Submitted by Thiago Alves de Souza null (thiagobasouza@hotmail.com) on 2017-12-01T02:38:57Z No. of bitstreams: 1 Dissertação_Thiago_Alves_de_Souza.pdf: 1361479 bytes, checksum: 607ce9719a0123dea1f4b2761a5e36e2 (MD5) / Submitted by Thiago Alves de Souza null (thiagobasouza@hotmail.com) on 2017-12-04T14:43:23Z No. of bitstreams: 1 Dissertação_Thiago_Alves_de_Souza.pdf: 1361479 bytes, checksum: 607ce9719a0123dea1f4b2761a5e36e2 (MD5) / Submitted by Thiago Alves de Souza null (thiagobasouza@hotmail.com) on 2017-12-05T14:11:15Z No. of bitstreams: 1 Dissertação_Thiago_Alves_de_Souza.pdf: 1361479 bytes, checksum: 607ce9719a0123dea1f4b2761a5e36e2 (MD5) / Submitted by Thiago Alves de Souza null (thiagobasouza@hotmail.com) on 2017-12-11T18:47:11Z No. of bitstreams: 1 Dissertação_Thiago_Alves_de_Souza.pdf: 1361479 bytes, checksum: 607ce9719a0123dea1f4b2761a5e36e2 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2017-12-13T12:06:42Z (GMT) No. of bitstreams: 1 souza_ta_me_jabo.pdf.pdf: 1361479 bytes, checksum: 607ce9719a0123dea1f4b2761a5e36e2 (MD5) / Made available in DSpace on 2017-12-13T12:06:42Z (GMT). No. of bitstreams: 1 souza_ta_me_jabo.pdf.pdf: 1361479 bytes, checksum: 607ce9719a0123dea1f4b2761a5e36e2 (MD5) Previous issue date: 2017-10-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As neoplasias mamárias constituem os tumores com maior incidência em cadelas. Dentre os fatores que contribuem para o desenvolvimento desta e de outras neoplasias, o microambiente tumoral inflamatório desempenha um papel crucial. Vários estudos relataram papéis importantes para linfócitos, macrófagos, plasmócitos, neutrófilos, eosinófilos e mastócitos neste contexto. No presente estudo, o objetivo foi avaliar a densidade de células inflamatórias e área de fibrose tumoral e sua relação com tumores mamários caninos com diferentes características histológicas e clínicas (tumor misto benigno, carcinoma em tumor misto, carcinoma sólido e carcinoma tubular). A análise de células inflamatórias e área de fibrose tumoral foram realizadas por meio de técnicas histoquímicas, enquanto a identificação de linfócitos Tregs foram realizadas por imuno-histoquímica. A análise estatística das densidades de células inflamatórias e áreas fibrose tumoral e sua relação com os tipos histológicos revelou diferença significativa para as plasmócitos (p = 0,035), neutrófilos (p = 0,0113), macrófagos (p = 0,0047) e fibrose tumoral (p = 0,05). Os dados encontrados sugerem associações entre alto número de neutrófilos e neoplasias mamárias agressivas, entre altas densidades de plasmócitos, macrófagos e células CD8+ e entre menor densidade de células CD4+ e neoplasias menos agressivas. Maiores áreas de fibrose tumoral mostraram relação com neoplasias mamárias caninas mais agressivas. / Mammary neoplasias constitute the tumors with higher incidence in bitches. Among the factors that contribute for the development of this and other neoplasias, the inflammatory tumor microenvironment plays a crucial role. Several studies reported important roles for lymphocytes, macrophages, plasma cells, neutrophils, eosinophils and mast cells in this context. In the present study, the goal was to evaluate density of inflammatory cells and area of tumor fibrosis and their relation with canine mammary tumors with different histologic and clinical presentation (benign mixed tumor, carcinoma in mixed tumor, solid carcinoma and tubular carcinoma) Counting and staining of inflammatory cells and tumor fibrosis were performed through histochemistry, while counting and staining of Tregs were performed through immunohistochemistry. Statistical analysis of the densities of inflammatory cells and tumor fibrosis related to histologic types revealed significant difference for plasma cells (p=0,035), neutrophils (p=0,0113), macrophages (p=0,0047), and tumor fibrosis (p=0,05). The found data suggest associations between high number of neutrophils and agressive mammary neoplasias, between high densities of plasma cells, macrophages and CD8+ cells and between low density of CD4+ cells and less aggressive neoplasias. Higher areas of tumor fibrosis showed relation with more aggressive canine mammary neoplasias.

Made available in DSpace on 2014-06-11T19:26:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-30Bitstream added on 2014-06-13T20:55:03Z : No. of bitstreams: 1 conceicao_dm_me_botfca.pdf: 648319 bytes, checksum: 402d279e48ec4dd5e8449a08fd3d9141 (MD5) / Outra / O objetivo do trabalho foi analisar, de forma comparativa, os efeitos dos fungos Puccinia menthae e Erysiphe biocellata na anatomia e histoquímica de Mentha spicata, Mentha piperita, Mentha suaveloens e Mentha arvensis, verificar o efeito de óleos essenciais de plantas medicinais sobre os fungos e constatá-los como fontes de controle alternativo e averiguar prováveis fatores de resistência às doenças presentes na estrutura anatômica e histoquímica. As plantas foram inoculadas com os esporos dos fungos na face abaxial de suas folhas e submetidas às condições adequadas ao desenvolvimento do patógeno. Após 10 a 15 dias da inoculação, folhas doentes e sadias foram submetidas às análises anatômicas e histoquímicas. Os reagentes empregados para evidenciar as substâncias apolares foram: sudan III para lipídios totais; sulfato azul do nilo para diferenciação de lipídios ácidos e neutros; reagente de NADI para identificação de óleos essenciais e oleoresinas; tricloreto de antimônio para identificação de esteróides e ácido sulfúrico para a identificação de lactonas sesquiterpênicas e para substâncias polares: vanilina clorídrica para a identificação de taninos; lugol para a identificação de amido; e azul brilhante de comassie para identificar proteínas totais. Os testes ‘in vitro’ constaram de óleos essenciais de Cymbopogan martini, Corymbia citriodora, Syzygium aromaticum e Citrus limonum nas concentrações 0; 0,01; 0,02; 0,03 e 0,04%, além do padrão químico, sobre esporos dos dois patógenos em 4 repetições. Os óleos essenciais foram homogeneizados em meio de cultura ágar-água com Tween 80 e vertidos em placas de Petri. Após a solidificação do meio os esporos foram inoculados e depois de 24 horas contados de 50 esporos germinados. O estudo anatômico das folhas infectadas por P.menthae revelou desestruturação da cutícula, colonização... / The objective of the study was to analyse, compared the effects of E. biocellata and P. menthae in the anatomy and in the histochemistry of the mentha species M. spicata, M. piperita, M. suavelones e M. arvensis, to verify the effects of essential oils on the fungi spores as sources of alternative control and to investigate possibles resistance factors present in the anatomy and in the histochemistry. The plants were inoculated with the fungi spores in the lower face of the leaf, they were submitted to appropriate conditions for the development of the pathogen. After 10 to 15 days of inoculation, the infected and health leaves were submitted to the anatomic cuts and histochemistry study. The employed reagents to evidence the apolar substances were: sudan III for identify total lipids, blue nilo sulfate for acid and neutral lipids, NADI reagent to identify resins and essential oils, antimony trichloride, to identify steroids and sulphuric acid/glycerine to identify sesquiterpenes lactones. For polar substances were used: vanillin hydrochloric, for identify tannins, lugol, to identify starches and comassie bright blue to identify proteins. The in vitro tests consisted in four treatments of essential oils of Cymbopogan martini, Corymbia citriodora, Syzygium aromaticum and Citrus limonum in concentrations of: 0%; 0,01%; 0,02%; 0,03% and 0,04%, chemical pattern, in 4 repetition. The essential oils were homogenized in agar-water medium with tween 80 and flowed in Petri plates. After the medium solidification, the spores were inoculated in the medium, by passed 24 hours, the germinated spores were counted. The anatomic study of health menthas, when compared with infected species, with P. menthae resulted in cuticule disrupting and tissue colonization, similar in all species. For E. biocellata there were no disruting of the tissues or visible anatomic changes. The histochemistry study identified... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-24Bitstream added on 2014-06-13T18:50:24Z : No. of bitstreams: 1 barreiro_dp_me_botib.pdf: 1501640 bytes, checksum: aa250655ae3cef104579c8c1bda9f123 (MD5) / A presença de coléteres na superfície adaxial de órgãos jovens constitui uma característica relevante de Rubiaceae. Alibertia sessilis (Vell.) K. Schum., conhecida como marmelinho ou marmelo-do-cerrado, é uma espécie subarbustiva de Rubiaceae nativa de cerrado e tem grande importância alimentícia e medicinal neste bioma. Os ápices vegetativo e reprodutivo apresentam-se recobertos por uma secreção hialina, de aspecto vítreo e hidrofóbico produzida por coléteres. Neste trabalho foram investigados a distribuição, estrutura, histoquímica e o modo de secreção dos coléteres presentes em ápices vegetativos e reprodutivos desta espécie. Para estudos convencionais, as amostras foram preparadas segundo técnicas usuais em microscopia de luz e microscopia eletrônica de varredura (MEV) e transmissão (MET); técnica especial foi aplicada para dissolução da secreção e observação dos coléteres ao MEV. A técnica do ZIO foi empregada para maior visualização do sistema de endomembranas ao MET. Testes histoquímicos foram feitos em secções de material recém coletado. Coléteres nesta espécie são do tipo dendróide e ocorrem em abundância na face adaxial das estípulas, brácteas e sépalas; consistem de um eixo central multicelular e multisseriado revestido por numerosas células epidérmicas digitiformes ou pontiagudas, de tamanhos irregulares, unidas entre si somente na porção proximal e separadas umas das outras na porção distal. As células colunares são axialmente alongadas e possuem paredes espessas, núcleo conspícuo, citoplasma abundante e vacúolos desenvolvidos. As células epidérmicas possuem paredes delgadas, núcleo conspícuo, citoplasma abundante e vacuoma pouco desenvolvido. Os coléteres, de ambos os ápices, não possuem cutícula. Lipídeos, proteínas e polissacarídeos foram detectados nas células epidérmicas e colunares; compostos... / The presence of colleters in the adaxial surface of young organs constitutes a relevant characteristic of Rubiaceae. Alibertia sessilis (Vell.) K. Schum., known as marmelinho or marmelo-docerrado, is a subshrub native Rubiaceae species of cerrado and it has great nutritious and medicinal importance in this biome. The vegetative and reproductive apices present recovered for a hyaline secretion, of glass aspect and hydrophobic produced by colleters. In this work the distribution, structure, histochemistry and the way of secretion of the colleters present in vegetative and reproductive apices of this species were investigated. For conventional studies, the samples were prepared according to usual techniques in light microscopy and scanning (SEM) and transmission electron microscopy (TEM); special technique was applied for dissolution of the secretion and observation of the colleters to MEV. The technique of ZIO was used for larger visualization of the endomembranes system to MET. Histochemical assays were made in fresh material sections. Colleters in this species are of the dendroid type and occur abundantly in the adaxial surface of the stipules, bracts and sepals; they consist of a multicellular and multiseriate central axis recovered by numerous digitiform or sharp epidermal cells of irregular sizes, joined among themselves only in the proximal portion and separated from each other in the distal portion. The columnar cells are axially elongated and present thick walls, conspicuous nucleus, abundant cytoplasm and developed vacuoles. The epidermal cells present thin walls, conspicuous nucleus, abundant cytoplasm and a few developed vacuome. The coléteres, of both apices, don't present cuticle. Lipids, proteins and polysaccharides were detected in the epidermal and columnar cells; phenolic compounds were only detected in the columnar cells... (Complete abstract click electronic access below)

Orientador: Silvia Rodrigues Machado / Banca: Rita de Cássia Sindronia Maimoni-Rodella / Banca: Joecildo Francisco Rocha / Resumo: A presença de coléteres na superfície adaxial de órgãos jovens constitui uma característica relevante de Rubiaceae. Alibertia sessilis (Vell.) K. Schum., conhecida como marmelinho ou marmelo-do-cerrado, é uma espécie subarbustiva de Rubiaceae nativa de cerrado e tem grande importância alimentícia e medicinal neste bioma. Os ápices vegetativo e reprodutivo apresentam-se recobertos por uma secreção hialina, de aspecto vítreo e hidrofóbico produzida por coléteres. Neste trabalho foram investigados a distribuição, estrutura, histoquímica e o modo de secreção dos coléteres presentes em ápices vegetativos e reprodutivos desta espécie. Para estudos convencionais, as amostras foram preparadas segundo técnicas usuais em microscopia de luz e microscopia eletrônica de varredura (MEV) e transmissão (MET); técnica especial foi aplicada para dissolução da secreção e observação dos coléteres ao MEV. A técnica do ZIO foi empregada para maior visualização do sistema de endomembranas ao MET. Testes histoquímicos foram feitos em secções de material recém coletado. Coléteres nesta espécie são do tipo dendróide e ocorrem em abundância na face adaxial das estípulas, brácteas e sépalas; consistem de um eixo central multicelular e multisseriado revestido por numerosas células epidérmicas digitiformes ou pontiagudas, de tamanhos irregulares, unidas entre si somente na porção proximal e separadas umas das outras na porção distal. As células colunares são axialmente alongadas e possuem paredes espessas, núcleo conspícuo, citoplasma abundante e vacúolos desenvolvidos. As células epidérmicas possuem paredes delgadas, núcleo conspícuo, citoplasma abundante e vacuoma pouco desenvolvido. Os coléteres, de ambos os ápices, não possuem cutícula. Lipídeos, proteínas e polissacarídeos foram detectados nas células epidérmicas e colunares; compostos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The presence of colleters in the adaxial surface of young organs constitutes a relevant characteristic of Rubiaceae. Alibertia sessilis (Vell.) K. Schum., known as "marmelinho" or "marmelo-docerrado", is a subshrub native Rubiaceae species of cerrado and it has great nutritious and medicinal importance in this biome. The vegetative and reproductive apices present recovered for a hyaline secretion, of glass aspect and hydrophobic produced by colleters. In this work the distribution, structure, histochemistry and the way of secretion of the colleters present in vegetative and reproductive apices of this species were investigated. For conventional studies, the samples were prepared according to usual techniques in light microscopy and scanning (SEM) and transmission electron microscopy (TEM); special technique was applied for dissolution of the secretion and observation of the colleters to MEV. The technique of ZIO was used for larger visualization of the endomembranes system to MET. Histochemical assays were made in fresh material sections. Colleters in this species are of the dendroid type and occur abundantly in the adaxial surface of the stipules, bracts and sepals; they consist of a multicellular and multiseriate central axis recovered by numerous digitiform or sharp epidermal cells of irregular sizes, joined among themselves only in the proximal portion and separated from each other in the distal portion. The columnar cells are axially elongated and present thick walls, conspicuous nucleus, abundant cytoplasm and developed vacuoles. The epidermal cells present thin walls, conspicuous nucleus, abundant cytoplasm and a few developed vacuome. The coléteres, of both apices, don't present cuticle. Lipids, proteins and polysaccharides were detected in the epidermal and columnar cells; phenolic compounds were only detected in the columnar cells... (Complete abstract click electronic access below) / Mestre

Orientadores: Marilia de Moraes Castro, Luiza Sumiko Kinoshita / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T04:14:47Z (GMT). No. of bitstreams: 1 Martins_FabianoMachado_D.pdf: 4467324 bytes, checksum: 1ebe227a237ea5dc6d9af203a5e3ebbf (MD5) Previous issue date: 2008 / Resumo: No Brasil, Apocynaceae está representada por 90 gêneros e cerca de 850 espécies distribuídas em diversas formações vegetais, incluindo o cerrado. Esta família destaca-se por ter grande importância econômica ou medicinal, característica relacionada à presença de vários tipos de estruturas secretoras em suas espécies. Vinculado ao projeto temático BIOTA/FAPESP (proc. nº 00/12469-3) que estudou a morfologia, anatomia, histoquímica e ultra-estrutura de plantas do cerrado do estado de São Paulo, este estudo elegeu Odontadenia lutea (Vell.) Markgr., Secondatia densiflora A.DC. e Temnadenia violacea (Vell.) Miers com o propósito de efetuar um levantamento de tipos de estruturas secretoras em órgãos vegetativos e reprodutivos. Os coléteres ocorrem nos ápices vegetativos e reprodutivos das três espécies estudadas. Coléteres foliares ocupam posição peciolar, interpeciolar e intraestipular; vascularização ocorre no coléter axilar de O. lutea e no coléter marginal distal interpeciolar de T. violacea. Tipos inéditos de coléteres - laminar tripartido e assimétrico - são relatados para os botões florais de O. lutea. A secreção produzida pela epiderme em paliçada do coléter calicinal de O. lutea é heterogênea, sendo sintetizada mucilagem em uma primeira fase e compostos fenólicos em etapa posterior. Idioblastos secretores são estruturas comuns nos órgãos vegetativos e florais de O. lutea e S. densiflora, estando ausentes em T. violacea, sendo encontrados no parênquima fundamental e associados ao floema. No caule secundário de S. densiflora, ocorre uma hipoderme secretora com secreção de aspecto denso semelhante ao dos idioblastos. Os laticíferos são do tipo anastomosado, sendo observados em todos os órgãos estudados. Eles têm diâmetro variável e sua parede é geralmente mais espessa que as das células parenquimáticas. A secreção do laticífero tem aspecto denso, sendo corada pela safranina. Em S. densiflora, os nectários florais têm origem receptacular, são unidos na base e livres na região apical constituindo cinco unidades. O nectário é composto por epiderme coberta por fina cutícula, parênquima nectarífero e feixes vasculares. Na região terminal, os estômatos liberam o néctar para o meio externo. Nas três espécies estudadas, o ginostégio apresenta diferenças morfológicas. Não foi observada adnação entre a cabeça do estilete e conectivo expandido em S. densiflora. O. lutea e T. violacea ocorre adnação da epiderme em paliçada do conectivo com a epiderme em paliçada do anel parenquimático da cabeça do estilete. As estruturas secretoras das três espécies em estudo mostram ser relevantes à taxonomia e podem auxiliar na delimitação de tribos da subfamília Apocynoideae. / Abstract: Apocynaceae is represented in Brazil by 90 genera and approximately 850 species distributed among diverse vegetation formations, including the cerrado. This family has significant economic and medicinal importance that is related to the presence of differents types of secretory structures (glands) among their species. The present study, linked to the thematic project BIOTA/FAPESP entitled "Morphology, anatomy, histochemistry, and ultrastructure of cerrado vegetation in the state of São Paulo, Brazil" (proc nº 00/12469-3), focuses on a survey of the glands found in vegetative and floral organs of Odontadenia lutea (Vell.) Markgr., Secondatia densiflora A.DC., and Temnadenia violacea (Vell.) Miers. The colleters are located on the vegetative and floral apices of the three species studied. Foliar colleters are found on the petiole, as well as the interpetiolar and intra-stipular regions; vascularization is observed in the axial colleter of O. lutea and in the marginal distal interpetiolar colleter of T. violacea. New types of colleters - laminar tripartite and asymmetrical - are observed on the floral buds of O. lutea. The palisade epidermis of the calycine colleter of O. lutea produces a heterogeneous secretion, composed of mucilage and phenolic compounds. The mucilage is synthesized in a primary phase and the phenolic compounds are produced in a second phase. Secretory idioblasts are common structures in vegetative and floral organs of O. lutea and S. densiflora, but absent in T. violacea. These idioblasts are found in fundamental parenchyma and associated with phloem. In secondary stem of S. densiflora, a secretory hypodermis remains as a continuous layer, which is interrupted only by stomatal complex regions, and the secretory cells are filled with dense material. The lactiferous are of the anastomose type, and are observed in all organs studied. They have variable diameters and their walls are generally thicker than those of the parenchyma cells. The secretion of the lactiferous is dense and stains with Safranin. The floral nectaries of S. densiflora originate from the receptacle and are united at the base but free in the apical region, forming five units. The nectary is composed of nectariferous epidermis, nectariferous parenchyma and vascular bundles. The nectar is liberated through stomata found on the terminal region of the nectaries. The gynostegium of the three species studied are morphologically different. Adnation between the style head and the expanded connective is only observed in O. lutea and T. violacea. The secretory structures of the three species examined are taxonomically relevant, and might aid the delimitation of the subfamily Apocynoideae tribes. / Doutorado / Doutor em Biologia Vegetal

In order to investigate the total number, relative proportions, size variations and distribution patterns of fibres of different types in human skeletal muscles, a technique was developed which allows whole muscle cross-sections to be collected and stained enzyme histochemically. Necropsy material was obtained from m. tibialis anterior and m. vastus lateralis of previously healthy young adults who had suffered sudden accidental deaths. The muscle specimens were extirpated within 72 hours post morten, frozen in liquid nitrogen, embedded in carboxymethylcellu- lose, sectioned and enzyme histochemically stained for myofibrillar ATPase in order to permit light microscopic identification of type 1 and type 2 muscle fibres. The present results show that there is a large variation in both the total number of fibres and the size of the whole muscle cross-sectional area between different individuals and, also, between different levels of the same muscle. The distribution of fibres of different types over the muscle cross-section was heterogeneous. Usually the relative proportion of type 2 fibres showed a peak along a medio- lateral line passing through the centre of the muscle cross-section. When comparing the left and right m. tibialis anterior of the same individual, one of the- muscles (usually the left one) was found to contain a larger number of fibres than the contralateral one. However, the pattern of distribution of fibres of different types was similar in the two muscles. The fibre sizes were also found to vary between different regions of the muscle cross-section. Both type 1 and type 2 fibres were significantly larger in the deep muscle region compared to the central or superficial sites. The mean fibre size as well as the total number of fibres correlated strongly with the whole muscle cross-sectional area. The female m. tibialis anterior contained fewer and smaller fibres than the corresponding male muscle, although the whole muscle cross-sectional area was of similar magnitude. The distribution of fibre types over the muscle cross-section differed somewhat between females and males. The variation in fibre sizes between different muscle sites was less pronounced in the females, but as in male muscles the type 2 fibres were always larger than the type 1 fibres. In conclusion, systematic variations in fibre type distribution and fibre size occurs over the muscle cross-section. The total number of fibres in the cross- section seems to vary with individual, sex, distance from muscle origin and left- right leg. The combination of fewer and smaller fibres in females compared to males leads to about 40 % smaller total muscle fibre cross-sectional area in female m. tibialis anterior. The results are discussed in relation to previous studies using muscle biopsy techniques. The possible contibution of variations in number, sizes or distribution patterns of muscle fibres to adaptation of muscle to varying functional demands is also discussed. / <p>S. 1-27: sammanfattning, s. 28-91: 5 uppsatser</p> / digitalisering@umu.se

All developing embryos contain an extracellular matrix (ECM) consisting of proteins, glycoproteins, and proteoglycans. These components are important for morphogenetic processes such as cell migration, cell differentiation and cell death. The ECM of the starfish, Pisaster ochraceus, consists of three major components: A hyaline layer which coats the external surface of the embryo; a basal lamina which lines the basal surfaces of the epithelia; and a blastocoelic component which fills the embryonic cavity or blastocoel. Observations of chemically fixed asteroid embryos have revealed the hyaline layer to contain five sub-layers of fibrous strands encrusted with amorphous material. Strands of a similar nature form a meshwork within the fluid-filled blastocoel. Recent studies of the living embryo, however, have suggested that the ECM within the blastocoel of echinoderms, including the asteroid, is a gel-like substance and not a fluid with extracellular fibres. Since artefacts imposed by chemicals such as aldehydes and osmium are well documented, a method of preservation, which does not involve the use of these chemicals, may resolve the apparent conflict over the nature of the ECM of the asteroid embryo. Freeze substitution, an expensive cryofixation technique which has proven successful in fixing vertebrate tissue, does not require the use of aldehydes and osmium. The initial objective of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pisaster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask filled with liquid nitrogen, a small cup was filled with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four different cryogens and four separate cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane was found to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Following freeze substitution, the blastocoel appears to contain a gel-like substance, rich in sulfated GAG's, with extracellular fibres and not a fluid with fibres. In addition, the hyaline layer was found to consist of at least six sub-layers of greater thickness than was seen in chemically fixed embryos. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Freeze substitution also revealed the presence of microvillus associated bodies, structures which may represent major attachment points of the hyaline layer to the epithelium. Although the fixation of asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, particular!ly of the ECM components, justifies its use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies. / Medicine, Faculty of / Graduate

Master of Science

The effect of forced molt on pituitary function and other endocrine parameters was investigated in three year old hens subjected to a dietary forced molting procedure. In addition to molting, fasting caused cessation of egg production, body and organ weight loss, alterations in hormone secretion and morphological changes in some endocrine glands. Body and ovary weights decreased but returned to normal with ad libitum feeding. Pituitary, thyroid and adrenal weights were not affected but serum hormone levels measured by RIA revealed a decrease in LH, FSH and PRL and increases in TSH, T₃ and GH all of which returned to higher levels with ad libitum feeding. Serum P₄ levels remained low (and egg-laying stopped) until ad libitum feeding was resumed and then increased and egg-laying returned to a typically productive level. Serum ACTH and T₄ increased with fasting and remained elevated. Gonadotrophs and corticotrophs increased in numbers with fasting and/or food restriction but thyrotrophs, somatotrophs and lactotrophs decreased. Correlations between cell populations and serum hormone levels was quite common. Colloid-filled follicles resembling a hypertrophic thyroid gland occurred throughout the pituitary pars distalis. Granules appear to be discharged into the follicular lumen through exocytotic pores in the apical plasmalemma of follicular cells. Lactotrophs, corticotrophs and somatotrophs are commonly arranged in follicles or clusters. PRL-containing granules are in the center of some follicles and are concentrated near pituitary cysts. Pituitary cysts, lined with ciliated epithelium and sparse mucous cells, are more prevalent in fasted hens and decline with the resumption of feeding. Reduced lactotroph populations and presumptively degenerated lactotrophs in cyst lumens are correlated with reduced serum PRL levels. Necrotic cells occurred in the pituitary parenchyma of fasted birds but dilated RER in the thyrotrophs of fasted hens indicate enhanced activity of these cells. Ultrastructural evidence presented here indicates that pituitary secretion by lactotrophs occurs both intraluminally and perivascularly.

The amygdala, in particular its basolateral part (BLA), plays a critical role in binding affective qualities to otherwise neutral stimuli, and in eliciting emotional behaviors. Plasticity of inputs to BLA projection neurons involved in emotional memory has been extensively studied. However, how BLA neurons collectively process sensory information to encode and stabilize emotional memories is unknown. Precise coordination of BLA network activities seems critical. Specifically, timed integration of salient stimuli, and synchrony with hippocampal theta oscillations appear to be important. Recent reports suggest that GABAergic neurons may be instrumental in shaping ensemble activity in the BLA. Studies of neocortex and hippocampus showed that diverse GABAergic interneuron types play highly specific roles in coordinating network operations. The presence of similar interneuron populations in the BLA suggests comparable mechanism may govern its activities. However, GABAergic cell types and their functions have not been characterized.