luminal proteins consisting of ER chaperones and accessory factors. Given this scenario, it is not unlikely that newly synthesized protein substrates transiently associate with this large pre-existing complex of chaperones and dissociate during late stages of their maturation. Conclusion In all, this study provides significant insights into some of the early events associated with the oxidative folding of RBP in the ER. The delineation of the disulfide oxidation pathway of RBP has been possible. The results obtained from this study suggest that RBP probably dissociates from the quality control quite early during its folding process and this step in its maturation might not be influenced by retinol. The stimulus for its ligant dependent secretion is likely to operate at a later stage of its sojourn in the ER, possibly consequent to positive cues from accessory binding factors such as TTR. Lastly, Perservation of the ER microenvironment in isolated microsomes, as evidenced from this study, augurs well for the use of this system to analyze mechanisms underlying folding, maturation, secretion and/or retention of secretory proteins.

The major sorting station of biosynthetic material destined for the cell surface or secretion is the trans Golgi Network, TGN. This organelle sorts proteins and lipids into vesicular transport carriers that are targeted via different pathways to distinct membrane compartments of the cell. The molecular principles that operate in cargo sorting at the TGN are still not very well understood. Especially, we know very little about the sorting of lipids. It was postulated that a sorting mechanism based on clustering of lipid rafts, dynamic membrane domains enriched in sphingolipids and sterols, could be an important part of the picture. My thesis study dealt with the elucidation of the molecular sorting principles at the TGN and their exploitation for cell surface polarity in the yeast Saccharomyces cerevisiae. To this end, we conducted a genome wide screen that identified yeast mutants defective in cell surface delivery of the model cargo protein FusMid-GFP. The most striking result of this screen was that mutant strains with defects in ergosterol (the major yeast sterol) and sphingolipid biosynthesis lost sorting competence. To elucidate a direct role for sphingolipids and ergosterol in cargo sorting and secretion we sought to characterize the lipid composition of secretory vesicles. Hence, we established a vesicle purification protocol based on an immunoisolation strategy. Additionally, in collaboration with the group of A. Shevchenko, we developed a mass spectrometry methodology that allows the comprehensive and quantitative lipid analysis of subcellular organelles. Preliminary results corroborate our genetic evidence. The data show that the vesicles are enriched in sphingolipids and decreased in phosphatidylcholine indicating a role for raft clustering in cargo sorting at the TGN. The studies of cell polarity during yeast mating also unraveled a role for raft clustering. We could identify that the lipid bilayer at the tip of the mating projection was more ordered than at the plasma membrane enclosing the cell body and that this was dependent on sphingolipid synthesis. The results of my thesis suggest that in the yeast Saccharomyces cerevisiae fundamental cell biological processes such as cargo sorting and vesicle formation at the TGN as well as cell surface polarity during mating employ raft clustering mechanisms.

Durch die höhere Verfügbarkeit von Schnittbildverfahren in der Kleintiermedizin treten Flüssigkeitsansammlungen in der Bulla tympanica immer häufiger als Zufallsbefund bei Hunden, insbesondere bei brachyzephalen Rassen, auf. Aufgrund der fehlenden klinischen Symptome der Patienten ist eine akute Entzündung unwahrscheinlich. Daher bezeichnen neuere Studien diese Flüssigkeitsansammlung als primär sekretorische Otitis media (PSOM). In der vorliegenden Studie wurde untersucht, ob Hunde der Rassen Mops und Französische Bulldogge mit einer primär sekretorischen Otitis media einen konduktiven Hörverlust für das betroffene Ohr im Vergleich zu „Ohr gesunden“ Hunden ihrer Rasse zeigen. Des Weiteren wurde untersucht, ob Französische Bulldoggen signifikant häufiger eine primär sekretorische Otitis media als Hunde der Rasse Mops zeigen. Es wurden bei jeweils 41 Hunde der Rasse Mops und Französische Bulldogge, die zur chirurgischen Versorgung des Brachyzephalen Syndroms vorgestellt wurden, die frühen akustisch evozierten Potentiale (FAEP) abgeleitet. Des Weiteren erfolgte bei allen Patienten eine Computertomographie des Kopfes, eine Otoskopie und ggf. eine Punktion der Bulla tympanica inklusive einer Zytologie und bakteriologischen Untersuchung des Sekrets. In der vorliegenden Studie konnte kein konduktiver Hörverlust für die Hunde mit einer PSOM nachgewiesen werden. Es zeigte sich nur eine Verlängerung der Latenz der Welle I bei Vorliegen einer PSOM, jedoch ohne dass eine signifikant erhöhte Hörschwelle für diese Patienten festgestellt werden konnte. Als Ursache für die Verlängerung der Latenz der Welle I kommt sowohl die beschriebene Füllung der Bulla als auch ein stenotischer Gehörgang in Frage. Die Hörschwelle ist zwar bei den Hunden mit einer PSOM tendenziell höher als bei den Patienten ohne Füllung der Bullae, allerdings konnte hierfür kein signifikanter Unterschied nachgewiesen werden. Bei 40 % der untersuchten Patienten mit einseitiger PSOM konnte für beide Ohren dieselbe Hörschwelle bestimmt werden, so dass eine Füllung der Bulla tympanica nicht immer zu einer vorhersagbaren Veränderung der Hörschwelle führen muss. Eine mögliche Erklärung für diesen Umstand ist ein sensorineuraler Hörverlust, der durch eine chronische Entzündung des Mittelohres zustande kommt. Dies ist bisher nur in der Humanmedizin beschrieben und die Pathogenese ist noch unklar. Die Ergebnisse zeigen zusätzlich, dass Französische Bulldoggen signifikant häufiger von einer PSOM betroffen sind. Die Ursache für diese Häufung könnte ein dickerer weicher Gaumen der Französischen Bulldoggen im Vergleich zu Hunden der Rasse Mops sein, durch den es zu einer Funktionsstörung der Tuba auditiva kommen könnte. Beim Vergleich der Hunde ohne Vorliegen einer Füllung konnte festgestellt werden, dass Französische Bulldoggen eine höhere Hörschwelle und eine längere Latenz der Welle I als Hunde der Rasse Mops aufweisen. Bei beiden Rassen ist zusätzlich auffällig, dass die Hörschwelle um 30 dB höher liegt als bei Hunden anderer Rassen (SHIU et al. 1997). Alle brachyzephalen Hunde dieser Studie zeigen somit ein im Vergleich zu anderen Rassen vermindertes Hörvermögen, das aber durch eine PSOM nicht weiter verschlechtert wird. Der Symptomenkomplex Brachyzephalen Syndrom muss nach der vorliegenden Studie durch die Symptome vermindertes Hörvermögen und das Vorliegen einer PSOM ergänzt werden. Die klinischen Auswirkungen des Hörverlusts, wie etwa eine starke Anhänglichkeit, könnten bei Hunden dieser Rassen übersehen werden, da dies als gewünschter Charakterzug der Rassen interpretiert wird und nicht als mögliches Symptom einer Erkrankung wahrgenommen wird. / Because of the increased availability of cross-sectional imaging modalities in small animal medicine the incidental finding of material in the middle ear is more common, especially in brachycephalic dogs. Because the animals show no clinical signs, an acute inflammation is unlikely. Therefore recent studies term it as primary secretory otitis media (PSOM). The aim of the current study was to determine whether brachycephalic dogs with PSOM show a conductive hearing loss compared to brachycephalic dogs without changes in the middle ear. Additionally it was evaluated whether French bulldogs suffer from PSOM more frequent than pugs. BAER was recorded in 41 pugs and 41 French bulldogs, which were under general anesthesia because of the surgical correction of the brachycephalic syndrome. In all patients a computed tomography of the head, an otoscopy and if possible a myringotomy with aspiration of the fluid in the middle ear was performed. If fluid was available a cytological examination and a bacterial culture of the fluid was initiated. In the current study no conductive hearing loss was detected in brachycephalic dogs with PSOM. The latencies of wave I were increased in patients with PSOM, although the thresholds of hearing were not increased. The increased latencies of wave I can be explained by the fluid in the middle ear as well as the stenotic external ear canal. The thresholds of hearing in dogs with PSOM were tendentially higher than in dogs without fluid in the middle ear, but the correlation was not significant. In 40 % of the patients with PSOM in one ear the threshold of hearing in both ears is at the same level. Therefore there is no strict correlation between fluid in the middle ear and an increased threshold of hearing on the affected side. This could be explained by a sensorineural hearing loss caused by chronic inflammation of the middle ear. Until now a chronic inflammation as a cause of sensorineural hearing loss has only been described in human medicine, not in veterinary medicine. The pathogenesis is still unknown. The results show that French bulldogs suffer more frequently from the PSOM than pugs. French bulldogs usually show a bigger soft palate than pugs which could result in a dysfunction of the tuba auditiva. Hence the bigger soft palate could be causing the increased prevalence of PSOM in French bulldogs. Compared to pugs without PSOM, French bulldogs without PSOM show an increased latency of wave I as well as an increased threshold of hearing. Additionally for both breeds an increase in the threshold of hearing by 30 dB compared to normocephalic breeds could be detected (SHIU et al. 1997). It is remarkable that French bulldogs as well as pugs show a hearing loss without correlation to the PSOM. In conclusion hearing loss has to be added to the characteristic triad of symptoms of the brachycephalic syndrome. Clinical signs of hearing loss like loyalty to the owner could be misinterpreted especially in these breeds, since this is a favored behavior of these dogs.

Prostate cancer remains one of the most commonly diagnosed cancers in men and is a leading cause of cancer death. While great success has been achieved at curing early stage prostate cancer, limited success has been obtained when treating late-stage hormone independent prostate cancer. This is due to the increased propensity for skeletal and non-skeletal metastases. Thus development of novel effective therapeutic modalities against late stage prostate cancer is of critical importance. / Towards these objectives, I have focused my attention on the role of prostate secretory protein (PSP-94) which is expressed in normal individuals and in patients with early stage prostate cancer. Using our well established in vivo models of prostate cancer, I have evaluated the ability of PSP-94 and its amino acids 31-45 required (PCK3145) to decrease tumor growth and skeletal metastases in vivo and evaluated the potential mechanism(s) associated with PCK3145 anti-cancer actions. / Prostatic cancer can also develop as a result of epigenetic activation of tumor promoting genes. To evaluate the role of methylation in prostate cancer, late stage prostate cancer cells were treated with the universal methylating agent S-adenosylmethionine (SAM) and an anti-sense oligonucleotide directed against MBD2 (AS). Scrambled oligonucleotide was included as a control (S). Both SAM and MBD2-AS resulted in inhibition in uPA, MMP-2 and VEGF production leading to decreased tumor cell invasive capacity. However, SAM and MBD2-AS were not able to either further repress partially methylated genes (GSTP1) or reactivate already methylated genes (AR). Furthermore, SAM and MBD2-AS treatment resulted in significant reduction in tumor growth in vivo . Immunohistochemical and RT-PCR analyses carried out on SAM and MBD2-AS tumors revealed decreased protein and mRNA expression of uPA and MMP-2 which was partially due to increased methylation of the respective promoters even after 10 weeks post in vitro treatment as analyzed by bisulfate sequencing. In addition decreased levels of angiogenesis and tumor survival markers were observed. / Collectively, these studies are aimed at the development of novel reliable approached to diagnose and treat advanced, hormone refractory prostate cancer to reduce tumor associated morbidity and mortality.

The biological, technical and computational aspects of this project collectively focused on using electron tomography (ET) for the high-resolution (10-20 nm) 3D reconstruction of entire insulin-secreting beta cells within islets of Langerhans isolated from mouse pancreata. Islets were cultured overnight to represent either steady-state (non-stimulated) or elevated glucose (stimulated) conditions, prior to fast-freezing, freeze-substitution, plastic embedment and cutting into 250-400 nm thick sections for tomographic imaging using intermediate voltage electron microscopy (EM). 3D images (tomograms) of each section were used to evaluate the performance of the new technical and computational approaches developed, and make biological comparisons of intercellular structure-function. Analysis focused on key compartments/organelles of the insulin-secretory pathway - Golgi apparatus, mitochondria, insulin secretory granules and multi-granular bodies. To allow the application of ET to entire mammalian cells, several technical limitations were addressed. Since segmenting (delimiting compartments of interest) tomograms manually, represented the major ërate-limiting stepí of ET, an interactive approach for 3D segmentation using novel interpolation algorithms (crude smooth, pointwise smooth and spherical interpolation) to iteratively predict the shape of 3D surfaces between user-drawn contours was developed. The performance of these tools in segmenting a range of compartment types was examined, and found to significantly enhance the speed and accuracy of manual segmentation. To better compensate for the physical collapse of plastic sections in the EM, a novel method was developed for estimating section collapse by analyzing approximately spherical organelles. Using this method on mature insulin granules in high-resolution datasets, coupled with measurements from the whole cell reconstructions, section collapse was found to be substantially less (~25%) than the value (40%) previously used to re-scale 3D models. Other new approaches developed to further improve the accuracy and quality of tomograms, included interactive tools for fiducial tracking, and the use of larger gold particles, a ëreduced second axisí to account for the missing wedge problem, and deformation grids to account for anisotropic deformation. As well as affording more efficient and precise mapping of cell ultrastructure in 3D for subsequent quantitative analysis, these developments provided new insights for future automated (hybrid) segmentation pipelines and new computational approaches for improving quality and isotropic accuracy of volumetric image data. The Interpolator and DrawingTools for segmentation, AnalysisTools for estimating section collapse and BeadHelper for tracking fiducial particles, written as plug-ins for the IMOD software package distributed by the University of Colorado, are now being used by the wider ET community with significant positive feedback. Using the novel approaches developed, four insulin-secreting beta cells - two from the periphery of an islet frozen 1 hr after stimulation with 11 mM glucose, and two from the periphery of another islet under steady-state 5.6 mM glucose conditions - were reconstructed in their entirety in 3D. Quantitative data on the key compartments/organelles provided new information regarding global changes in cellular organization, and enabled robust comparisons of each pair of functionally equivalent cells at unprecedented spatial resolution. Relative differences in the number, dimensions, architecture and distribution of organelles per cubic micron of cellular volume (including mitochondrial branching) reflected differences in the cellsí individual capacity/readiness to respond to secretagogue stimulation. In the two stimulated cells this was reflected by inverse relationships between the number/size of mature granules versus immature granules, the number/size of mitochondria, and the volume of the trans-Golgi network relative to the entire Golgi ribbon. Complementary stereological analysis of whole islets indicated which cells were the most representative under stimulated versus non-stimulated conditions, and revealed a marked natural heterogeneity between cells both within and between individual islets. Overall, this project led to significant improvements in efficiency and accuracy for segmenting cellular compartments/organelles, and in image quality and accuracy for tomogram computation and reconstruction through use of the newly developed techniques. The improved 3D reconstruction and analysis of pancreatic beta cells in toto in native tissue provided a powerful approach for quantitatively mapping the organelles involved in insulin synthesis/secretion at unprecedented detail, and afforded a level of insight into the complex 3D organization of mammalian cells not previously achieved by any other analytical technique or imaging method.

Orientador: Silvia Rodrigues Machado / Banca: Elza Maria Guimarães dos Santos / Banca: Adriana Hissae Hayashi / Resumo: Uma característica da família Verbenaceae é a presença de tricomas secretores, geralmente produtores de óleos essenciais de grande valor medicinal. Muitas espécies de Lippia Houst. são utilizadas em programas fitoterápicos e de complementação alimentar no Brasil, sendo L. alba e L. sidoides as mais utilizadas e estudadas. O cerrado possui aproximadamente 33 espécies de Lippia cujo potencial farmacológico é desconhecido. Apesar da importância econômica como fonte de medicamentos e da representatividade de Lippia na flora aromática nativa, pouco se conhece sobre os aspectos estruturais e da secreção dos seus tricomas secretores. Neste trabalho foram estudados os tricomas secretores presentes em órgãos vegetativos e reprodutivos de L. stachyoides Cham. sob o ponto de vista morfológico, ontogenético, histoquímico e ultra-estrutural. Foram utilizadas técnicas convencionais em estudos anatômicos e ultra-estruturais; a identificação in situ da composição química das substâncias presentes nos tricomas foi realizada por meio de testes histoquímicos. Em L. stachyoides, os tricomas secretores são capitados e foram classificados em cinco tipos morfológicos, os quais são amplamente distribuídos no caule, folhas e inflorescências. A histoquímica revelou que nos tipos I e IV predominam substâncias hidrofílicas e nos tipos II e V, substâncias lipofílicas. De um modo geral, as características ultraestruturais observadas em cada tipo de tricoma corroboraram os resultados das análises histoquímicas. Os resultados do presente trabalho sugerem que as variações morfológicas dos tricomas secretores em L. stachyoides estão associadas com a composição química da secreção / Abstract: A feature of the family Verbenaceae is the presence of secretory trichomes, producers of essential oils with great medicinal value. Many species of Lippia Houst. are employed in phytotherapic and food complementation programs in Brazil, being L. alba and L. sidoides the most employed and studied species. The Brazilian cerrado has approximately 33 species of Lippia, whose pharmacological potential is unknown. Despite the economic importance of Lippia as a source of medicinal drugs and its representation in native aromatic flora, little is known about the structural and secretion features of its trichomes. In this work, the secretory trichomes, occurring in vegetative and reproductive organs of Lippia stachyoides Cham., were studied on the morphological, ontogenetic, histochemical and ultrastructural views. Usual techniques of vegetal anatomy and ultrastructure were employed; in situ identification of the chemical composition of the substances present in the trichomes was performed according to histochemical tests. In L. stachyoides, the secretory trichomes are capitate and classified according to five morphological types which are widely distributed in the shoot, leaves and inflorescences. The histochemistry revealed that in types I and IV, hydrophilic compounds are predominant and that lipophilic substances predominate in the types II and V. In general, the ultrastructural features observed in each trichome type corroborate the histochemical analysis. The results of this work suggest that morphological variations of secretory trichomes in L. stachyoides are associated to the chemical composition of the secretion / Mestre

Thesis deals with relationship between histomorphological and molecular-genetic findings of selected salivary gland tumors. Author, as a molecular-cytogeneticist mainly focused on detection of tumor-specific translocations of the salivary gland tumors which can serve as differential diagnostic markers. The thesis is composed as a commented files of authors own publications, and it is divided into four parts. First part deepens the knowledge of salivary adenoid cystic carcinoma. It was proved, that t(6;9)(q22-23;p23-24) resulting in fusion of transcription factors MYB-NFIB, or more rarely t(8;9) resulting in MYBL1-NFIB fusion represent robust differential diagnostic marker of adenoid cystic carcinoma. Further it was proved, that the 1p36 deletion can serve as an unfavorable prognostic indicator of adenoid cystic carcinoma, as the patients with 1p36 deletion had significantly lower survival. Second part summarizes new developments about mammary analogue secretory carcinoma (MASC), which was described by our group as a new salivary tumor entity characterized by translocation t(12;15)(p13;q25) resulting in ETV6-NTRK3 fusion. Another novel observation is a discovery of ETV6-RET fusion in a subset of MASC cases. Further, the first two MASCs of nasal mucosa origin have been described. Third part consists...

A presença de sistemas subterrâneos em plantas do Cerrado é uma característica que há algum tempo vem atraindo a atenção de pesquisadores. Suas funções adaptativas e ecológicas já são bem conhecidas, mas devido à diversidade de tipos a denominação desses órgãos nem sempre é aplicada corretamente, já que muitas vezes são realizados apenas estudos morfológicos que se mostram insuficientes. Portanto, análises anatômicas representam uma ferramenta indispensável na correta aplicação terminológica dessas estruturas. A família Asteraceae, muito bem representada no Cerrado paulista, apresenta grande quantidade de espécies que possuem sistemas subterrâneos espessados com capacidade gemífera e que acumulam frutanos como fonte de reservas. Essas características permitem a sobrevivência das plantas nesse ecossistema garantindo a regeneração da parte aérea que é eliminada devido a episódios de fogo ou seca prolongada, fenômenos comuns no Cerrado, ressaltando a importância desses órgãos subterrâneos na contribuição para o banco de gemas. Em sistemas subterrâneos espessados de Asteraceae, apesar de haver poucos estudos que enfoquem este assunto, observa-se a ocorrência de estruturas secretoras com valor diagnóstico que auxiliam estudos taxonômicos da família e possuem importante papel ecológico, já que constituem estratégia de defesa contra herbivoria. No entanto, devido à realização de análises incompletas verificada na literatura, muitas vezes os espaços secretores internos são genericamente denominados como canais, quando em observações em secções longitudinais, verifica-se que na verdade não são estruturas alongadas ou sua formação ocorre de maneira diversa. Alguns sistemas subterrâneos permitem a propagação vegetativa das plantas e podem constituir importante estratégia adaptativa no Cerrado. A relação entre a taxa germinativa e a capacidade de propagar-se vegetativamente explica os diferentes resultados obtidos em ensaios de germinação de sementes de espécies que possuem ou não a capacidade de reprodução clonal através de suas estruturas subterrâneas. O conjunto de informações obtidas, não só contribui para ampliar o conhecimento da flora do Cerrado, mas também fornece argumentos que justifiquem a sua conservação. / It has some time that the presence of underground systems in plants from Cerrado is a feature that has been attracting the researchers attention. Their ecological and adaptive functions are already well-known, but due to the diversity of underground organs types the denomination of these organs is not always correctly applied, since many times only morphological studies are performed what have shown unsatisfactory. Therefore anatomical analyses represent an essential tool to the correct terminological application of these structures. The family Asteraceae, very well represented in the Cerrado of the São Paulo state, exhibit great amount of species that possess bud bearing thickened underground systems that accumulate fructans of the inulin type as reserve compounds. These features allow the plants survival in this ecosystem assuring the regeneration of the aerial part that is eliminated due to fire episodes or extended dry, common phenomenon in Cerrado, reinforcing in this way the importance of these underground organs in the contribution to the bud bank. In thickened underground systems of Asteraceae in spite of having few studies which focus this subject, it is observed the occurrence of secretory structures with diagnosis value that aid taxonomic studies of the family and possess important ecological role since they constitute protection strategy against herbivory. Nevertheless due to the achievement of incomplete analysis as verified in the literature, many times the internal secretory spaces are generically named as canals, while in longitudinal sections observations it is verified that they are not actually elongated structures or their formation occurs of diverse way. Some underground systems allow the vegetative propagation of the plants and they can constitute important adaptation strategy in Cerrado. The relation between the germination rates and the vegetative propagation capacity explains the different results obtained in seed germination experiments of species that possess or not clonal reproduction capacity through their underground structures. The whole of information obtained contribute to extend the knowledge about the Cerrado flora and also provides arguments that justify its conservation.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-29T12:26:38Z No. of bitstreams: 1 marciojosedasilvacampos.pdf: 1176619 bytes, checksum: 2a8507a8f5f473e63daeeccfac1ec957 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-30T11:19:31Z (GMT) No. of bitstreams: 1 marciojosedasilvacampos.pdf: 1176619 bytes, checksum: 2a8507a8f5f473e63daeeccfac1ec957 (MD5) / Made available in DSpace on 2017-03-30T11:19:31Z (GMT). No. of bitstreams: 1 marciojosedasilvacampos.pdf: 1176619 bytes, checksum: 2a8507a8f5f473e63daeeccfac1ec957 (MD5) Previous issue date: 2010-02-09 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Dor é comumente relatada durante o tratamento ortodôntico, sendo proveniente da força aplicada aos dentes e de lesões traumáticas na mucosa bucal, onde a Imunoglobulina A secretora (sIgA) é a principal proteção e pode participar na manutenção da sua integridade. A dor pode aumentar o estresse psicológico, que relaciona-se com a alfa-amilase salivar. A intensidade da dor pode variar segundo a idade do paciente e sua motivação ao tratamento. O objetivo foi avaliar a intensidade de dor e sua relação com a motivação dos pacientes e com as concentrações salivares de sIgA e alfa-amilase, em adultos e crianças durante duas fases do tratamento ortodôntico. Vinte indivíduos (10 crianças e 10 adultos) responderam à um questionário de avaliação da motivação ao tratamento. Amostras de saliva foram coletadas e a intensidade de dor foi registrada diariamente, antes e após a colagem dos bráquetes e após a inserção do arco inicial. Apenas uma questão, relacionada à percepção da severidade da má oclusão, apresentou correlação com a intensidade de dor. Não houve diferença significante na intensidade de dor e na concentração sIgA entre adultos e crianças. De modo geral as crianças exibiram menor prevalência de dor, porém com maior intensidade. Houve uma tendência de correlação negativa entre a dor na mucosa bucal e a concentração de sIgA nas crianças, o que pode indicar a importância da sIgA na proteção da mucosa bucal. A concentração de alfa-amilase não teve correlação significante com a intensidade de dor, porém apresentou um aumento progressivo durante o período de avaliação, provavelmente devido ao estresse psicológico causado pela presença e ativação do aparelho fixo. / Pain is usually reported during orthodontic treatment. It comes from the strength applied to the teeth and traumatic lesions in the buccal mucosa, where secretory immunoglobulin A (sIgA) is the main protection and plays a role in integrity maintenance. Pain can increase the psychological stress, which induces changes in salivary alpha amylase. Pain intensity can vary according to patient’s age and his/her motivation towards treatment. The aim of this study was to evaluate pain intensity and its relation with patient’s motivation and salivary levels of slgA and alpha amylase, in adults and children, during two stages of orthodontic treatment. Twenty individuals (10 children and 10 adults) answered a questionnaire regarding their motivation towards treatment. Saliva samples were collected and pain intensity was evaluated on a daily basis, after and before the bonding of brackets and after the insertion of the initial arch. Only one question regarding the perception of malocclusion severity correlated with pain intensity. There was no significant difference in pain intensity and in slgA concentrations between adults and children. In general, pain prevalence in children was lower, yet reported pain was more intense. Pain in the buccal mucosa was negatively correlated with slgA concentrations in children, a finding that suggests a protective effect of slgA in the buccal mucosa. Although there was no significant correlation between the concentrations of alpha amylase and pain intensity, the levels of this enzyme increased during the evaluation period, probably due to the psychological stress caused by the presence and activation of the fixed braces.