Dissertations / Theses on the topic 'Tissue Treg'

Depuis les thérapies antirétrovirales (ART), les principales causes de mortalité et de morbidité sont liées à des pathologies non liées aux SIDA qui sont dûes à un niveau d’inflammation chronique de bas bruit. Cette inflammation est liée entre autre à la persistance de la perte d’homéostasie intestinale qui conduit à des translocations microbiennes dans la circulation systémique. En effet, les sujets traités et infectés par le VIH présentent une déplétion sévère et massive des lymphocytes T CD4+, en particulier dans le tissu lymphoïde associé à l'intestin (GALT) une altération de la barrière épithéliale et une fibrose au niveau des ganglions intestinales.Le récepteur sST2, un récepteur leurre de l'alarmine IL-33, constitue un facteur prédictif important de la mortalité toutes causes confondues, chez les patients séropositifs sous traitement antirétroviral. L'IL-33, précédemment connue comme un moteur des réponses immunitaires Th2, est désormais reconnue comme un adjuvant cytokinique de type "switch-hitting". Libérée par les cellules endommagées, elle favorise l'homéostasie tissulaire; notamment IL-33 permet de restaurer l'intégrité de la muqueuse intestinale après des lésions épithéliales d'origine virale. De plus, IL-33 renforce les réponses immunitaires Th1 pour éliminer les agents pathogènes, elle agit sur les lymphocyes T CD8+ qui expriment ST2. Les Tregs tissulaires induites par l'IL-33 médient la réparation par la libération d'amphiréguline (AREG), un facteur de croissance semblable à l'EGF.Dans cette thèse, nous sommes intéressés à l’axe IL-33/ST2 dans la réparation tissulaire et sur son rôle sur les lymphocytes CD8+ chez des patients traités et infectés par le VIH.Dans une première étude, nous avons analysé si la persistance des lésions intestinales pouvait s'expliquer par la dérégulation de la réparation tissulaire. Dans une deuxième étude, nous avons cherché à caractériser les cellules T CD8 exprimant ST2 et à évaluer le rôle de l'IL-33 sur la réponse T CD8 spécifique du VIH. Les taux plasmatiques de sST2 étaient élevés. Les expressions de l'ARNm et de la protéine IL-33 sont élevées dans la muqueuse intestinale des patients VIH, alors qu'elles étaient indétectables dans leur plasma. L'IL-33 était associée à une augmentation de la fibrose et de l'activation immunitaire tandis que la restauration des T CD4+ a diminué . La caractérisation des Tregs tissulaires a révélé deux populations. Au sein des lymphocytes de la lamina propria (LPL), la fréquence des Tregs ST2+ était augmentés chez les patients HIV traités et cette population de Treg est celle qui produit majoritairement de l’'AREG. Nous avons observé une absence des Tregs qui produisent de l'AREG parmi les LPL des patients HIV traités.Dans une deuxième étude, la caractérisation des cellules T CD8+ exprimant ST2 dans les cellules mononucléées sanguines périphériques chez les patients infectés par le VIH a permis de déterminer que cette population est cytotoxique avec un phénotype effecteur (CD45RA+ CCR7-) et une forte capacité à proliférer après stimulation du TCR. Nous avons montré que ces cellules conservaient une forte expression de ST2 par rapport aux donneurs sains après 5 jours de culture. Ensuite, nous avons observé une augmentation des réponses cytokines et cytotoxiques spécifiques de GAG et de CEF chez les patients VIH traités après culture avec l'IL-33 comme l’attestait l'augmentation de la concentration de l'IFNg, du Granzyme A, du Granzyme B et du sFAS-L dans les surnageants de culture.En résumé, nos résultats mettent en évidence un rôle double de l'IL-33 dans l'infection chronique par le VIH : i) un rôle délétère contribuant à la fibrose dans l'intestin de l'infection et ii) un rôle positif renforçant les réponses spécifiques aux peptides GAG et CEF chez les patients infectés par le VIH sous traitement antirétroviral, indiquant son potentiel en tant qu'immunoadjuvant pour améliorer les réponses vaccinales
HIV has transformed into a chronic disease, since the advent of ART. There is persistence of immune activation and inflammation. It leads to a severe and massive CD4+T cell depletion, particularly in the gut associated lymphoid tissue (GALT). In addition, persistent inflammation exacerbates tissue damage, particularly in the GI tract. Epithelial barrier damage is a prerequisite for leaky gut and microbial translocation, contributing to persistent immune activation in individuals with chronic HIV infection. This is a potential mechanism of impaired CD4 reconstitution by contributing to fibrosis. Moreover, persistent antigen exposure, negative co-stimulation and chronic inflammation despite ART induced viral suppression, leads to CD8 T cell dysfunction.sST2, a decoy receptor of the alarmin, IL-33, is reported to be a significant predictor of all-cause mortality in HIV patients on HAART. IL-33, previously known as a driver of Th2 immune responses, is now recognized as a switch-hitting cytokine adjuvant. Released from damaged cells, it promotes tissue homeostasis and repair. IL-33 functions to restore gut mucosal integrity following viral- or commensals- induced epithelial damage. It enhances Th1 immune responses attempting to eliminate the pathogens, followed by ILCs- and tissue Tregs- induced repair. IL-33 induces protective immunity against viral infections by boosting CD8+ T cell response. IL-33 induced tissue Tregs play a role in tissue repair mediated by the release of Amphiregulin, an EGF-like growth factor.In this thesis, we assessed the involvement of the IL-33/ST2 axis in epithelial tissue repair and its role on CD8+ T cell function. In a first study, we analyzed whether the persistence of gut damage might be explained by the dysregulated tissue repair involving IL-33/ST2 and tissue Treg/Amphiregulin pathways. In a second study, we aimed to characterize CD8 T cells expressing ST2 and to assess the role of IL-33 on HIV specific CD8 T response.Investigations were carried out on mucosal and blood samples from HIV infected patients on c-ART and seronegative healthy controls. Plasma sST2 levels were elevated. IL-33 mRNA and protein expressions revealed elevated expression in the gut mucosa of HIV patients, whereas it was undetectable in the plasma. IL-33 was associated with increased fibrosis and immune activation while decreased CD4 restoration. Phenotypic and functional characterization of tissue Tregs revealed two distinct subsets. ST2+ Tregs were upregulated in the LPL of HIV infected patients and identified as the source of AREG-producing Tregs. However, we observed a functional defect of these cells with a decrease of AREG-producing Tregs in the HIV LPL. Overall, these results suggest that the profound defect of AREG production by Tregs may contribute to the persistence of gut barrier dysfunction despite ART in HIV infected patients.Phenotypic and functional characterization of ST2 expressing CD8 T cells in the PBMCs, deciphered this subset to be a cytotoxic population of effector (RA+ CCR7-) CD8 T cells, with a high capacity to proliferate with TCR stimulation. Their characterization did not differ between HIV infected and healthy controls. CD8 T cells from blood of HIV infected patients on c-ART were shown to maintain a high expression of ST2 compared to healthy donors. These cells were negatively associated with sST2 levels in the plasma. We observed that, after 5 days of culture with IL-33, GAG- and CEF specific CD8 T cells displayed more cytolytic and non-cytolytic responses with an increased concentration of IFNg, Granzyme A, Granzyme B and sFAS-Lin the culture supernatant.To summarize, our results highlight the dual role of IL-33 in chronic HIV infection: i) a deleterious one contributing to fibrosis in the gut of HIV infection and ii) a positive one enhancing GAG- and CEF specific responses in HIV infected patients on c-ART, indicating its potential as an immunoadjuvant for enhancing vaccine responses

In recent years, there has been a worldwide increase in obesity, which parallels a rise in pathologies, including type 2 diabetes, collectively termed the metabolic syndrome. Chronic, low-grade inflammation has been implicated as a major link between these diseases. Recent work showed the presence of a unique subset of CD4+Foxp3+ regulatory T cells residing in visceral adipose tissue (VAT Treg) with PPAR-g being the key transcription factor responsible for their phenotype and function in controlling adipose tissue inflammation and, thereby, insulin sensitivity. VAT Tregs inversely correlated with insulin resistance. In contrast, there was a dramatic age-associated increase in frequency of VAT Tregs in lean animals, correlating with continued insulin sensitivity, despite significant increases in body and adipose tissue weights. This increase in Treg frequencies was not observed in other lymphoid and non-lymphoid tissues, including the subcutaneous fat depot. We characterized this unique age-associated increase in VAT Tregs through the use of adoptive transfer models, in vivo labeling and tracking systems, parabiosis, and analysis of the T cell receptor (TCR) repertoire used by VAT Tregs. Our findings indicate that the progressive increase in VAT Tregs is not due to conversion of conventional CD4+ T cells nor to substantial infiltration of Tregs from the circulation and secondary lymphoid organs. However, by analyzing the TCR repertoire on a single-cell level we uncovered a striking oligo-clonal expansion of VAT Tregs, suggesting their accumulation results from in situ proliferation. We further showed that this accumulation is dependent on major histocompatibility complex (MHC) class II, but not on CD1d. Finally, we showed that IL-33 was able to induce proliferation of VAT Tregs. In parallel, we extended our analysis of TCR repertoire to the Treg population residing in skeletal muscle. In acute and chronic models of muscle injury, muscle-resident Tregs underwent a substantial clonal expansion, with a particular clone being detected in multiple individuals. Taken together these studies highlight the importance of proliferation as a mechanism of Treg accumulation in tissues in response to acute and chronic inflammation.

ADAM10, ADAM17, and SPAG6 contribute significantly to humoral immunity and secondary lymphoid tissue architecture. ADAM10 and ADAM17 are two closely related zinc-metalloproteinases. Through cleavage of their ligands CD23 and TNF, respectively, they greatly influence IgE production and secondary lymphoid tissue architecture maintenance. Th1 prone WT strains initially exhibit increased ADAM17 and TNF yet reduced ADAM10 relative to Th2 prone WT strains. In the absence of B cell ADAM10, a compensatory increase in ADAM17 and TNF cleavage is noted only in Th1 prone C57Bl/6, not Th2 prone Balb/c. B cell TNF homeostasis is important for maintaining secondary lymphoid tissue architecture. We show for the first time that excessive B cell TNF production in C57-ADAM10B-/- lymph nodes contributes to loss of B/T segregation, increased HEV number and size, fibrosis, loss of FDC networks, and impaired germinal center formation. Furthermore, B cell ADAM10, which enhances IgE production through CD23 cleavage, is shown to be a marker of Th2 susceptibility. B cell ADAM10 is elevated in Th2 prone mouse strains and allergic patients compared to Th1 prone controls and as B cell ADAM10 level increases, so does IgE production. Lastly, the B cell profile of allergic patients is determined to be B cell ADAM10highADAM17lowTNFlow. Furthermore, the mechanism underlying reduced class-switched antibody production in C57-ADAM10B-/- mice is explored. C57-ADAM10B-/- B cells exhibit a B10, or IL-10 producing, phenotype, which is linked to reduced antibody production. Furthermore, increased Tregs noted in C57-ADAM10B-/- mice contributed to reduced class switched IgE production and disease parameters following a house dust mite airway inflammation challenge. SPAG6, a component of the central apparatus of the “9+2” axoneme, plays a central role in flagellar stability and motility. Immune cells lack cilia, but the immunological synapse is a surrogate cilium as it utilizes the same machinery as ciliogenesis including the nucleation of microtubules at the centrosome. We demonstrate that Spag6 localizes in the centrosome and is critical for centrosome polarization at and actin clearance away from the synapse between CTL and target cells. Furthermore, improper synapse formation and function likely explains reduced CTL function and class-switched antibody production in Spag6KO mice.

Streptococcus pneumoniae (pneumococcus) is a gram-positive bacterium that can cause significant morbidity and mortality in humans especially in children and elderly. T regulatory cells (Treg) have an important role in modulation of immune responses to microbial infection. Although Th17 cells are involved in autoimmune diseases, these cells may play a protective role against pathogens. In this PhD project, Th17 and Treg cells in nasal-associated lymphoid tissue were characterised and their relationship with nasopharyngeal carriage of pneumococcus studied in children and adults. Frequencies of Th17 and Treg in tonsillar tissue and peripheral blood samples obtained from children and adults were analysed for intracellular expression of IL17A and Foxp3 by flow cytometry. Also, tonsillar MNC and PBMC were stimulated by pneumococcal culture supernatant (CCS) derived from wild type stain D39. The ratio of Th17/Treg cells in NALT was studied in both children and adults together with their association with pneumococcal carriage. Numbers of Th17 and Treg cells in in tonsillar tissues were shown to be significantly higher than in peripheral blood in both children and adults. The ratio of tonsillar Th17/Treg was shown to increase with age and tended to be higher in pneumococcal culture negative children than in culture-positive. It is suggested that the balance of Th17/Treg is a crucial determinant of pneumococcal clearance or persistence/carriage in human nasopharynx. A significant increase in numbers of Th17 and Treg cells were shown following pneumococcal CCS stimulation. CCS derived from isogenic mutant strains (i.e., Ply- and CbpA-) elicited lower numbers of Th17 and Treg cells. It is suggested that pneumococcal proteins including Ply and CbpA may activate Th17 and Treg cells in human NALT, and therefore may contribute to the regulation of pneumococcal carriage or clearance in human nasopharynx. Induction of Th17 and Treg from tonsillar MNC were studied using tonsillar MNC depleted of activated and memory T cells. Stimulation with pneumococcal CCS induced Th17 from naïve T cells in tonsillar MNC in the presence of exogenous cytokines (i.e., TGF-β/IL21/IL1-β). TGFβ was shown to be crucial in Treg induction. Thus, the induction of both Th17 and Treg in human tonsillar tissue may be common in humans especially in children during natural infection/carriage, and the balance of the two may determine the clearance or carriage of pneumococcus in nasopharynx. Pneumococcal proteins including pneumolysin (Ply), its toxoid (PdB) and choline binding proteins (CBP) were shown to activate and promote Treg and Th17 cells in tonsillar MNC, thus they may play an important part in modulation of pneumococcal carriage in human nasopharynx. Understanding the development of natural immunity to pneumococcus and to pneumococcal proteins in particular may provide important information in the development of protein-based vaccines against pneumococcal infection in humans. Key words: Pneumococcus, Tonsillar MNC, Treg cells and Th17 cells.

Toxoplasmosis is an important cause of congenital disease, and it is one of the most common opportunistic infections in patients with acquired immunodeficiency syndrome. The need for a reliable experimental model of this infection is crucial not only for achieving a better understanding of the patho-physiology of the disease, but also for developing better methods for evaluating new therapeutic regimens. The purpose of the present study was to investigate the role of CD4+CD25+ T regulatory lymphocytes in mice infected with Toxoplasma gondii. T regulatory (Treg) cells have been shown to play an important role in our immune system in controlling the activity of other T lymphocytes. These cells are differentiated from other T lymphocyte populations based on the co-expression of CD4 and CD25 and expression of the Foxp3 gene. The results of several recent studies have suggested that certain pathogens may be able to increase their survival in the host by exploiting T reg cell activity. T regulatory cells have been shown to control the persistence of the protozoan parasite, Leishmania major, in mice; however, this population of cells plays only a limited role during murine infection with Trypanosoma cruzi. In the present study we have investigated the role of Treg cells during murine infection with the ME49 strain of T. gondii. In vivo depletion of Treg cells was accomplished by injecting mice with a monoclonal antibody (Mab) isolated from the 7D4 rat hybridoma cell line. This Mab is specific for the interleukin-2 receptor chain (also known as CD25). Female Swiss Webster mice of approximately 6-7 weeks of age were depleted of Treg cells by intraperitoneal injection of 400µg of Mab, mice were injected once 7days prior to infection, and a second time 1 day prior to infection, with 20 tissue cysts of T. gondii. Mouse weight and tissue cyst numbers were monitored to evaluate the impact of Treg depletion on the outcome of infection. Our results suggest that depletion of Treg cells has little measurable impact during the acute stage of infection with the ME49 strain of T. gondii. Further studies will be required to determine what role, if any, these cells play in the chronic stage of murine toxoplasmosis.

Tissue resident memory T cells are a subset of memory T cells that reside in tissue permanently, without entering circulation. Their strategic location at sites of common pathogen entry or reinfection, provides a critical advantage for controlling infections. While their role in infection control has become clear, whether tissue-resident T cells can contribute to immune homeostasis or play a role in cancer is unclear. In this study, we investigated the role of tissue-resident T cells in tissue homeostasis and cancer. We show that tissue-resident regulatory T cells exist in human lymphoid tissues and their numbers are greater at sites of recurrent infections. When compared to their circulating counterparts, tissue-resident regulatory T cells are better equipped to maintain tissue homeostasis and functionally are able to suppress T cell proliferation. The study also examined the role of tumour-resident CD8+ T (TR8) cells in oropharyngeal squamous cell carcinomas (OPSCCs). Here we found that TR8 cells were significantly higher in HPV+ OPSCCs when compared to HPV- OPSCCs. Interestingly, we found that increased TR8 cell numbers were associated with better patient survival in both cancers. Importantly, our data shows that TR8 cells could underlie improved survival in patients with HPV+ OPSCCs when compared to patients with HPV- OPSCCs. However, when we examined the role of HPV-oncoprotein E6 in modulating the expression of negative regulators within OPSCCs we found no evidence. Finally, we also examined what role tissue-resident T cells may have in recurrent miscarriage (RM). We found higher proportions of tissue-resident CD8+ T cells in women with RM, suggesting a potential role. In summary, our study shows that tissue-resident T cells could play a critical role not only in infections and cancer but also in tissue homeostasis. Understanding the role of these cells in disease is critical for developing future therapies and vaccines.

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

An in vitro system of generating protoplasts from their callus cultures was established. The friable callus was more productive in terms of producing protoplasts than the green compact callus. The concentration of the various cell wall degrading enzymes had an effect on the viability of the protoplasts in the medium. The protoplast system developed from the experiments was stable and could be used for the transformation experiments of Albizia lebek and for other plant improvement practices.

Over recent years there has been growing focus on the potential of tissue engineering approaches to produce functional bone tissue for clinical use. This thesis highlights the potential for using magnetic particle based techniques for tissue engineering applications. This study demonstrates that magnetic micro-- and nano scale particles are well tolerated by human osteoblasts over prolonged periods of time, and can be used to stimulate calcium signalling pathways in these cells. via the application offorces to integrin rc<:eptors and the internal cytoskeleton. Furthennore, this study demonstrates for the first time that human osteoblasts express the mechanosensitive 2PK+ channel TREK-I, and investigates the potential for using magnetic particle based techniques to directly and specifically activate this class of ion channel. A model system is reported that pennits the application of mechanical forces directly to distinct regions of the TREK-l channel structure and data is presented showing the direct activation of TREK-l channels following manipulation of magnetic p~icles targeted against the extended extracellular loop region of the channel structure. In addition initial data is presented concerning the effect of static magnetic fields and targeted magnetic particle manipulation on the bacterial mechanosensitive ion channel MscL. This thesis represents the first rejX>rts of targeted mechanical stimulation of mechanosensitive ion channels with magnetic particles. This approach has huge potential applications in the field of ion channel research, and offers for the first time a method to directly investigate the structural basis ofmechanosensitivity within ion channels. Furthennore the ability to manipulate ion channel activity without the need for phannacological drugs has huge jX>tential applications in tissue engineering and the treatment of conditions and diseases caused by ion channel dysfunction

The biliary tree is a series of ductular tissues responsible for the drainage of bile produced by the liver and pancreatic secretions from the pancreas. The biliary tree is affected by a diversity of life- threatening diseases collectively called cholangiopathies. Cholangiopathies show regionalization, with some diseases such as biliary atresia predominantly targeting extrahepatic bile ducts (EHBDs) outside of the liver. Despite this, little is known on whether anatomical location within the biliary tree contributes to differences in functionality of biliary epithelium, especially in the EHBD compartment. Additionally, reports have demonstrated the possibility for in vitro culture of bile duct stem/progenitor cell organoids from both intrahepatic (IHBD) and EHBD sources. The relation of these organoid systems to each other, and to their tissue of origin, is largely unknown. In this dissertation, I address these major questions by combining transcriptional analyses and in vitro culture of human bile duct organoids derived from primary IHBD and EHBD epithelium. First, I show that in vitro organoids can be derived from four regions of the human biliary tree: gallbladder, common bile duct, pancreatic duct, and intrahepatic bile ducts. Characterization of these organoids demonstrated expression of adult stem cell (LGR5/PROM1) and ductal (KRT19/KRT7) markers suggesting these cultures contained cells with a biliary stem/progenitor phenotype. Further, I show that IHBD organoids are distinct from EHBD organoids requiring different conditions for sustained growth. Using RNA-Sequencing, I demonstrate that primary tissues from different regions of the extrahepatic biliary tree display unique expression profiles and identify novel tissue-specific markers. I also show that only a limited number of these tissue specific differences are maintained in the in vitro organoids and that the organoids are very different from their tissue of origin. Finally, I demonstrate that IHBD, but not EHBD organoids, express a low-level of hepatocyte-specific markers under differentiation conditions. Taken together, the work in this dissertation has uncovered regional specific markers for different anatomical regions of the human biliary tree. Further, I demonstrate that major differences exist between IHBD organoids and EHBD organoids in vitro and discover that only IHBD organoids have the capacity to express hepatocyte markers under differentiation conditions. Ultimately, these results may help to identify new targets for therapeutic development for cholangiopathies and regenerative medicine. They have also provided important insight to the understanding of both basic biliary physiology and also the field of biliary stem/progenitor cell organoids.

La thèse, intitulée "Tre miti ovidiani in Dante : Piramo e Tisbe, Narciso e Semele" est une analyse de la présence de ces trois fabulae des Métamorphoses dans l'œuvre de Dante. La sélection des mythes a été dictée par la nécessité de définir un champ aussi vaste et par l'intention de mettre en évidence la relation entre l'Eros et la Connaissances, qui est si important dans le système poétique et philosophique de l'auteur florentin.Cette étude se fie par ailleurs l'objective de souligner la manière dont Dante utilise le modèle d'Ovide: la rencontre avec la source classique prévoit un dialogue continu, à la fois textuel et théorique-conceptuel. Le mythe devient ainsi un récit qui faut démêler dans ses applications multiples et parfois contradictoires. En ce qui concerne les sources, une attention particulière a été accordée aux commentaires médiévaux sur les Métamorphoses. En particulier, les œuvres suivantes ont été gardées à l'esprit : le Allegoriae super Ovidii Metamorphosi de Arnolfo d'Orléans, le Integumenta Ovidii de Jean de Garland, l'exégèse de Giovanni del Virgilio et l'Ovide moralisé. Ce poème mythographique de la première moitié du XIVe siècle, même si postérieur, s'est révélé utile en vue d'une reconstruction de la réception du mythe après la Commedia. On a donc essayé de reconstruire la perception réelle que le Moyen Age avait du texte d'Ovide, en prenant également en compte la présence des fabulae dans la littérature vernaculaire antérieure ou contemporaine de Dante.L'ensemble de l'analyse tend à montrer comment, dans le voyage de perfectionnement de Dante, le concept d'Eros soit intimement lié à la possibilité d'une vraie connaissance, à condition qu'elle soit soutenue par la raison et la foi
Analysis of three ovidian myths in Dante's Comedy

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.

Ce mémoire présente une approche par Microscopie Ionique Analytique (MIA) des relations ioniques dans l'activité cambiale et la xylogénèse chez deux modèles expérimentaux : le pin sylvestre (pinus sylvestris L. ) Et le hêtre (fagus sylvatica L. ). Ce travail comporte des mises au point méthodologiques concernant la préparation des échantillons ligneux et les conditions instrumentales de l'analyse en MIA. Nous avons mis au point une nouvelle méthode de préparation des échantillons végétaux, la fixation - déshydratation en phase vapeur, qui assure une bonne conservation des ultrastructures cellulaires et une bonne rétention des éléments diffusibles, comparativement à la technique de préparation par précipitation au pyroantimoniate. Les conditions de mise en oeuvre de l'analyseur ionique ont été spécifiquement établies sur notre matériel biologique et ont permis d'effectuer des analyses semi-quantitatives des distributions ioniques à l'échelle tissulaire et cellulaire. Les distributions saisonnières de Ca et de Na dans le tissu cambial et les tissus conducteurs voisins ont été étudiées sur des rameaux de hêtre et de pin sylvestre. La valeur du rapport de concentration Ca/Na est fortement corrélée à l'âge des organes caulinaires, un rapport Ca/Na élevé caractérisant les tissus des organes matures à activité cambiale intense, un rapport Ca/Na faible les tissu des organes jeunes à activité cambiale modérée. Nous avons montré qu'une augmentation temporaire du calcium dans le cambium et le liber est impliquée dans les processus conduisant à la reprise de l'activité cambiale chez le hêtre. La distribution de Ca, Na, Mg et K a été explorée lors des processus de maturation pariétale secondaire chez des plantules de hêtre se développant sur des milieux minéraux de composition variés. Lors d'une culture sur un milieu normal, les résultats obtenus par MIA ont montré que les cellules à épaississements pariétaux secondaires présentaient des teneurs en calcium beaucoup plus faibles que celles observées dans les cellules ne présentant que des parois primaires. Par contre, les teneurs en calcium dans les structures pariétales secondaires sont augmentées lorsque l'on fait varier les concentrations en sodium dans le milieu de culture (privation ou apport de Na). Dans le cas d'un apport de NaCl de 20 mM, les dépôts secondaires au niveau des parois des cellules du xylème secondaire et des fibres libériennes, sont absents. De plus, nous avons enregistré des augmentations importantes des teneurs en sodium, mais aussi en potassium, magnésium et calcium dans ces cellules où les dépôts pariétaux secondaires se seraient normalement formés. Bien que l'implication du calcium dans les processus de différenciation pariétale soit déjà connue, cette approche par MIA révèle une étroite interdépendance entre le calcium et les autres cations majeurs (sodium, potassium et magnésium) dans ces processus. L'ensemble de ces ions fait partie d'un système intégré, dans lequel des modifications d'un composant perturbe l'intervention des autres dans les mécanismes physiologiques étudiés.

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Cette ?tude propose une lecture du sport d endurance, prenant comme perspective th?orique l imaginaire radical et consid?rant les dimensions socio-historiques e subjectives de la pratique de courses de longue distance. D abord, l ?chantillon la recherche a ?t? compos? de huit sujets-atl?tes du groupe de courreurs de rue Sport Vida. Ainsi, em m?me temps que nous faisons une analyse socio-historique de cette pratique sportive, nous consid?rons l ensemble des aspects s?cio-culturels et poursuivons la recherche avec comme objectif de comprendre les sens qui lui sont attribu?s par les sjuets-atl?tes, au-del? de l aspect ?conomique et de la consommation. Nous observons que, m?me si l alt?tismo qui est pratiqu? a des aspects competitifs (economiques), les atl?tes cr?ent d autres sens pour continuer a pratiquer ce sport, comme les amiti?s, ?tre ensemble avec les amis. Ils rompent avec la logique d?terministe du sport d?passer la limite du corps, vaincre ? n importe quel prix, d?passer les coll?gues -, en cherchant des moments de solidarit?, un sport sans violence et affectif. Nous percevons n?anmoins des contradictions dans le discours de quelques atl?tes quand confessent que le plus important est l amour du sport, les amiti?s, mais r?clament du manque de sponsorts et d appui pour pouvoir s entrainer tranquillement. Cette recherche a aussi montr? que dans la pratique de ce sport, les atl?tes construisent une obstination, sachant le sacrifice qu il impose au corps, mais cela se transforme en plaisir, excitation et recherche d ?motions fortes. Valeurs ?thiques sont aussi construites et valoris?es dans l atl?tisme, ce qui est observ? lorsque que les sujets-atl?tes critiquent avec v?emence a propos de l usage de substances chimiques par les sportifs. En choisissant l imaginaire radical comme principale inspiration th?orique pour cette recherche, il devient ?vident que le sport peut ?tre ressignifi?, ? partir du moment que cet imaginaire est potencialis? dans l enseignement de l ?ducation physique, porvocant chez les ?l?ves une r?flexion critique sur la soci?t? et sur le sport, qui passe ? ?tre redimensionn? vers la solidarit?, avec d?mocratie et autonomie. Enfin, l ?tude a r?v?l? que le sport d endurance est capable de cr?er des liens sociaux et structurer des relations ? partir de cette pratique
Este estudo prop?e uma leitura do esporte de rendimento, com o aporte te?rico do imagin?rio radical, considerando as dimens?es s?cio-hist?ricas e subjetivas na pr?tica de corridas de longa dist?ncia. De in?cio, a amostra da pesquisa foi composta por oito sujeitos-atletas do grupo de corredores de rua Sport Vida. Assim, ao fazermos uma an?lise s?cio-hist?rica dessa pr?tica esportiva, consideramos em conjunto os aspectos socioculturais e seguimos com o objetivo de compreender os sentidos a ela atribu?dos pelos sujeitos-atletas, para al?m do aspecto econ?mico e do consumo. Observamos que, mesmo o atletismo envolvendo aspectos relacionados ao rendimento, os atletas criam outros sentidos para continuarem desenvolvendo essa pr?tica, como as amizades, o estar juntos com os amigos. Eles rompem com a l?gica determinista do esporte ultrapassar o limite do corpo, vencer a qualquer pre?o, sobrepujar os colegas , buscando momentos de solidariedade, um esporte sem viol?ncia e afetivo. Percebemos, por?m, contradi??es, no discurso de alguns atletas, quando confessam que o mais importante ? o amor pelo esporte, as amizades, mas reclamam da falta de patroc?nio e de apoio para poderem treinar com mais tranq?ilidade. Esta pesquisa tamb?m revelou que, nessa pr?tica, os atletas constroem uma obstina??o, devido ao sacrif?cio que ela imp?e ao corpo, por?m isso ? transformado em prazer, excita??o e busca de fortes emo??es. Valores ?ticos tamb?m s?o constru?dos e valorizados no atletismo, o que ? observado quando os sujeitos-atletas fazem cr?ticas contundentes ao uso de subst?ncias qu?micas por aqueles(as) que o praticam. Ao tomarmos o imagin?rio radical como principal fonte te?rica para esta pesquisa, fica evidente que o esporte pode ser ressignificado, desde que esse imagin?rio seja potencializado no ensino da educa??o f?sica, provocando nos alunos uma reflex?o cr?tica sobre a sociedade e sobre o esporte, que passa a ser direcionado para a solidariedade, com democracia e autonomia. Enfim, o estudo revelou que o esporte de rendimento ? capaz de criar la?os sociais e estruturar rela??es ? sua volta

A transformação genética possibilita a introdução de genes de interesse nos genomas, podendo assim ser empregada na tentativa de melhorar características agronômicas e florestais. No entanto, para a obtenção de plantas transgênicas são necessários protocolos eficientes de regeneração de plantas in vitro. Em teca, dados sobre cultura de tecidos são escassos, havendo a necessidade de determinar condições ótimas para a mesma. Com isso, o trabalho teve por objetivos estudar a organogênese in vitro de teca visando desenvolver um método de regeneração eficiente, avaliar condições para o processo de transformação e testar a susceptibilidade da lagarta Hyblaea puera a toxinas produzidas pelo Bacillus thuringiensis. Foram avaliadas a influência de TDZ e BAP na indução da competência organogenética em hipocótilos, nó cotiledonar e cotilédones de teca. Os biorreguladores AIB, BAP, NAA e GA3 foram utilizados na regeneração de segmentos de hipocótilo, nó cotiledonar, raiz, epicótilo e cotilédone. Antibióticos supressores de Agrobacterium tumefaciens e a higromicina (seleção de células transgênicas), foram também avaliados. Finalmente, testes com o inseticida biológico DipelTM e esporos de B. thuringiensis crescidos em laboratório foram realizados com as lagartas de Hyblaea puera. Na aquisição de competência organogenética o TDZ proporcionou um aumento de 46% na regeneração e o BAP 26% quando comparados ao controle. Para a organogênese in vitro foi avaliado um máximo de 70% de regeneração em nó cotiledonares em meio MS adicionado de 1 mg.L-1 de BAP + 0,5 mg.L-1 de GA3. Entretanto, em outras concentrações dos meios de regeneração hipocótilos, raiz, cotilédones e epicótilos tiveram máximas frequências de regeneração em torno de 60%, 60%, 30% e 10%, respectivamente. Os antibióticos supressores da Agrobacterium tumefaciens tiveram efeitos diferentes para cada explante. Timentin e cefotaxima na concentração de 300 mg.L-1 aumentaram o número de brotos em hipocótilos e nó cotiledonar em 1,6 e 2,0 vezes, respectivamente. Em cotilédone esses antibióticos tiveram efeitos negativos no número de brotos. Carbenicilina em todas as doses influenciou negativamente a regeneração em todos os explantes utilizados. A higromicina a 2,5 mg.L-1 inibe em 100% a regeneração de cotilédones, nó cotiledonar e hipocótilo. Os ínstares mais novos de H. puera são susceptíveis tanto ao produto comercial DipelTM quanto aos esporos crescidos em laboratório, apresentando 100% de mortalidade a concentrações de 2x105 UFC após 24 horas de ingestão. Mostrando assim seu potencial na transgenia visando à expressão de genes de Bt para a resistência a insetos. Os resultados apresentados nesse trabalho contribuem para o ganho de informação sobre os fatores que influenciam a organogênese desta espécie, bem como, definir parâmetros que possam ser utilizados em experimentos futuros visando à transformação genética da espécie.
Genetic transformation allows the introduction of genes in host genomes and can therefore be used to improve forestry and agronomic traits like insect resistence. However, efficient plant regeneration protocols are necessary to obtain transgenic plants. Thus far, information about in vitro teak (Tectona grandis L. f) organogenesis is scarce. Therefore, the aims of this study were: develop an efficient protocol for in vitro organogenesis of teak, assess conditions for its genetic transformation and test the susceptibility of the caterpillar Hyblaea puera to toxins produced by Bacillus thuringiensis. We evaluated the influence of TDZ and BAP on the induction of organogenic competence in hypocotyl, cotyledonary nodes and cotyledons. Growth regulators IBA, BAP, NAA and GA3 were used in the regeneration of the hypocotyl, cotyledonary node, root, epicotyl and cotyledon. Antibiotics for suppression of Agrobacterium tumefacien (timentin, cefotaxime and carbenicillin) and for selection of transgenic cells (hygromycin) were also evaluated. Finally, tests with the biological insecticide DipelTM and spores of B. thuringiensis grown in laboratory were performed with the caterpillar of Hyblaea puera. TDZ increases 46% the regeneration frequency and BAP 26% when compared to controls. Cotyledonary nodes showed the best regeneration frequency (70%) growing on MS medium added of 1 mg.L-1 BAP + 0.5 mg.L-1 GA3. Hypocotyls, roots, cotyledons and epicotyls presented variable frequency of regeneration (60%, 60%, 30%, and 10% respectively) growing on distinct concentrations of grown regulators. We tested three antibiotics (timentin, cefotaxime, and carbenicillin) to suppress A. tumefaciens in vitro growth and they presented different effects on the organogenesis of each explants used in this study. Timentin and cefotaxime at concentration of 300 mg.L-1 increased the number of buds on hypocotyls and cotyledonary nodes. Conversely, these antibiotics had negative effects on the number of shoots of cotyledonary explants. Carbenicillin at all doses presented a negative influence on regeneration of all explants. Hygromycin at concentration of 2.5 mg.L-1 inhibits 100% of regeneration of cotyledons, cotyledonary nodes, and hypocotyls. The young instars of H. puera are susceptible to likely both commercial product DipelTM and spores grown in the laboratory, presented 100% mortality at concentrations of 2x105 CFU after 24 hours of ingestion. These findings suggest its potential to be used in teak transgenic approaches for insect resistance. Our results contribute to information about factors that influence the organogenesis of this specie, as well as define parameters that can be used in future experiments aimed at the genetic transformation of the specie.

Les travaux relatant l'embryogenese somatique chez les gymnospermes sont encore recents et n'ont ete realises qu'a partir de materiel tres juvenile. C'est ce genre d'explant que nous avons utilise comme temoin pour caracteriser et visualiser l'embryogenese somatique. Nous avons par la suite obtenu ce phenomene a partir de cotyledons de jeunes plantes agees de 3 a 7 jours apres germination, places directement en conditions de callogenese. Cependant, le taux de cals embryogenes est ameliore quand les cotyledons subissent un traitement caulogene d'une semaine prealablement; dans nos meilleures conditions experimentales 22,7% de cotyledons forment des cals embryogenes. Ceux-ci, places sur un milieu de multiplication, doivent etre subdivises et repiques tous les 10 a 12 jours afin de favoriser leur proliferation

Les traits hydrauliques sont reliés aux fonctions fondamentales du transport de l’eau dans la plante et de la résistance à la sécheresse, déterminant l’écologie, l’évolution, et les processus en lien avec le changement climatique pour les plantes. Cependant, ces traits restent que peu documentés pour les forêts tropicales humides, empêchant de mieux comprendre l’écologie des arbres tropicaux et le futur des forêts tropicales. À partir d’une approche générale s’intéressant à la structure (morphologie et anatomie) et au fonctionnement (physiologie) de la pousse feuillée au sein des arbres, cette thèse analyse la structure et le fonctionnement de la pousse feuillée des arbres, notamment dans la perspective de la résistance à la sécheresse. Cette thèse s’appuie sur un jeu de données général qui concerne 42 espèces d’arbres de canopée échantillonnées en forêt tropicale humide de Guyane, et qui illustre une large gamme de tailles de feuilles. Un second jeu de données, destiné à mieux comprendre les sources de variabilité intraspécifique, porte de l’espèce pionnière Cecropia obtusa Trécul (Urticaceae). Une première partie de la thèse vise à comprendre la coordination entre la feuille et la tige pour une large gamme de dimension foliaire, en analysant les changements morpho-anatomiques et fonctionnels soulignant la relation entre dimensions de la feuille et dimensions de la tige à l’échelle interspécifique. Ce cadre est aussi appliqué à l’échelle intraspécifique pour le modèle C. obtusa pour la relation pétiole-limbe. Comprendre les changements liés aux dimensions de la feuille permet ainsi d’investir correctement les effets ontogénétiques et saisonniers sur les traits foliaires vasculaires et hydrauliques pour C. obtusa. La seconde partie vise à mieux comprendre les mécanismes de résistance à la sécheresse de la pousse feuillée. En retenant 25 espèces, les déterminants anatomiques de la résistance à l’embolie par sécheresse des tiges et sont étudiés. Les différentes propriétés et mécanismes déjà connus pour des plantes tempérées sont abordés conjointement afin de conforter ou non leur existence et comprendre leurs contributions relatives. La segmentation de vulnérabilité (c.-a.d. la différenciation de résistance à l’embolie) à l’interface tige-feuille est également étudiée pour 20 espèces. Sa coordination avec la segmentation hydraulique (c.-à-d. différenciation de résistance hydraulique), qui est dérivée des études anatomiques réalisées dans la première partie, est étudiée. Je montre ainsi (i) un large spectre de variation pour la segmentation de vulnérabilité entre espèces, (ii) une relation positive avec la segmentation hydraulique, suggérant qu’il y a des espèces promouvant à la fois la segmentation hydraulique et la segmentation de vulnérabilité pour découpler le fonctionnement la feuille de la tige du point de vue hydraulique, et (iii) que la segmentation de vulnérabilité a un impact important sur le temps de dessiccation théorique de la pousse feuillée, confirmant que la segmentation de vulnérabilité est un mécanisme de résistance à la sécheresse. Dans une dernière partie, je teste le pouvoir prédictif des différents traits hydrauliques étudiés sur la distribution hydro-topographique des espèces, ainsi que l’impact du stress hydrique sur la croissance des arbres, à une échelle locale. Dans la Discussion générale, je discute de la signification fonctionnelle de la surface de la feuille et des traits de résistance à la sécheresse en forêt tropicale humide, ainsi que la relation potentielle entre la surface de la feuille et la résistance à la sécheresse
Hydraulic traits are related to the fundamental functions of conduction and drought resistance, driving plant ecology, evolution, and processes related to climate change. However, these traits are still poorly documented for tropical rainforest, hindering our understanding of tropical tree ecology and the future of tropical forests. Through a trait-based approach combining leaf and stem in-depth anatomy and physiology, this thesis analyses the structure and the functioning of tree leafy shoots, a compartment exposed to the most severe water stress within a tree. This thesis is based on a dataset of 42 canopy tree species sampled in a rainforest in French Guiana, exemplifying a large range of leaf size. A second dataset, aiming at better understanding the sources of intraspecific variability, focus on the pioneer tree species Cecropia obtusa Trécul (Urticaceae). The first part aims at understanding the coordination between leaf and stem across a large leaf size range, by analysing the morpho-anatomical and functional changes underlying the leaf size-stem size relationship for 42 tropical rainforest canopy trees. This framework is also applied at the intraspecific level for the model C. obtusa, for the petiole-lamina relationship. Understanding leaf size-related changes allows to properly investigate ontogenetic and seasonal effects on leaf vascular and hydraulic traits for C. obtusa. The second part aims at better understanding the drought resistance of the leafy shoot. By retaining 25 species, the anatomical determinants of drought-induced embolism resistance for stems are studied. The different properties and mechanisms already known for temperate plants are investigated jointly to consolidate or not their existence, and understand their relative contribution. Vulnerability segmentation (i.e. differentiation of embolism resistance) at the leaf-stem interface is investigated across 20 species. I further evidenced (i) a large spectrum of variation for vulnerability segmentation across species, (ii) a positive relation with hydraulic segmentation (i.e. differentiation of hydraulic resistance), suggesting that some species promote both hydraulic and vulnerability segmentations to decouple hydraulically leaf and stem, and (iii) that vulnerability segmentation has a massive role in enhancing the theoretical desiccation time of shoots, confirming vulnerability segmentation as a drought resistance mechanism for tropical trees. In a final part, all traits are merged to test their predictive power for species hydro-topographic distribution and growth-mediated response to water stress, at a local scale. In the general discussion, I discuss the functional signification of leaf size and drought resistance traits for tropical rainforest, as also potential relation between leaf size and drought resistance

Surexploitee pour son bois, infeodee pour son developpement a une mycorhization, dependant d'une production de semences episodique et d'une germination aleatoire, shorea curtisii, incapable en outre de multiplication vegetative spontanee ou par les procedes traditionnels, est tres representatif des dipterocarpacees asiatiques menacees d'extinction rapide. Une mise au point d'un procede de micropropagation in vitro contribuerait a sauvegarder l'espece. L'observation, chez shorea curtisii, de galles epiphylles a organisation de bourgeon abortif suggerant une certaine aptitude des feuilles a la neoformation de meristemes caulinaires, privilegie celles-ci pour larecherche d'une methode de clonage in vitro (. . . )

La multiplication des microboutures au cours des subcultures est caractérisée par une évolution de l'état physiologique qui dépend de l’âge, des réserves nutritives et du milieu de culture. Parmi les problèmes qui limitent la multiplication végétative: l'hyperhydrie et le gonflement des tiges des microboutures. Le premier est caractérisé par une teneur en eau élevée, par des rapports p/na, k/na et cytokinines/auxine élevés. Le deuxième est caractérisé par une teneur élevé en zéatine+zéatine-riboside. Les méristèmes de plantes d'âges différents ont une réactivité distincte aux niveaux morphologique et physiologique.

Expression de la resistance selon les races et les hybrides vm et vf. Caracterisation de l'hypersensibilite codee par le genie vm. Etude comparative des classes de symptomes liees a la resistance codee par le gene vf. Caracterisation des differences d'hypersensibilite dans 2 cas

Ulcerative colitis (UC) is an inflammatory bowel disease that results in a chronic inflammation and ulcers of the mucosa of the colon. In UC, two important issues, specifically related to the prognosis, remain to be clarified. A) Extension of disease greatly influences the prognosis and biological factors involved in the limitation of the disease extent are presently unknown B) Due to increased availability of biological drugs targeting different cytokines, biological therapy need to be optimized on the key cytokine(s) relevant for the inflammatory process. Aim: A) We hypothesized that regulatory T cells might be relevant in limiting the extension of inflammation and that B) UC patients might be stratified according to distinctive cytokine profiles. Methods: In a cross-sectional study, we evaluated the relationship between UC extension and the proportions of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3-Tregs in the colonic lamina propria (LP) in UC patients undergoing colonoscopy for clinical relapse and controls undergoing colonoscopy for non-inflammatory conditions. The role of these cells in UC extension was also investigated in the murine oxazolone-induced colitis model. B) At the same time, we analyzed tissue cytokine by RTqPCR in endoscopic biopsies from uninvolved and involved tissue of UC patients and controls. Mucosal microbiota analysis was also performed. Results: A) Involved and uninvolved tissue show different and opposite frequency of LP CD3+CD4+Foxp3+ and LP CD3+CD4+LAP+ Tregs with the latter significantly increased in uninvolved vs involved tissue. In mice with oxazolone-induced distal colitis, treatment with α-LAP-depleting antibody was associated with the development of extensive colitis; B) We quantified by RTqPCR TNF-α, IFN-γ, IL-10, IL-6, IL-17, and IL-13 and analyzed the results by r square of the k means for cluster solution. IL-17A and IL-13 mRNA tissue content show classificatory power and their distribution points to the existence of discrete clusters of subjects. Six clusters define the optimal partition of data explaining 91% of the total information present in the data set (R-square=0.91). UC patients were distributed in two clusters characterized by high/low IL-13 mRNA tissue content in the context of high IL-17A mRNA tissue content. The two subsets differ in clinical-pathological characteristics. High IL-13mRNA patients are younger at diagnosis and show higher prevalence of extensive colitis than low IL-13mRNA ones. They also show a more frequent use of steroid/immunosuppressant/anti-TNFα therapy during a one-year follow-up. The two subgroups show a differential enrichment of mucosa associated microbiota genera with prevalence of Prevotella in patients with high IL-13mRNA tissue content and Sutterella and Acidaminococcus in patients with low IL-13mRNA tissue content. Conclusion: A) LP CD3+CD4+LAP+ Tregs efficiently limit the extension of inflammatory lesions. Therapeutic strategy aimed at their expansion may help in containing the spreading of inflammation and possibly in preventing inflammatory relapse. B) Evaluation of IL-13 and IL-17 mRNA content allows the identification of different UC patients’ subsets with associated different clinic phenotypes. Therapeutic options might be optimized according to the patients’ tissue cytokine profile.

The mechanisms that control growth processes in biology tissues have attracted continuous research interest despite their complexity. With the emergence of big data experimental approaches there is an urgent need to develop statistical and computational models to fit the experimental data and that can be used to make predictions to guide future research. In this work we apply statistical methods on growth process of different biological tissues, focusing on development of neuron dendrites and tumor cells. We first examine the neuron cell growth process, which has implications in neural tissue regenerations, by using a computational model with uniform branching probability and a maximum overall length constraint. One crucial outcome is that we can relate the parameter fits from our model to real data from our experimental collaborators, in order to examine the usefulness of our model under different biological conditions. Our methods can now directly compare branching probabilities of different experimental conditions and provide confidence intervals for these population-level measures. In addition, we have obtained analytical results that show that the underlying probability distribution for this process follows a geometrical progression increase at nearby distances and an approximately geometrical series decrease for far away regions, which can be used to estimate the spatial location of the maximum of the probability distribution. This result is important, since we would expect maximum number of dendrites in this region; this estimate is related to the probability of success for finding a neural target at that distance during a blind search. We then examined tumor growth processes which have similar evolutional evolution in the sense that they have an initial rapid growth that eventually becomes limited by the resource constraint. For the tumor cells evolution, we found an exponential growth model best describes the experimental data, based on the accuracy and robustness of models. Furthermore, we incorporated this growth rate model into logistic regression models that predict the growth rate of each patient with biomarkers; this formulation can be very useful for clinical trials. Overall, this study aimed to assess the molecular and clinic pathological determinants of breast cancer (BC) growth rate in vivo.

碩士
臺灣大學
園藝學研究所
95
The fringe tree (Chionanthus retusus Lindl.) is a graceful flowering tree which tree crown is beautiful. It would like snowing on tree when fringe is flowing. Fringe tree is considered as a rare species and limited population in Taiwan. There is epicotyl dormancy on the seed and hard to rooting through cutting. The purpose of this research is to induce callus formation from endosperm, young floret, derived embryogenic cell for suspension culture, and embryo tissue, to study embryonic tissue cultures and somatic embryogenesis. Endosperm culture before glutinous stage was hard to success because the endosperm tissue is like liquid. It could not be affixed on the medium and differentiate on it. There was dedifferentiation and reproliferation in the solid stage endospermic tissue ( intacted with embryo culture for 10 days) on MS medium supplemented with 2,4-D 5 mg/L. The dedifferentiation frequency of endospermic tissue was 16.7%. There was transparent callus formation in calyx and ovary on WPM medium supplemented with different auxins(2,4-D or picloram) and BA. Callus formation was more and frequency of callus induced is higher by picloram and BA. Frequency of callus induced was 100% by picloram 2 mg/L and BA 1 mg/L but the callus was friable. Giving control extracellular pH 4.4-5.0 level, could made the young floret-derived cell suspension culture of fringe tree change the phase. The cell masses would be induced to free cells and cell cluster stage and had less somatic embryogenesis. Giving control extracellular pH 5.5-6.1 level could made it somatic embryogenesis faster. The cell suspension culture of fringe tree under SH (added NAA 0.4 mg/L) would appeared dense cell mass and some globular structures by 20 days after suspension. The callus formation from abaxial side of cotyledon and hypocotyl surface from zygote embryo tissue stored 6 month and then indirectly regenerate somatic embryogenesis on MS medium supplemented with picloram and dicamba 1-2 mg/L.The callus formation was more from zygote embryo tissue of fringe tree by MS medium and picloram 2 mg/L. Indirect somatic embryogenesis were from the yellow or white embryonic callus after 1 month. The frequency of somatic embryogenesis was 35% and average of somatic embryogenesis was 2.35 (somatic embryo/per explent) by picloram 2 mg/L after 2 month. The frequency of somatic embryogenesis was 30% and average of somatic embryogenesis was 24.45 (somatic embryo/per explent) by dicamba 1 mg/L. Small somatic embryo (< 3 mm) of fringe tree would be suppressed by ABA 0.1-0.2 mg/L on normal development and enlarge with callus. 33.8% and 38.89% of somatic embryo could be maturation. ABA 0.5 mg/L made embryo to turn green slowly and ABA 1.0 mg/L would suppress development of somatic embryos or made them to become abnormal. ABA 2 mg/L and 5 mg/L would induce secondary cotyledon formation, somatic embryo growing fast, maintain the color of cotyledon in white and transparent. 81.82% and 100% of somatic embryos were maturation. Somatic embryo development of fringe tree was better in MS medium without PGR. They began to turn green and rooting. Strong auxin like 2,4-D 0.1 mg/L could let bigger somatic embryo development but others would dedifferentiation to become embryonic callus. The embryonic callus could have somatic embryo again.

Physiologically based pharmacokinetic (PBPK) modeling is a tool used in drug discovery and human health risk assessment. PBPK models are mathematical representations of the anatomy, physiology and biochemistry of an organism. PBPK models, using both compound and physiologic inputs, are used to predict a drug’s pharmacokinetics in various situations. Tissue to plasma partition coefficients (Kp), a key PBPK model input, define the steady state concentration differential between the tissue and plasma and are used to predict the volume of distribution. Experimental determination of these parameters once limited the development of PBPK models however in silico prediction methods were introduced to overcome this issue. The developed algorithms vary in input parameters and prediction accuracy and none are considered standard, warranting further research. Chapter 2 presents a newly developed Kp prediction algorithm that requires only readily available input parameters. Using a test dataset, this Kp prediction algorithm demonstrated good prediction accuracy and greater prediction accuracy than preexisting algorithms. Chapter 3 introduced a decision tree based Kp prediction method. In this novel approach, six previously published algorithms, including the one developed in Chapter 2, were utilized. The aim of the developed classifier was to identify the most accurate tissue-specific Kp prediction algorithm for a new drug. A dataset consisting of 122 drugs was used to train the classifier and identify the most accurate Kp prediction algorithm for a certain physico-chemical space. Three versions of tissue specific classifiers were developed and were dependent on the necessary inputs. The use of the classifier resulted in a better prediction accuracy as compared to the use of any single Kp prediction algorithm for all tissues; the current mode of use in PBPK model building. With built-in estimation equations for those input parameters not necessarily available, this Kp prediction tool will provide Kp prediction when only limited input parameters are available. The two presented innovative methods will improve tissue distribution prediction accuracy thus enhancing the confidence in PBPK modeling outputs.

Eucalyptus trees are a significant source of fuelwood, timber and raw material for the paper and pulp industry. In South Africa, Eucalyptus grandis and its hybrids are in high demand due to their fast growth and suitability of their timber for a wide range of products. Breeding and selection for superior quality eucalypts could sustain this high demand through selection and subsequent multiplication of superior genotypes and their use in controlled crosses. However, for this to be successful, a wide genepool must be available and maintained. Germplasm conservation of both vegetative and sexual material is therefore an integral part of such activities. However, in the case of trees, in vivo conservation practices are expensive and hazardous. The aim of this project was therefore to establish alternative conservation strategies for short-to-medium and long-term use of Eucalyptus spp. to facilitate on-going breeding and clonal programmes. For short-to-medium term storage, shoot cultures were subjected to various minimal growth conditions. Of the investigated treatments, reducing nutrients was the best storage method and shoots were maintained for 10 months with multiplication rates of 13.75 ± 7.05 shoots/explant. Encapsulated axillary buds were stored in jars at 10 °C or 28 °C. Of these two treatments, viability was sustained for longer (6 months) at 4 °C. Before establishing pollen storage regimes, viability assessment methods were evaluated and these consisted of in vitro germination on a BK (Brewbaker and Kwack, 1963) medium for 24 hours (26 ± 3.0%) and staining with two tetrazolium salts. Medium-term storage of pollen was best achieved by maintenance in the fridge (4 °C) without any desiccating substance (8 months at 6.73 ± 1.21%). Cryopreservation protocols were investigated for axillary buds and pollen. Buds that were 2 mm long were pretreated with chemical cryoprotectants, and a mixture of DMSO (dimethylsulphoxide) and glycerol was found to induce high survival rates (63%) after washing with MS (Murashige and Skoog, 1962) and 4 g.l-1 sucrose solution. Explants precultured in 1M sucrose showed increased tolerance (explant retained high survival rates of 80%) when desiccated to 20% moisture content fresh weight basis (fwb). Although pretreatments were successfully established, explants did not survive storage in liquid nitrogen indicating the need for further optimization of protocols. Pollen was successfully cryopreserved for 12 months with 23% survival rates. Applications and future research strategies of the developed protocols to Eucalyptus breeding programmes are discussed.
Thesis (M.Sc.)-University of Natal, Durban, 1998.