A relação da podoplanina com a proliferação celular tem sido demonstrada, porém ainda é tema de controvérsia. O objetivo deste estudo foi investigar a expressão da podoplanina em ameloblastomas e remanescentes de epitélio odontogênico (REO) de folículos pericoronários de dentes não-irrompidos e verificar a relação da imunomarcação da podoplanina com a proliferação celular. A amostra incluiu 33 pacientes submetidos à biópsia incisional ou à exérese de ameloblastomas e 32 folículos de pacientes submetidos à extração de dentes não-irrompidos. Todos os espécimes selecionados foram corados pela técnica imuno-histoquímica com os anticorpos anti-podoplanina e anti-Ki-67. A expressão da podoplanina foi avaliada por um método semi-quantitativo de escores. Já o índice de proliferação celular foi obtido pela porcentagem de células positivas (no mínimo 500 células/ameloblastoma e todas as células do REO foram avaliadas). Todos os ameloblastomas apresentaram imunomarcação pela podoplanina nas células periféricas. Nas células centrais, a expressão foi ausente ou fraca. Nos REO, a podoplanina apresentou expressão predominantemente nas células em contato com o tecido conjuntivo. A expressão membranosa da podoplanina foi superior nos ameloblastomas em relação aos REO (!=0,001). Também houve diferença significante entre a imunomarcação citoplasmática e membranosa da podoplanina nos REO (!=0,001). A expressão do Ki-67 foi mais acentuada nos ameloblastomas do que nos REO (!<0,001). A correlação entre a expressão citoplasmática (r= -0,15, != 0, 396) e membranosa (r= 0,00, != 0, 989) da podoplanina e do Ki-67 nos ameloblastomas não foi estatisticamente significante, tampouco entre a imunomarcação citoplasmática (r= 0,15, != 0, 421) e membranosa (r= -0, 09, ! = 0, 629) da podoplanina e do Ki-67 nos REO. Os resultados sugerem que não há associação da podoplanina com a proliferação celular e reforçam a necessidade da elucidação do papel desta proteína em tumores odontogênicos benignos. / The association between podoplanin and cellular proliferative activity has been demonstrated but is still source of debate. The aim of this study was to investigate the expression of podoplanin in ameloblastomas and remnants of odontogenic epithelium (ROE) from dental follicles of unerupted teeth and verify the relation between the podoplanin expression and proliferative activity of the odontogenic cells. Thirty-three patients submitted to the incisional biopsy or resection of ameloblastoma and 32 patients submitted to the extraction of unerupted tooth were selected for analysis. Archived paraffin-embedded ameloblastomas and dental follicles specimens were sectioned and stained with anti-human podoplanin and anti-Ki-67 antibodies. The podoplanin expression by odontogenic epithelial cells was evaluated using a score method and the Ki-67 labelling index was obtained by the percentage of positive odontogenic cells (at least 500 cells/ameloblastoma and all ROE cells). All ameloblastomas showed podoplanin expression in ameloblast-like cells of the epithelial islands. Weak or absent immunostaining was observed in the central cells resembling stellate reticulum. Podoplanin expression in ROE was mainly found in the cells in contact with the connective tissue. Membranous expression of podoplanin in ameloblastomas was stronger than its expression in ROE (!=0.001). Statistically significant difference between cytoplasmic and membranous expression of podoplanin in ROE was observed (!=0.001). The index of cellular odontogenic proliferative activity, verified by Ki-67 expression, was highest in ameloblastomas when compared with ROES (!<0.001). No statistically significant correlation between podoplanin and cellular odontogenic proliferative activity in ameloblastomas and dental follicles was found (!>0.05). These results support the evidence that there is no connection between podoplanin expression and odontogenic cellular proliferative activity in ameloblastomas and reinforce that the exact role of this protein in benign odontogenic tumor needs to be elucidated.

O saco vitelino é uma das membranas extra-embrionárias que desempenha um papel importante para a sobrevivência inicial do embrião, atua como fonte de nutrição durante o período em que a placenta verdadeira ainda não está completamente formada. É uma provável fonte de células tronco, o qual abriga as primeiras células do sangue durante o desenvolvimento em mamíferos, os eritrócitos, os quais expressam fatores de transcrição que especificam estas células a seu destino hematopoiético. O objetivo deste trabalho foi caracterizar as células tronco hematopoéticas provenientes do saco vitelino de embriões bovinos, em diferentes fases gestacionais, sendo estes coletados em abatedouro local. Para descrição da análise macroscópica e cultivo celular das células do saco vitelino, os embriões bovinos foram divididos em grupos de idade gestacional: Grupo I (25 a 29 dias), Grupo II (30 a 34 dias), Grupo III (35 a 39 dias), Grupo IV (40 a 44 dias) e Grupo V (45 a 50 dias) em que permaneceram mais tempo em cultura e apresentaram a formação de aglomerados celulares, diferente dos grupos IV e V (40 a 45 dias) em que permaneceram poucos dias em cultura e não apresentaram aglomerados celulares. Esta divergência relaciona-se à idade gestacional (45 a 50 dias), período em que se inicia a regressão do saco vitelino. Em citometria de fluxo os grupos I, II e III (25 a 39 dias) obtiveram características semelhantes, alta expressão de marcadores hematopoéticos (CD34, CD90 e CD117). Para os grupos IV e V (40 a 50 dias) observa-se um declínio da expressão de CD34 e CD117 (marcadores hematopoéticos) e no grupo V houve um acréscimo da expressão de CD45 (marcador para leucócito) confirmando que estas células não estão mantendo-se indiferenciadas As células demonstraram ser resistentes a criopreservação, capazes de formar colônias em matriz de Metilcelulose, mostraram a formação de colônias após 14 dias em cultivo e a morfologia para células sanguíneas (linfócitos e monócitos) foi confirmada na citologia celular. Na expressão gênica obteve-se baixa expressão do gene GATA3, níveis diferentes de expressão entre os grupos para o marcador RUNX1 e ANXA5. Dessa forma, nossos achados mais significativos comprovaram o isolamento de células hematopoéticas a partir do saco vitelino de embriões bovinos, sugerindo que este é uma fonte laboriosa, porém viável e eficaz para a obtenção de células tronco para futuras aplicações na terapia celular e gênica. / The yolk sac is one of the extra-embryonic membranes which plays an important role in early embryonic survival and serves as source of nutrition during the period where in the placenta is not completely true formed. The yolk sac is a likely source of stem cells, which have first blood cells during development in mammals, the red blood cells, which express transcription factors that specify these hematopoietic cells to their destination. This study aimed to identify and characterize hematopoietic stem cells from the yolk sac of bovine embryos at different stages of pregnancy, which are collected at a local slaughterhouse. For a description of the macroscopic and cellular culture of yolk sac cells, are as follows bovine embryos were divided into groups of gestational age: Group I (25 to 29 days), Group II (30 to 34 days), Group III (35 to 39 days ), Group IV (40 to 44 days) and Group V (45 to 50 days) which stayed longer in culture and showed the formation of cell clusters, different from groups IV and V (40-45 days) in few that remained days in culture and showed no cell clumps. This divergence is related to gestational age (45 to 50 days), during which begins regression of the yolk sac. In flow cytometry groups I, II and III (25 to 39 days) had similar characteristics, high expression of hematopoietic markers (CD34, CD90 and CD117). For the groups IV and V (40 to 50 days) it is observed a decrease in expression of CD117 and CD34 (hematopoietic markers) and in group V were increased expression of CD45 (leukocyte marker), confirming that these cells are not keeping Undifferentiated cells are shown to be resistant to cryopreservation, capable of forming colonies in methylcellulose matrix showed the formation of colonies after 14 days in culture morphology and to blood cells (lymphocytes and monocytes) was confirmed by cytology cell. In gene expression was low GATA3 gene expression, different levels of expression between the groups for the marker and RUNX1 ANXA5. Our most significant findings confirmed the isolation and identification of hematopoietic cells from the bovine embryo yolk sac, therefore, it is feasible and an effective way of obtaining stem cells for future applications in cell therapy and gene.

Oviparous amniotes produce a large yolky egg that gives rise to a free-living hatchling. Structural characteristics and functional attributes of the egg are best known for birds, which have a large mass of fluid yolk surrounded by an extraembryonic yolk sac. Yolk nutrients are delivered to the embryo via the vascular yolk sac. This developmental pattern and nutrient transport mechanism is thought to be representative of all other lineages of amniotes. Recent discovery of a snake with cellularized yolk organized around a meshwork of blood vessels reveals an additional pattern for yolk mobilization, which may also occur in other squamate reptiles (lizards and snakes). This complex yolk sac raises interesting questions about developmental mechanisms and suggests a possible model for the transition between the egg of anamniotes and that of amniotes.

The amniote egg was a key innovation in vertebrate evolution because it supports an independent existence in terrestrial environments. The egg is provisioned with yolk, and development depends on the yolk sac for the mobilization of nutrients. We have examined the yolk sac of the corn snake Pantherophis guttatus by the dissection of living eggs. In contrast to the familiar fluid-filled sac of birds, the corn snake yolk sac invades the yolk mass to become a solid tissue. There is extensive proliferation of yolk-filled endodermal cells, which associate with a meshwork of blood vessels. These novel attributes of the yolk sac of corn snakes compared with birds suggest new pathways for the evolution of the amniote egg.

Reproductive mode has been remarkably labile among squamate reptiles and the evolutionary transition from oviparity to viviparity commonly has been accompanied by a shift in the pattern of embryonic nutrition. Structural specializations for placental transfer of nutrients during intrauterine gestation are highly diverse and many features of the extraembryonic membranes of viviparous species differ markedly from those of oviparous species. However, because of a high degree of evolutionary divergence between the species used for comparisons it is likely that the observed differences arose secondarily to the evolution of viviparity. We studied development of the extraembryonic membranes and placentation in the reproductively bimodal lizard Lacerta vivipara because the influence of reproductive mode on the structural/functional relationship between mothers and embryos can best be understood by studying the most recent evolutionary events. Lecithotrophic viviparity has evolved recently within this species and, although populations with different reproductive modes are allopatric, oviparous and viviparous forms interbreed in the laboratory and share many life history characteristics. In contrast to prior comparisons between oviparous and viviparous species, we found no differences in ontogeny or structure of the extraembryonic membranes between populations with different reproductive modes within L. vivipara. However, we did confirm conclusions from previous studies that the tertiary envelope of the egg, the eggshell, is much reduced in the viviparous population. These conclusions support a widely accepted model for the evolution of squamate placentation. We also found support for work published nearly 80 years ago that the pattern of development of the yolk sac of L. vivipara is unusual and that a function of a unique structure of squamate development, the yolk cleft, is hematopoiesis. The structure of the yolk sac splanchnopleure of L. vivipara is inconsistent with a commonly accepted model for amniote yolk sac function and we suggest that a long standing hypothesis that cells from the yolk cleft participate in yolk digestion requires further study.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17801号 / 生博第289号 / 新制||生||37(附属図書館) / 30608 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 松本 智裕, 教授 石川 冬木, 教授 西田 栄介 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The purpose of this study is to examine if the Swedish syndicalist movement SAC´s ideology also is able to function as a worldview. Worldview is a contested concept and the meaning may shift depending on who you are talking to or what study you are reading. Therefore, I aim to create a deeper understanding of the concept of worldview in our time with this study. The material is a booklet from SAC that explains their ideology and trade union work. I will analyse the material through a qualitative content analysis combined with Stenmark and Franck´s (2016) worldview definition as a theory. The research questions for this study are based on Stenmark and Franck´s (2016) worldview definition, which of the smaller worldview components are possible to find in the material? And given the answer to research question 1, to what extent can SAC´s ideology and union work be understood as a worldview based on Stenmark and Franck´s definition? The study concludes that SAC´s ideology and trade union work are able to function as a worldview. Several of the main components of a worldview is present in the material like SAC´s view of soceity and central valuation system among others. The description of how a worldview functions allows the ideology to be seen as a worldview.

Objective: To describe foreign bodies (FBs) in the nasolacrimal sac of dogs, the history, and simple diagnostic and therapeutic approaches. Animals studied Fourteen dogs of different breeds, ages, and sexes were presented with unilateral dacryocystitis and had been treated without success for over 1–8 months. Procedures: Patient history, including prior treatment, was obtained from medical records. Slit-lamp examination was performed in all cases (SL 17, Kowa Company Ltd.). Jones tests 1 and/or 2 were performed in 13/14 cases. Dacryocystotomy was initiated with an incision into one canaliculus until the lacrimal sac was exposed and could be explored. After extracting the FB from the nasolacrimal sac, the surgical wound and canaliculus were left open. Aftercare included the administration of antibiotic eye drops with or without dexamethasone and systemic analgesia. Results: All 14 dogs were mesocephalic. Four of them were Dachshunds. Dacryocystotomy revealed plant-related FBs in all cases. The purulent discharge disappeared immediately after removal and did not recur during follow-up. Conclusions: A simple dacryocystotomy is recommended for dogs with a strong suspicion of a foreign body in the lacrimal drainage system. Dacryocystorhinography appears to be an optional tool in these cases.

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption posthatch. Nutrient uptake is mediated by digestive enzymes and membrane bound transporter proteins. The objective of this study was to determine expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and small intestine of Leghorn and Cobb chickens derived from 22–30 wk (young) and 45–50 wk (old) breeder flocks. Genes examined included peptide transporter PepT1, amino acid transporters EAAT3, CAT1, and B0AT, monosaccharide transporters SGLT1 and GLUT5, and digestive enzymes APN and SI. Expression of these genes was measured in YSM at embryonic day (e) 11, 13, 15, 17, 19, 20, and 21 and small intestine at e15, e17, e19, e20, and e21. Absolute quantification real-time PCR assessed gene expression. PepT1, APN, and B0AT expression in YSM peaked between e15 and e17 and then decreased until e21, while expression increased over time in the small intestine. SGLT1 and EAAT3 expression increased over time in the small intestine and YSM. There was minimal gene expression of SI in the YSM, while the small intestine had high expression. GLUT5 and CAT1 expression decreased in the YSM, while peaking at e19 then decreasing in the small intestine. Breed and flock age affected expression levels in some genes. These results demonstrate that these genes show tissue- and development-specific patterns of expression and that the YSM expresses many digestive enzymes and nutrient transporters associated with the small intestine. / Master of Science

Pendant l'ontogenèse, les progéniteurs hématopoïétiques sont générés en 3 vagues indépendantes. Les 2 premières (primitive, puis transitoire définitive) ont lieu dans le sac vitellin (SV), avant la génération des Cellules Souches Hématopoïétiques (CSH), qui apparaissent plus tard au niveau de la région "Aorta‐Gonad‐Mesonephros" (AGM), lors de la 3ème vague, dite définitive. Tal1/SCL et son paralogue Lyl‐1 appartiennent à un complexe transcriptionnel qui régule le développement hématopoïétique. Tal‐1/SCL est indispensable à la spécification des progéniteurs hématopoïétiques des 3 vagues. Par contre, le rôle de Lyl‐1 lors du développement hématopoïétique est mal connu. Grâce au gène rapporteur lacZ des souris Lyl‐1lacZ, nous montrons que Lyl‐1 marque et régule les progéniteurs macrophagiques primitifs (MΦPrim) du SV, ainsi que la microglie. Notre analyse en RNA‐seq montre que l'ensemble des gènes exprimés par les progéniteurs MΦPrim est bien distinct de celui des progéniteurs MΦ transitoire définitifs, plus tardifs, reflétant le statut primitif des MΦPrim. De plus, l'invalidation de Lyl‐1 influence les voies de signalisations de l'inflammation et conduit à une activation anormale des gènes de la microglie impliqués dans la régulation synaptique. Lors de la 3ème vague du développement hématopoïétique, nous montrons que l'invalidation de Lyl‐1 conduit à une diminution de l'activité des reconstitutions à long terme des CSH de l'AGM à E10 et à une réduction de la population de CSH dans le foie fœtal à E12 et E14. La réduction de la population de CSH semble liée uniquement à un taux accru d'apoptose des CSH Lyl‐1LacZ/LacZ uniquement au stade de l'AGM. En conclusion, nos résultats montrent que Lyl‐1 régule la production des progéniteurs MΦ primitifs, le développement de la microglie et celui des CSH, dont il contrôle la taille de la population, peu après leur génération. / During ontogeny, hematopoietic progenitors are generated in three independent waves, the first two (primitive and transient definitive) develop from the yolk sac (YS), before the appearance of Hematopoietic Stem Cells (HSC) that occurs later in the Aorta‐Gonad‐Mesonephros (AGM), in the third and definitive wave. Both Tal1/SCL and its paralog Lyl‐1 belong to the transcriptional complex that regulates hematopoietic progenitor development. While Tal‐1/SCL is mandatory for the specification of hematopoietic progenitors from the three embryonic waves, to date the functions of Lyl‐1 during developmental hematopoiesis remains largely unknown. By making use of the lacZ reporter from the Lyl‐1lacZ mice, we previously found that Lyl‐1 marks and regulates YS macrophage progenitors from the primitive wave (MΦPrim), and embryonic microglia. In a RNA‐seq analysis, we show that MΦPrim gene expression landscape is clearly distinct from later transient definitive MΦ　progenitors, reflecting their primitive status. Lyl‐1 invalidation also influences some inflammatory signalling pathways and also leads to the abnormal activation of microglia genes involved in synaptic regulation. In the definitive wave, we found that Lyl‐1 disruption leads to a reduced efficiency of long‐term reconstitution by HSC from embryonic day (E)10 AGM and reduced HSC pool size in E12 and E14 fetal liver. The reduction of the HSC pool results from a higher apoptosis level in Lyl‐1LacZ/LacZ HSCs restricted to the AGM stage. Together, our data establish that Lyl‐1 regulates the development of MΦPrim progenitors and HSC pool size soon after they are generated.