A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22353号 / 医博第4594号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 佐藤 俊哉, 教授 湊谷 謙司, 教授 福原 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The ophthalmic trigeminal placode (opV) is the birth place of one cell type of sensory neurons contributing to the trigeminal ganglion. Signals from the neural tube induce placodal identity within the surface ectoderm. Specified opV placode cells then up-regulate neuron differentiation markers and migrate to the ganglion. Several molecular pathways have been shown to act in opV placode cell development. Despite this, signals that specify individual neurons from within the opV placode remain unknown. However, it is known that components of the Notch signaling pathway are expressed in the opV placode. I tested the role of Notch signaling in opV placode development by separately inhibiting and over-activating the pathway. Using DAPT, an inhibitor of gamma-secretase, I inhibited Notch signaling in 13-15 somite stage chick embryo heads. Attenuated Notch signaling caused increased neuronal differentiation of opV cells at 13-15 somites. I also observed an increase in migratory opV placode (Pax3+) cells in the mesenchyme and expression of neuronal marker Islet1 in the ectoderm. Further, I activated Notch signaling by misexpressing the Notch intracellular domain (NICD) by in ovo electroporation of 10-12 somite stage chick embryos. This resulted in Pax3+ targeted cells failing to differentiate and remain instead in the ectoderm. Thus, Notch/Delta signaling plays an important role in selecting ophthalmic trigeminal cells to differentiate and migrate to the trigeminal ganglion.

The thesis aims at methodological developments in Nuclear Magnetic Resonance (NMR) and study of oriented samples like liquid crystals and single crystals and powder samples. Though methodological development in solid state NMR (ss-NMR) has gone far ahead, this work attempts to contribute some novel methods in this direction. The work presented here falls into two categories (i) methodological developments for obtaining information on anisotropic interactions and (ii) experiments which utilize the existing methodologies to study systems of interest under static condition and also under sample spinning at the Magic Angle. In the solid state, dipolar couplings play a crucial role. On the one hand these couplings could be used to transfer polarization from an abundant nucleus to a rare nucleus and increase the sensitivity of the rare nucleus. On the other hand, the measurement of dipolar couplings itself is crucial for extracting structural and dynamic information. A third aspect is that dipolar couplings could be used to obtain correlation, say between two different nuclear species or between the same kind of nuclei as in an exchange experiment. A major part of this thesis deals with all three aspects mentioned above. The thesis presents a new heteronuclear polarization transfer scheme which is devoid of some of the short comings of the existing and well-known polarization transfer schemes. This pulse sequence has been found to be useful in different contexts involving both spin ½ and spin 1 nucleus. The use of dipolar couplings for obtaining correlation in both static oriented systems and in powder samples has been illustrated. In the case of the powder sample, the study has been useful in obtaining useful orientation information. Finally, chemical shifts are known to be indicators of finer structural features of molecules in solution and solid state. 13 C MAS NMR studies have been exploited in understanding these structural features of short peptides containing prolines in the solid state and for comparing with their structures in solution. Chapter 1 covers the theoretical aspects required for the experimental work described in the thesis. A brief description of NMR has been followed by the explicit description of various interaction Hamiltonian’s in ss-NMR. Subsequently the experimental and the theoretical tools needed for ss-NMR study like Magic Angle Spinning (MAS), Cross-Polarization, Homo/Heteronuclear decoupling schemes have also been discussed. Chapter 2, describes a new heteronuclear polarization transfer scheme for oriented samples – named DAPT (Dipolar Assisted Polarization Transfer) and its application to different systems. DAPT uses a homonuclear decoupling sequence such as BLEW-12 for effecting heteronuclear polarization transfer. The chapter has been divided into five related parts. Section 2(A) starts with an introduction to the existing heteronuclear polarization transfer schemes. Subsequently the theoretical background of the new sequence is presented. Experimental implementation of the sequence in an oriented system, liquid crystal is presented and is compared with the well-known polarization transfer scheme, Hartmann-Hahn Cross Polarization (HH-CP). In 2(B) the implementation of the sequence as a local field spectroscopy for measuring heteronuclear dipolar couplings is presented. After initial discussion about local field spectroscopy and its relevance in ss-NMR, the improvements made in the earlier mentioned sequence along with its 2D implementation in a liquid crystal sample are described. A comparative study is also presented using DAPT with various other homonuclear decoupling sequences. Chapter 2(C) deals with the extension of DAPT to spin-1 systems. The difficulties in setting up the HH-CP in spin-1 systems are highlighted. Experimental demonstrations on a test sample of oriented CD3 I and also on a deuterated liquid crystal is described. The sequence has been incorporated as part of a 2D correlation experiment, where the F1 dimension provides the quadrupolar couplings of deuterium and the F2 the chemical shifts of the attached carbons. The comparison of the sequence with HH-CP, its merits and demerits are discussed and the potential applications are highlighted. Chapter 2(D) deals with the relatively less studied transition in 14N nucleus, known as the Overtone-Transition (OT). An introduction to OT and its relevance is provided in the beginning followed by the extension of DAPT in exciting and detecting OT. The experiments have been done on a single crystal of a model peptide, N-Acetyl-DL-Valine and are compared with the conventional method. Amide proton chemical shifts are also measured using DAPT in an indirect way. The advantages and the future application in studying OT are also discussed. Chapter 2(E) discusses the extension of DAPT to the single crystal of NAV and in identifying the molecules in the unit cell. The SLF spectrum of NAV is complicated due to the presence of two magnetically in-equivalent molecules in the unit cell and with pairs of splitting for each C - 1H and C - 1H pairs. The dipolar couplings are extracted from the experiment and with the aid of a MATLAB program and by incorporating the crystal coordinates, identification of C-1H and C-1H pairs belonging to a particular molecule have been carried out. Chapter 3 describes a novel and useful modification of the well-known Separated Local Field (SLF) sequence in solid state known as PISEMA (Polarization Spin Exchange at the Magic Angle). PISEMA is a popular technique for measuring heteronuclear dipolar couplings in oriented in oriented biological membranes and in liquid crystals. While it has several advantages such as a large dipolar scaling factor, narrow line-widths in the dipolar dimension and ease of setting up etc it suffers from a major problem. The technique is highly sensitive to the proton off-sets which affect the measured dipolar couplings. In the present chapter the origin of this problem has been analyzed in detail and a solution has been proposed. The modification to the experiment has been implemented on a liquid crystal and the off-set independence of the new sequence has been demonstrated. Further studies on a more rigid system such as a solid single crystal has been used to verify the effect of the modification on homonuclear decoupling efficiency and the consequent effects on the line widths in addition to off-set independence. The advantages of the proposed method over the existing one in terms of line-width and robustness in measuring heteronuclear dipolar couplings are demonstrated. Chapter 4 presents a study of deuterium exchange on a di-peptide. Deuterium as well as carbon-13 NMR spectroscopy has been extensively used earlier on static powder samples for studying exchange phenomena. In the present study we have applied the methodology for obtaining relative N-D vector orientation in a di-peptide. The magnetization exchange between deuterium nuclei through the dipolar couplings between them has been monitored. The need to match the quadrupolar split energy levels of two different deuterium’s differently oriented in the magnetic field requires that the sample be spun slowly. Characteristic exchange powder patterns were obtained which were used to infer relative orientation information. Comparison with the crystal structure indicates that the magnetization is likely to be inter-molecular rather than intra-molecular. The chapter follows the following sequence. A brief description about the importance of exchange studies in ss-NMR is presented. A theoretical approach is followed by a discussion of the angular dependence of the frequencies and the visualization of the mutual re-orientation angles. The motivation of the project followed by the experimental techniques, especially the use of slow MAS ~ 100 Hz in exchange studies are also presented. Initial studies have been carried out on di-methyl sulphone to check the reproducibility of the earlier reported results and later the sequence is extended to amide proton deuterated di-glycine. The 2D exchange spectrum recorded under slow MAS is then discussed in the context of the crystal structure and possible amide deuteriums involved in the exchange process are inferred. Chapter 5 discusses the natural abundant 13C and 15N NMR spectroscopy in the solid state in designed tri-peptides containing prolines. Proline is a unique amino acid because of it torsion angle values and is responsible for the turns and the globularity of the proteins. The well studied SH3 domain protein often binds to short peptides containing prolines and hence these study gains importance. Three peptides containing prolines were taken up for study. For peptide (1), the conformation was observed as cis/trans in the solution state and for the other two peptides it was all trans. The X-ray studies showed that peptide (1) has two molecules in the unit cell with both cis conformation. This motivated us to look at the solid state spectra of the peptides. Chemical shifts are signatures of conformers and it was established from the chemical shift differences that there exist two molecules in the unit cell for peptide (1), both in cis conformation. The conformers for the other two peptides predicted by NMR chemical shifts also matched with those obtained from X-ray studies. This opens up the possibility of using simple NMR measurements in the solid state as tools for secondary structure determination in larger peptides and proteins.

Otic neuronal precursors are specified in the otic placode but interestingly do so only in the anterior domain of the otic placode, the proneural domain. In the present study, we have explored why only this territory has the competence to undergo neurogenesis, this means the early events of otic proneural regionalization and neural commitment. The proneural and non-neural domains presented complementary gene expression patterns of transcription factors and members of the Notch pathway. Overall, we propose that proneural character is acquired in the anterior territory by the action of localized ectodermal FGF8-FGF10 signaling that enhances Sox3 function. FGF signals through Sox3 activity would be essential for the specification of the proneural domain versus a non-neural territory, while Notch would be involved in refining this early regionalization. / Els precursors neurals de la oïda són especificats dins un subdomini de la placoda acústica, el domini proneural. En aquesta tesis doctoral, s'ha explorat la raó per la qual la competència neural només és adquirida en aquest subdomini. Els dominis neural i no-neural del territori presenten dominis d'expressió genètica complementaris. Aquest treball proposa que el caràcter proneural és adquirit en el subdomini anterior del territori per l'acció de FGF8 i FGF10 localitzats en l�ectoderm �tic que promouen l�acci� de Sox3. De manera que, les senyals FGF i l�activitat Sox3 s�n essencials per l�especificaci� del domini proneural, mentre que la via Notch �s necess�ria per refinar la regionalitzaci� final entre un domini neural complementari a un domini no-neural.