Propôs-se a caracterização morfológica, funcional e de ocorrência das tonsilas linguais laterais. Compondo o Grupo experimental I, 25 espécimes de línguas humanas foram fixados em solução de formol a dez por cento e assim mantidos até o processamento. Os espécimes foram submetidos à exposição radiográfica para detecção de algum tecido mineralizado. As peças anatômicas foram examinadas a olho nu e com a utilização do estereomicroscópio. Obtiveram-se três blocos de tecido para a análise microscópica de cada espécime: um advindo da tonsila lingual e outros dois das margens laterais da região posterior do terço médio lingual, direito e esquerdo, respectivamente. Após o processamento histotécnico de rotina para coloração em hematoxilina-eosina de Harris, os espécimes foram analisados microscopicamente. Compondo-se o Grupo experimental II, procedeu-se à análise clínica dos tecidos moles bucais de 420 crianças em idade escolar, advindas da rede pública de ensino da cidade de Monte Negro, Estado de Rondônia, com o objetivo de se detectar a presença de tonsilas linguais laterais. Todas as análises, laboratoriais e clínicas, foram realizadas de forma descritiva e os dados obtidos, organizados e demonstrados comparativamente em tabelas e gráficos, com dados de freqüências absolutas e relativas. A análise clínica teve seus dados coletados correlacionados com a utilização do teste do quiquadrado. Aplicou-se a estatística kappa. Ao exame macroscópico: dos 25 espécimes de línguas humanas, nove apresentavam tonsilas linguais laterais (36%); o número de cristas nas papilas folhadas variou de um a seis. Ao microscópio óptico notou-se a presença de 16 espécimes com presença de tonsilas linguais laterais (64%, n=25); epitélio estratificado pavimentoso não queratinizado, com formação de criptas tonsilares e presença subepitelial de folículos linfóides. Na análise clínica, Grupo experimental II, o percentual de ocorrência de tonsilas linguais laterais (3,09%, n=420) foi proporcionalmente menor do que aquele obtido nos espécimes do Grupo experimental I (36%, n=25). Conclui-se que a ocorrência de tonsilas linguais laterais é maior quando obtida por análise microscópica, em comparação com a análise clínica. A presença e a morfologia das papilas folhadas linguais são inconstantes, quer no mesmo espécime, quer de um espécime para outro e podem mascarar a presença de tonsilas linguais laterais. / This study aimed to characterize the lateral lingual tonsils as to their morphology, function and occurrence. The experimental group I was composed of 25 specimens of human tongues fixated and kept in 10% formalin until processing. The specimens were radiographed for detection of any mineralized tissue. The specimens were examined by naked eye and with aid of a stereomicroscope. Three blocks of tissue were obtained from each specimen for microscopic analysis: one from the lingual tonsil and two from the lateral borders of the posterior region of medium, right and left thirds, respectively. After routine histotechnical processing and staining with Harris hematoxylin and eosin, the specimens were microscopically analyzed. The experimental group II was achieved by clinical analysis of oral soft tissues of 420 schoolchildren attending public schools at the city of Monte Negro, State of Rondônia, to investigate the presence of lateral lingual tonsils. All laboratory and clinical analyses were performed descriptively and data were organized and comparatively demonstrated on tables and graphs, presenting absolute and relative frequencies. The results of clinical analysis were correlated by utilization of the chisquare test. The kappa statistics was applied. Macroscopic examination of 25 specimens of human tongues revealed that nine presented lateral lingual tonsils (36%); the number of crests in each foliate papilla ranged from one to six. At the microscopic analysis of 25 specimens revealed that 16 presented lateral lingual tonsils (64%, n=25); non-keratinized stratified squamous epithelium with formation of tonsillar crypts and subepithelial presence of lymphoid follicles. With regard to clinical analysis of experimental group II, the percentage of occurrence of lateral lingual tonsils (3.09%, n=420) was proportionally lower than observed on specimens in experimental group I (36%, n=25). It was concluded that the occurrence of lateral lingual tonsils is higher when analyzed by microscopic analysis compared to clinical analysis. The presence and morphology of foliate papillae of the tongue are inconstant, both within and between specimens. They can mask lateral lingual tonsils.

Propôs-se a caracterização morfológica, funcional e de ocorrência das tonsilas linguais laterais. Compondo o Grupo experimental I, 25 espécimes de línguas humanas foram fixados em solução de formol a dez por cento e assim mantidos até o processamento. Os espécimes foram submetidos à exposição radiográfica para detecção de algum tecido mineralizado. As peças anatômicas foram examinadas a olho nu e com a utilização do estereomicroscópio. Obtiveram-se três blocos de tecido para a análise microscópica de cada espécime: um advindo da tonsila lingual e outros dois das margens laterais da região posterior do terço médio lingual, direito e esquerdo, respectivamente. Após o processamento histotécnico de rotina para coloração em hematoxilina-eosina de Harris, os espécimes foram analisados microscopicamente. Compondo-se o Grupo experimental II, procedeu-se à análise clínica dos tecidos moles bucais de 420 crianças em idade escolar, advindas da rede pública de ensino da cidade de Monte Negro, Estado de Rondônia, com o objetivo de se detectar a presença de tonsilas linguais laterais. Todas as análises, laboratoriais e clínicas, foram realizadas de forma descritiva e os dados obtidos, organizados e demonstrados comparativamente em tabelas e gráficos, com dados de freqüências absolutas e relativas. A análise clínica teve seus dados coletados correlacionados com a utilização do teste do quiquadrado. Aplicou-se a estatística kappa. Ao exame macroscópico: dos 25 espécimes de línguas humanas, nove apresentavam tonsilas linguais laterais (36%); o número de cristas nas papilas folhadas variou de um a seis. Ao microscópio óptico notou-se a presença de 16 espécimes com presença de tonsilas linguais laterais (64%, n=25); epitélio estratificado pavimentoso não queratinizado, com formação de criptas tonsilares e presença subepitelial de folículos linfóides. Na análise clínica, Grupo experimental II, o percentual de ocorrência de tonsilas linguais laterais (3,09%, n=420) foi proporcionalmente menor do que aquele obtido nos espécimes do Grupo experimental I (36%, n=25). Conclui-se que a ocorrência de tonsilas linguais laterais é maior quando obtida por análise microscópica, em comparação com a análise clínica. A presença e a morfologia das papilas folhadas linguais são inconstantes, quer no mesmo espécime, quer de um espécime para outro e podem mascarar a presença de tonsilas linguais laterais. / This study aimed to characterize the lateral lingual tonsils as to their morphology, function and occurrence. The experimental group I was composed of 25 specimens of human tongues fixated and kept in 10% formalin until processing. The specimens were radiographed for detection of any mineralized tissue. The specimens were examined by naked eye and with aid of a stereomicroscope. Three blocks of tissue were obtained from each specimen for microscopic analysis: one from the lingual tonsil and two from the lateral borders of the posterior region of medium, right and left thirds, respectively. After routine histotechnical processing and staining with Harris hematoxylin and eosin, the specimens were microscopically analyzed. The experimental group II was achieved by clinical analysis of oral soft tissues of 420 schoolchildren attending public schools at the city of Monte Negro, State of Rondônia, to investigate the presence of lateral lingual tonsils. All laboratory and clinical analyses were performed descriptively and data were organized and comparatively demonstrated on tables and graphs, presenting absolute and relative frequencies. The results of clinical analysis were correlated by utilization of the chisquare test. The kappa statistics was applied. Macroscopic examination of 25 specimens of human tongues revealed that nine presented lateral lingual tonsils (36%); the number of crests in each foliate papilla ranged from one to six. At the microscopic analysis of 25 specimens revealed that 16 presented lateral lingual tonsils (64%, n=25); non-keratinized stratified squamous epithelium with formation of tonsillar crypts and subepithelial presence of lymphoid follicles. With regard to clinical analysis of experimental group II, the percentage of occurrence of lateral lingual tonsils (3.09%, n=420) was proportionally lower than observed on specimens in experimental group I (36%, n=25). It was concluded that the occurrence of lateral lingual tonsils is higher when analyzed by microscopic analysis compared to clinical analysis. The presence and morphology of foliate papillae of the tongue are inconstant, both within and between specimens. They can mask lateral lingual tonsils.

DNA damage from both normal metabolic activities and environmental factors such as UV and radiation can cause as many as 1 million individual lesions to the DNA per cell per day (Lodish et al 2004). Cells respond to this continuous damage by employing many, highly efficient DNA repair mechanisms and undergo apoptosis when normal DNA repair fails. Of the many types of DNA damage that can occur, double strand breaks (DSBs) are the most toxic (Featherstone & Jackson 1999). A single unrepaired DSB is enough to induce cellular apoptosis and several mechanisms have developed to repair DSBs. The recognition, signalling and repair of DSBs involve large multi-­‐subunit complexes that bind to both the DNA and modified histone tails, which require modification of the chromatin in order to access their bind sites and function effectively (Allard et al 2004). Consequently several chromatin-­‐remodelling proteins have been implicated in DSB repair (van Attikum et al 2004, Chai et al 2005). LSH (Lymphoid specific helicase) is a putative chromatin-­‐remodelling enzyme that interacts with DNA methyltransferases and has been connected to DNA methylation (Myant & Stancheva, 2008). Knockouts of LSH or its homologues in A. thaliana and M. musculus show a reduction in DNA methylation of 60-­‐70% (Jeddeloh et al 1999, Dennis et al 2001). However in addition to this phenotype, knockout A. thaliana also have an increased sensitivity to DNA damage (Shaked et al 2006). A homologue of LSH has also been identified in S. cerevisiae, which interacts with known repair proteins (Collins et al 2007) and may be involved in DSB repair. Although the majority of Lsh-­‐/-­‐ mice die shortly after birth, 40% of the line produced by Sun et al survive and show unexplained premature aging (Sun et al 2004). As premature aging is a hallmark of increased acquisition of DNA damage there is the possibility of a conserved role for LSH in mammalian DNA damage repair. Here I show that LSH depleted mammalian cells have an increased sensitivity specifically to DSB inducing agents and show increased levels of apoptosis. Further analysis shows that cells lacking LSH repair DSBs slower, indicating a novel role for LSH in mammalian repair of DSB. I performed an in depth analysis of the DSB defects in LSH depleted cells in an attempt to elucidate the function of LSH in DSB repair. I found that LSH depleted cells can correctly recognise DSBs but recruit downstream signalling and repair factors, such as γH2AX, less efficiently. I show that reduced recruitment of downstream DSB repair factors is not accompanied by extended cell cycle checkpoint signalling. This suggests that LSH depleted cells continue through the mitosis with unrepaired DSBs, which most likely leads to apoptosis and the increased sensitivity to DSB inducing agents. These experiments also showed that recruitment of DSB signalling and repair factors is not impaired equally at all breaks, and I present a model system created to quantitatively compare individually breaks between WT and LSH depleted cells to identify DSB that require LSH for efficient repair. I also preformed an analysis of Lsh-­/-­ MEFs containing WT or catalytic null mutant LSH rescue constructs and I show that WT but not catalytic null LSH can restore efficient DSB repair. These studies identify a novel role for LSH in mammalian DSB repair and demonstrate the importance of its catalytic activity.

Les cellules lymphoïdes innées (ILC) représentent une famille de cellules hématopoïétiques récemment identifiée, qui joue un rôle essentiel dans la réponse immunitaire précoce via la production rapide de cytokines. Trois groupes - ou types - d’ILC ont été définis selon l’expression de certaines molécules membranaires ou intracellulaires, ainsi que la production différentielle de cytokines. Les ILC du groupe 1 (ILC1) expriment le facteur de transcription(FT) T-BET et sécrètent des cytokines inflammatoires de la réponse immune de type 1, l’IFN-? et le TNF-?. Les ILC2 sécrètent des cytokines associées à la réponse immune de type 2,notamment l’IL-5 et l’IL-13, et ce de façon dépendante du FT GATA-3. Enfin, les ILC3 se caractérisent par la production de cytokines telles que l’IL-17 et l’IL-22, et expriment le FTROR?t. J’ai étudié en utilisant des techniques de biologie moléculaire et cellulaire, et à partir d’échantillons sanguins et tissulaire de donneurs sains ou de patients atteints de maladies inflammatoires chroniques, la fonction de ces trois groupes d’ILC chez l’homme. Ces travaux ont permis la construction d’un nouveau modèle de développement de ces cellules à partir de précurseurs. / Innate lymphoid cells (ILC) represent a novel family of hematopoietic effectors that serve essential roles in early immune response by rapid cytokines production. Three distinct groups of ILC subsets have been described. Group 1 ILC include cytotoxic natural killer (NK) cells and other type-1 cytokines (IFN-? and TNF-?) producing cells that regulated by T-BET. Group 2 ILC (ILC2) express GATA-3 and ROR?, secrete type-2 cytokines, IL-5 and IL-13. Group 3 ILC (ILC3) utilize ROR?t to drive production of the TH17-associated cytokines, IL-17 and/or IL-22. In this thesis, I have performed series of experiments to uncover the developmental pathway and function of human ILC that may allow us to harness ILC in diverse clinical settings. First, I analyzed the phenotypic and functional heterogeneity of human peripheral blood ILC2. I found human IL-13+ ILC2 can acquire the capacity to produce IFN-?, thereby generating ÔplasticÕ ILC2. ILC2 cultures demonstrated that IFN-?+ ILC2 clones could be derived and were stably associated with increased T-BET expression. The inductive mechanism for ILC2 plasticity was mapped to the IL-12/IL-12R signaling pathway and was confirmed through analysis of patients with Mendelian susceptibility to mycobacterial disease (MSMD) due to IL-12R?1 deficiencies that failed to generate plastic ILC2. This IL-13+IFN-?+ ILC2 are detected ex vivo in gut tissues from CrohnÕs patients. Second, I identified and isolated ILC precursors (ILCP) in peripheral blood of healthy donors. This circulating ILCP can give rise to four lineages of mature ILC including cytotoxic NK cells and helper ILC1, 2 and 3 in vitro and in vivo. Transcirptomic and epigenetic analysis showed ILCP have ILC-committed transcription factor profiles but have mature ILC signature locus at the epigenetics poised states. We further identified ILCP in various tissues including fetal liver, cord blood, postnatal lung and tonsil. Our result proposed a new model of ÒILC-poiesisÓ where circulating ILCP serve as cellular substrates to generate mature ILC subsets in tissues. Understanding the role of IL-12 on driving ILC2 to ILC1 plasticity may allow us to target plastic ILC2 in various diseases. The identification and isolation of ILCP from circulating blood allow further transfer into clinical setting for cellular therapy, especially for various diseases that ILC has been shown to be importance including infection, allergy, cancer and metabolic diseases.

Erratum is pasted onto back end-paper. / Bibliography: leaves 164-207. / xvii, 207, [26] leaves, [23] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Explores the hypothesis that physiological inflammation in the small intestine and the mesenteric lymph node is upregulated during the weaning period. Aims to determine changes in the number, phenotype, and activation status (using interleukin-2 receptor expression) of intraepithelial lymphocytes, lamina propria lymphocytes, mucosal mast cells, and mesenteric lymph node cells from preweaning to post weaning rats. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1998

Thesis (M.S.)--University of Michigan, 1982.

Thesis (M.S.)--University of Michigan, 1982.

Thesis (Ph. D.)--University of Virginia, 2001. / Spine title: Yersinia uptake by mammalian cells. Includes bibliographical references (leaves 214-240). Also available online through Digital Dissertations.

Chromatin remodelling proteins support a variety of cellular functions and utilise the energy from ATP hydrolysis to either reposition or evict nucleosomes. One such protein, Lymphoid specific helicase (LSH), regulates DNA methylation in mammalian cells cooperatively with DNA Methyltransferase 3B (DNMT3B) through binding of the N-terminal domain of LSH. The correct functioning of LSH is essential for heterochromatin formation, with a knockout of LSH causing perinatal lethality or severe developmental abnormalities. There is little biochemical data and no structural data on LSH. Therefore, we aim to determine the structural characteristics and regulatory mechanism of LSH in vitro. LSH was expressed in an optimised insect cell system which increased protein yield 25-fold with greater than 95% purity. LSH is monomeric with increased thermal stability upon ATP or ADP binding. Full length LSH could not be crystallised therefore a core ATPase region of LSH missing the N-terminal domain was identified through limited proteolysis. This also provided evidence the N-terminal domain of LSH is disordered, which was proven through biophysical characterisation of LSH1-176. Expression of the LSH ATPase region was weak and the protein was unstable; suggesting the N-terminal domain of LSH is required for LSH stability. Therefore, complementary structural methods were used to study LSH. Crosslinking mass-spectrometry revealed the N and C termini are in close proximity, suggesting flexible linking regions, which was supported by limited proteolysis experiments. Negative staining Electron Microscopy defined LSH as a tri-lobal and elongated structure which could harbour the ATPase region in the two spherical lobes. 3D modelling of SAXS data obtained of LSH was in agreement with EM data. To understand molecular mechanisms of LSH, functional studies investigating LSH:DNA and LSH:DNMT3B interactions were performed. LSH had a KD for dsDNA of 0.4 μM in solution. LSH does not bind ssDNA nor does it have a greater affinity for methylated dsDNA. LSH was found to bind the dsDNA overhangs of nucleosomes but not to core nucleosomes, suggesting LSH solely interacts with DNA in chromatin and not histones. A stable complex of LSH:DNMT3B could not be achieved in vitro, however, other components for complex formation may have been missing. This study has improved our understanding of LSH structure, biophysical properties and its biochemical interaction with DNA and nucleosomes. This study has laid the foundations for the structural investigations of a LSH:nucleosome and potentially a LSH:DNMT3B complex in vitro to gain a greater understanding of how functional domains of LSH regulates its enzymatic function.

Made available in DSpace on 2014-08-13T14:50:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-04Bitstream added on 2014-08-13T18:00:11Z : No. of bitstreams: 1 000764397.pdf: 797747 bytes, checksum: 6a89fbc6b7371005e020d9f2b9ab35ec (MD5) / A Leucemia Linfocítica Crônica (LLC) é uma neoplasia de linfócitos B maduros, de curso clínico heterogêneo, com alguns pacientes necessitando de tratamento imediato e outros vivendo sem tratamento por décadas. Por essa característica, se faz necessária a identificação de marcadores prognósticos acessíveis e de valor clínico. O objetivo principal foi avaliar retrospectivamente os indicativos prognósticos de progressão da doença (ZAP-70, CD38, ploidia de DNA e Fase S) em pacientes portadores de LLC, frente a sua evolução clínica. Como objetivos secundários, foram comparadas as metodologias de análise de ZAP-70 por citometria de fluxo(CF) ISO Método e Análise em Tubo Único, assim como as técnicas de análise de conteúdo de DNA pelo modelo matemático (ModFit LT™) e estratégia de gating boleano (Infinicyt™). Na cauística foram incluídos 27 pacientes que possuíam disponíveis os dados clínicos e laboratoriais em estudo e que concordaram e preencheram o termo de consentimento livre e esclarecido. A avaliação da situação clínica foi feita uma única vez, e assim como os resultados das expressões de CD 38 e ZAP 70, foi realizada através de revisão de prontuário médico. A análise do conteúdo de DNA por CF foi avaliada pelos dois métodos supracitados, a partir de arquivos do laboratório de citometria de fluxo da instituição. Como resultados, não se observou relação das expressões de ZAP-70 e CD38 com o estado clínico dos pacientes. Em relação à análise de índice de DNA e Fase S, não houve diferença estatística (p=0,69 e p=0,08 respectivamente utilizando-se os softwares ModFit LT™ e Infinicyt™). Também foi encontrado que os dados obtidos na análise de DNA indicam a possibilidade de utilização da Fase S na avaliação prognóstica dessa série de pacientes. Todos os três fatores estudados são considerados prognósticos. Não houve relação dos marcadores estudados com a evolução ... / The Chronic Lymphocytic Leukemia (CLL) is a mature B lymphocytes malignancy, on heterogeneous clinical course, where some patients require immediate treatment and others live for decades without treatment. For this peculiarity, it’s required the identification of prognostic markers and clinical value. The main objective was to retrospectively, evaluate the prognostic indicator of the disease progression (ZAP- 70, CD38, DNA ploidy and S phase) in patients under CLL, facing the clinical evolution. As secondary purposes, methodologies were compared to ZAP- 70 analysis by flow cytometry (FC) ISO Method and Single-Tube Analysis, as well as technical analysis of DNA content by the mathematical model (ModFit LT ™) and boolean gating principle (Infinicyt ™ ). On the registration of the cases observed, there were included 27 patients under the clinical and laboratory data who had available and filled in the term consenting agreement. The evaluation of the clinical situation was made only once, and the results of expressions and CD38 and ZAP 70, were held by reviewing medical records. The analysis of DNA content by CF was evaluated by the above two methods files from a laboratorial flow cytometry institution. As a result, no relation of the expressions of CD38 and ZAP- 70 with the clinical status of the patients was observed. Concerning the analysis of DNA index and S phase, there wasn’t any statistical difference (p = 0.69 and p = 0.08 respectively using the ModFit LT ™ and Infinicyt ™ software ). It was also found that the data obtained in the analysis of DNA indicates the possibility of using the S phase in the prognostic evaluation of those patients. Conclusion: all the three factors studied are considered prognostic. There was no relation of the markers studied to the clinical course of patients. To correlate these data with clinical follow-up of those patients, it is necessary a longer period of time