Thesis (Ph. D.)--University of Massachusetts Amherst, 2009. / Includes bibliographical references (p. 93-106). Print copy also available.

Thesis (Ph.D.) -- University of Rhode Island, 2008. / Typescript. Includes bibliographical references (leaves 106-116).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1971. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Akademisk avhandling- Karölinska Institutet, Stockholm. / Extra t.p., with thesis statement, inserted. Bibliography: p. 27-30.

As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. Unlike tracheophytes that have stomata on anatomically complex leaves and stems, mosses bear stomata exclusively on spore-bearing organs (capsules). However, stomata do not occur in all mosses and, indeed, are absence in the earliest-divergent mosses (Takakia, Andreaea, Andreaeobryum and Sphagnum), suggesting that stomata originated in mosses independently of other plants. The occurrence of structurally unique pseudostomata in Sphagnum further confounds the resolution of homology of moss stomata with those of other plants. The five studies included in this dissertation are aimed at clarifying the structure, development and evolution of moss stomata. The first study focuses on the sporophyte anatomy and stomatal ultrastructure in two structurally and phylogenetically divergent mosses, Oedipodium and Ephemerum. Oedipodium is the sister to peristomate mosses and the first extant moss with true stomata. This monospecific genus has an elaborated capsule with an extended apophysis bearing numerous long-pored stomata. In contrast, Ephemerum nests within the peristomate mosses and has a reduced capsule that lacks an apophysis and has a few round-pored stomata. Ultrastructure of stomata is similar in these two mosses and comparable to that of tracheophytes, except that the stomata of mosses are not as structurally distinct from epidermal cells as are tracheophyte stomata. Anatomical features such as the presence of a cuticle, water-conducting cells, and spongy tissues with large areas for gas exchange are more pronounced in Oedipodium sporophytes and support the role of stomata in gas exchange and water transport during development and maturation. The second study examines changes in pectin composition during development in the model moss Funaria. Stomatal movement in tracheophytes requires guard cell walls to be strong, yet flexible, because they have to undergo reversible deformation to open and close the pore. Pectins are necessary for wall flexibility and proper stomatal functioning in seed plants. In this study of Funaria, immunogold-labeling using five antibodies to pectin epitopes was conducted on guard cell walls during development to relate these features to the limited movement of stomata in moss. Movement of Funaria stomata coincides with capsule expansion when guard cell walls are thin and pectinaceous. Walls dramatically increase in thickness after pore formation and the pectin content significantly decreases in mature guard cell walls, suggesting that a decrease in flexibility is responsible for the inability to open a close previously reported in older moss guard cells. Because this was the first study to demonstrate changes in pectin composition during stomatal development in any plant, a similar study was done on Arabidopsis to identify the main types of pectins in guard cell walls. Localization of pectins in guard cell walls of Arabidopsis is similar to mosses in the stage they can move, with homogeneous walls rich in arabinan pectins that are required for wall flexibility. This study extends knowledge of pectin composition from stomata of the moss Funaria with limited stomatal movement to an angiosperm in which stomatal activity is crucial to the physiological health of the plant. The fourth study describes stomata development and internal changes in sporophyte anatomy that lead to formation of air spaces in the moss Funaria. Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold-labeling was used to investigate the presence of pectin in the newly formed cavities. Stomata in mosses do not develop from a self-generating meristemoid like in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e., there are no stomatal lineage ground cells. This developmental pattern is congruent with the presence of a gene ortholog of FAMA, but not SPCH and MUTE, in Physcomitrella. The final study in this dissertation focuses on the enigmatic Sphagnum. Although true stomata are absent in early-divergent mosses, Sphagnum has specialized epidermal cells, pseudostomata, that partially separate but do not open to the inside. To further understand the structure, function and evolution of pseudostomata, capsule anatomy and ultrastructure of pseudostomata were detailed. As in moss stomata, pseudostomata wall architecture and behavior facilitate capsule dehydration, shape change, and dehiscence, supporting this common function. Unlike other moss stomata, pseudostomata collapse along their ventral walls and they lack a substomatal cavity. Similarities to true stomata include two modified epidermal cells with specialized cell walls that separate by cuticle deposition and respond to drying. Pseudostomata may be interpreted as modified stomata that suppressed substomatal cavity formation, which in turn eliminated pore development. However, clarification of the homology of pseudostomata and moss stomata will require genomic studies integrated with physiological and structural data. The studies described in this dissertation significantly advance our understanding of moss stomatal development and structure, and provide a comparison point to better evaluate the evolution of stomata. Moss capsule anatomy coupled with the exclusive existence of stomata on capsules supports the concept that stomata in moss are involve in gas exchange but also facilitate drying and dispersal of spores. Changes in wall architecture coupled with a decrease in total pectin explain the inability of mature stomata to move. Development and distribution of stomata in Funaria provides evidence of a direct and less elaborated mechanism for stomatal development than described in Arabidopsis. Resolving relationships among early land plants, especially hornworts and mosses, the only bryophyte groups with stomata, is critical to understanding stomata evolution. Evaluated together, the results of this dissertation are consistent with a single origin of stomata in land plants.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-30Bitstream added on 2014-06-13T19:45:11Z : No. of bitstreams: 1 villaverde_aisb_dr_botfmvz.pdf: 3478993 bytes, checksum: d5ea0090ec4786ded2155ff7802d56fe (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo da proteína fosfolipase C zeta (PLCζ), considerada o fator espermático ativador do oócito em mamíferos, pode beneficiar algumas técnicas de reprodução assistida, como a injeção espermática intracitoplasmática e transferência nuclear, por propiciar conhecimento a respeito do processo de fertilização e a possibilidade de utilização da PLCζ presente nos extratos espermáticos. Portanto, o objetivo deste estudo foi localizar a PLCζ em extratos provenientes do citosol e matriz perinuclear de espermatozóides de gatos domésticos normospérmicos e teratospérmicos. Amostras de sêmen foram colhidas de seis gatos adultos: normospérmicos (n=3), teratospérmicos (n=2) e de qualidade seminal intermediária (n=1). As proteínas do citosol foram extraídas utilizando os procedimentos de sonicação, ultracentrifugação, ultrafiltração e precipitação em sulfato de amônio. Por sua vez, as proteínas da matriz perinuclear foram extraídas após incubação em Na2CO3, sonicação, ultracentrifugação, ultrafiltração e precipitação em sulfato de amônio. Com base na avaliação ultraestrutural dos espermatozóides e dosagem de proteína total, foi confirmada a eficiência de ambos os protocolos de extração. As amostras da matriz perinuclear apresentaram 3,3 vezes menos proteína total e diferente perfil protéico na eletroforese unidimensional e bidimensional quando comparadas as amostras obtidas do citosol. Após análise das proteínas encontradas, foi concluído que nos extratos espermáticos do citosol e matriz perinuclear de gatos domésticos normospérmicos e teratospérmicos estão presentes proteínas de peso molecular semelhante ao previamente descrito para a proteína PLCζ em outras espécies de mamíferos / The study of phospholipase C zeta protein (PLCζ), considered as the oocyte activating sperm factor in mammals, could benefit some assisted reproductive techniques, such as intracytoplasmic sperm injection and nuclear transfer, by providing knowledge about the process of fertilization and the possibility of using the PLCζ contained in the sperm extracts. Thus, the aim of this study was to localize the phospholipase C zeta (PLCζ) protein in extracts from the spermatozoa cytosol and perinuclear matrix of normospermic and teratospermic domestic cat. Sperm samples were collected from six adult male cats: normospermic (n=3), teratospermic (n=2) and with intermediate sperm quality (n=1). Proteins from the cytosol were extracted using the procedures of sonication, ultracentrifugation, ultrafiltration and precipitation in ammonium sulfate. On the other hand, proteins from perinuclear matrix were extracted after incubation in Na2CO3, sonication, ultracentrifugation, ultrafiltration and precipitation in ammonium sulfate. Based on the ultrastructural analysis of the spermatozoa and total protein determination, the efficiency of both extraction protocols was confirmed. Samples from perinuclear matrix showed 3.3 times less total recovered protein and different protein profile in the uni- and bidimensional electrophoresis when compared to the samples obtained from the cytosol. After protein profile analysis, it can be concluded that several proteins showing similar molecular weight to that previously described for PLCζ protein in other mammalian species are presented in both sperm extracts from cytosol and perinuclear matrix of normospermic and teratospermic domestic cats

abstract: The distinguishing feature of the filamentous fungi is the hyphae - tube-like microscopic cells that exhibit polarized growth via apical extension and allow the fungus to interact with its environment. Fungi elongate at the hyphal apex, through the localized construction of new plasma membrane and cell wall through the exocytosis of secretory vesicles. One population of these vesicles have been identified as chitosomes, containing chitin synthase isoenzymes, which are responsible for the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin, the primary fibrillar component of the fungal cell wall. The chitosomes, in addition to other vesicles, can be observed aggregating in the hyphal tip in most filamentous fungi. In the Ascomycota and Basidiomycota, this collection of vesicles exhibits discrete organization and has been termed a Spitzenkörper. Although accumulations of vesicles can be observed in the hyphal tip of many growing filamentous fungi, some debate continues as to what precisely defines a Spitzenkörper. This study reports the details of three separate projects: first, to document the effects of deleting a single chitin synthase, CHS-1 and CHS-6 in Neurospora crassa with regards to hyphal ultrastructure, cytoplasmic organization, and growth in comparison to the wild-type. Given the importance of chitin synthesis in fungal cell growth, deletion of a critical chitin synthase presumably impacts cell wall structure, fungal growth and cytoplasmic organization. Second, an examination of the ultrastructure of four zygomycetous fungi - Coemansia reversa, Mortierella verticillata, Mucor indicus, and Gilbertella persicaria has been conducted. Utilization of cryofixation and freeze-substitution techniques for electron microscopy has produced improved preservation of cytoplasmic ultrastructure, particularly at the hyphal apex, allowing detailed analysis of vesicle size, contents, and organization. Lastly, hyphal tip organization was reviewed in a broad range of fungi. Previous studies had either focused on a few select fungi or representative groups. Vesicle organization, composition and size do appear to vary among the classes of fungi, but some trends, like the vesicle crescent in the zygomycetous fungi have been documented. / Dissertation/Thesis / Doctoral Dissertation Biology 2015

>Magister Scientiae - MSc / The infants of smoking mothers (compared to non-smoking mothers) have been shown to have a lower birth mass, a lower brain mass, an increased perinatal mortality rate as well as a predisposition to respiratory abnormalities in later life. Evidence suggests that one of the reasons for the latter is abnormal lung structure due to changes in the connective tissue skeleton. This study evaluated the in vivo effects of maternal nicotine exposure (lmg/kg/day subcutaneously - designated the experimental group), which is equivalent to smoking 32 cigarettes per day, on the connective tissue status of the neonatal (7, 14 and 21 day old) wistar rat lung. The control group received sterile saline as a placebo. The specific aspects investigated were: (1) the morphological changes in lung structure and connective tissue (collagen, elastic tissue and reticulin) distribution by means of light microscopy. (2) the quantities of collagen and Emphysema-like morphological changes are present at all ages. The histochemical appearance of collagen is not affected while reticular fibres appear to be abnormal in structure. On day 7 there appears to be no elastic tissue in the nicotine-exposed lung compared to the control lung. This difference is notelastic tissue in the lung. (3) the ultrastructure of the lung connective tissue skeleton by means of scanning electron microscopy. noticeable on days 14 and 21. Biochemical quantitation indicated that, for the three age groups studied, there was no significant difference in collagen content between experimental and control animals. Elastic tissue was significantly higher in 7 day old experimental lungs than in the control group, contradictory to the results of the histochemical studies. This difference was not significant for 14 and 21 day old lungs Ultrastructural studies of the lung connective tissue skeletons hoed abnormal fibres in the experimental group. Changes included fibre breaks, a beaded appearance of certain fibres and a deficiency in normal fibre arrangement due to the direct or indirect effects of nicotine The effects of nicotine on neonatal rat lung after maternal nicotine exposure is described. The direct mechanisms for these events are still not known but speculation as to this are presented here. Further studies which could explain these mechanisms are also suggested.

Many heavy metals are essential for biological functions; however some of these metals, especially at high concentrations, can have serious adverse effects on humans. The main sources of heavy metal exposure are through agriculture, transport, mining and related operations. South Africa has a thriving mining industry and is known for its rich mineral resources, but due to the incorrect method of disposal of the waste from these mines, substances, including heavy metals, get into the water and air supply, affecting the people living in close proximity to these mines. Exposure is through inhalation of contaminated air and consumption of contaminated food and water. The most vulnerable to heavy metals are the developing fetus, because of the high rate of cell division and differentiation. In the current study, two heavy metals cadmium (Cd) and chromium (Cr) were chosen based on the possibility of being exposed to them in South Africa. Thus, the aim of this study was to investigate the possible cellular effects of the heavy metals Cd and Cr alone and in combination, at different concentrations, on brain, liver and kidney tissue by using a chick embryo model. This model was successfully implemented over a 14 day period after which the embryos were terminated and the brains, livers and kidneys removed and processed for light- and transmission electron microscopy (with energy dispersive spectroscopy and electron energyloss spectroscopy). In addition, the effect of Cd and Cr alone and in combination on DNA structure and micronuclei formation was evaluated. The levels of the major antioxidant component, glutathione was determined in the brains of the chick embryos. At low concentration of Cd and Cr alone and in combination, a hormesis effect was observed in the survival rates and weights of the chick embryos, while at x1000 physiological dose (PD) Cr and Cd alone and in combination the effects were toxic. The majority of viable embryos did not have any macro-anatomy abnormalities. Morphological evaluation of the brain, liver and kidney samples revealed that Cd caused severe alterations at its highest concentration with minor alterations at the lower concentrations. Cr and the metal combination groups on the other hand, only caused minimal alterations throughout the concentration ranges evaluated. The presence of Cd and Cr alone and in combination in the liver tissue was confirmed with the electron energy-loss spectroscopy analysis that detected these metals in the nuclei, mitochondria and Golgi complexes of the hepatocytes. This might contribute to the ultrastructural changes observed in this organ. The genotoxicity testing on the red blood cells revealed no substantial differences, as only a few micronuclei were present. Although heavy metals cause DNA damage through an indirect mechanism of oxidative damage, the presence of Cd and Cr in the nucleus and mitochondria indicates that these metals may have a direct effect on DNA structure. With DNA agarose gel electrophoresis it was found that Cd and Cr alone and in combination caused DNA fragmentation. In the brain, GSH levels were normal; however changes may be the result of Cd and Cr causing the depletion of other antioxidant elements such as glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase. In conclusion, this study indicates that Cd and Cr alone and in combination are toxic to the chick embryo. Cd is more toxic than Cr, and both metals accumulate in the nuclei and mitochondria where they induce damage either through oxidative and/or other mechanisms associated directly with DNA damage. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Anatomy / MSc / Unrestricted

The preputial glands of male nude (nu/nu) mice were analyzed by a combination of electron microscopy and gas chromatography to determine whether or not they are affected, like developing hairs and nails, by the nu/nu genotype. Results of the analyses revealed no differences between the glands of nude and normal male mice in either their ultrastructural characteristics or lipid secretory products.