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Thesis (Ph. D.)--University of Wyoming, 2005. / Title from PDF title page (viewed on Nov. 7, 2007). Includes bibliographical references (p. 156-170).

Thesis (M.S.)--Villanova University, 2008. / Psychology Dept. Includes bibliographical references.

Orientador: Edmundo Medici Filho / Banca: Elisa Emi Tanaka Carloto / Banca: Antônio Francisco David / Banca: Lucia Teramoto / Banca: Julio Cezar de Melo Castilho / Resumo: As alterações anatômicas das vértebras cervicais têm sido empregadas na análise de maturação óssea em Ortodontia e Ortopedia Funcional dos Maxilares. Como essas mudanças são sutis, o objetivo neste estudo foi desenvolver e avaliar um método de análise de maturação óssea empregando-se medidas dessas estruturas. Foram utilizados 246 pares de radiografias cefalométricas laterais e de mão/punho, 135 de indivíduos do sexo feminino e 111 do masculino, divididos em 5 grupos segundo a Curva de Crescimento de Mercadante. Nas radiografias cefalométricas laterais, 15 razões foram obtidas das vértebras C2, C3 e C4, utilizando o programa Radiocef 4.0®. A média de cada razão foi comparada entre os grupos por meio das análises ANOVA e Tukey, que apontaram 10 razões como as que melhor diferem cada fase e que foram utilizadas na criação do novo método. Neste método, denominado Maturação por Razões em Vértebras Cervicais (MRVC), a maturação óssea é classificada em 7 estágios a partir dos resultados das 10 razões. Para sua validação, utilizou-se outra amostra de 58 pares de radiografias cefalométricas laterais e de mão/punho, 28 do sexo feminino e 30 do masculino, submetidas à avaliação de 4 examinadores por três métodos: Curva de Crescimento, Hassel e Farman e método MRVC. Os resultados apontaram melhor desempenho intra e inter-examinadores para a Curva de Crescimento e desempenho semelhante entre o método MRVC e o de Hassel e Farman. Por outro lado, o método MRVC proporcionou resultados próximos ao da Curva de Crescimento na determinação da maturação óssea do indivíduo em relação ao pico do surto de crescimento puberal. Concluiu-se que foi possível criar um método de análise de maturação óssea por medidas das vértebras cervicais em radiografias cefalométricas laterais com eficácia comparável à da Curva de Crescimento. / Abstract: Anatomic alterations of the cervical vertebrae have been used on the assessment of bone maturation in Orthodontic/Functional orthopedics. Since these changes are subtle, our aim was to create and analyze a method for the assessment of skeletal maturation using measurements of the cervical vertebrae. Two hundred and forty six pairs of lateral cephalometric and hand/wrist radiographs (135 male and 111 female) were used on this study. The sample was separated in 5 groups according to the stage of Mercadante's Pubertal Growth Curve. Fifteen ratios were calculated with the measurements of C2, C3 and C4 vertebrae. The mean for each ratio was compared among the groups with the ANOVA and Tukey tests, which indicated 10 ratios that better distinguish each stage. These ratios were used on the development of the method Maturation by Ratios on Cervical Vertebrae (MRCV). This method ranks the maturation in 7 stages based on the results of the ratios. For the validation of the method, four examiners assessed 58 pairs of lateral cephalometric and hand/wrist radiographs using 3 methods: Pubertal Growth Curve, Hassel and Farman and MRCV. Better intra and inter-examiner performance were observed for Pubertal Growth Curve method, while the performance with Hassel and Farman and MRCV were almost the same. However, the results for MRCV were close to the Pubertal Growth Curve on the determination of skeletal maturation related to the peak of the pubertal growth. Based on the results, we concluded that it was possible to create a method for skeletal maturation assessment with measurements of the cervical vertebrae in lateral cephalometric radiographs with efficacy comparable to the Pubertal Growth Curve's. / Doutor

Bone dysplasia’s affect every 1 in 5,000 babies; most of these dysplasia’s are incurable, and some are even lethal (Stembalska et al., 2021). Hundreds of skeletal dysplasia’s are heritable, yet the genes involved are not well defined (Krakow, 2015). Most of the skeleton forms through a process called endochondral ossification (EO). There are three parts of EO: chondrogenesis, maturation, and ossification. During chondrogenesis, mesenchymal progenitor cells condense and then differentiate into chondrocytes. After differentiation, chondrocytes will elongate, then proliferate and mature to set up for primary ossification. We know that this process happens through many activated genes, but the sequential steps through which this is achieved has yet to be elucidated. In order to understand the cause of skeletal dysplasia’s and find new treatments, the molecular mechanisms controlling EO requires further investigation. This study focuses on one gene, CXXC1 Finger Protein, Cfp1, and its role in chondrocyte maturation during skeletal mouse development. Cfp1 was specifically deleted in chondrocytes, and the resultant effects on cartilage and bone were analyzed. A mild phenotype was observed in the knockout mouse model. It was found that loss of Cfp1 in chondrocytes leads to delayed ossification in the vertebrae, tibias, metatarsals, and metacarpals. Therefore, Cfp1 is necessary for normal chondrocyte maturation.

Glucocorticoids are steroid hormones that affect a variety of physiological and pathological processes both throughout development and in adult life. During mammalian fetal growth, the late gestation rise in fetal glucocorticoid levels is essential for the maturation of tissues and organs in preparation for birth. In humans, glucocorticoids are routinely administered to women threatened by a preterm labour to accelerate fetal lung maturation and prevent neonatal respiratory distress and mice lacking glucocorticoid receptor (GR-/- mice) die neonatally as they are unable to inflate their lungs due to severe pulmonary immaturity. Apart from their importance for proper lung maturation, the physiological role of glucocorticoids in the development of other organs and tissues is not well known. However, prenatal exposure to excess glucocorticoids was shown to elicit detrimental “programming” effects, raising the susceptibility to adult diseases such as hypertension, obesity and metabolic disturbances in both humans and animal models. I therefore used global and conditional GR knock out mouse models to investigate the role and importance of adequate glucocorticoid signalling in fetal heart development and maturation. I further confirmed the direct effects of glucocorticoids on the cardiomyocyte structure and function in an in vitro setting. GR-/- fetuses are under-represented in late gestation (>50% of the number of GR+/+ littermates) but are present in the expected mendelian ratio at E14.5. At E17.5, GR-/- fetuses show edema (increased fluid accumulation and body sodium content). Excess extracellular fluid accumulation could be a result of a congenital heart failure. During development, corticosterone levels sharply increase within the fetal hearts at E15.5-E16.5, coincident with nuclear translocation of GR. Consistent with activation of GR only after this time, the phenotypic consequences of GR deficiency can be seen after E16.5 and not before. At E17.5, hearts of GR-/- fetuses are smaller than in GR+/+ but display no structural abnormalities. Cardiac function however is severely impaired, with left ventricular systolic and diastolic performance inferior in GR-/- fetuses compared to their wild-type littermates. Microscopically, at E17.5, the structure of the cardiac muscle and individual cardiomyocytes are affected by the lack of GR. The normal outer muscle layer, with characteristic rod-shaped, aligned cardiomyocytes is not discernable in the GR-/- heart. Within the cardiomyocytes, myofibrils are short, undefined and randomly scattered within the cell. Lack of the maturational progression in the GR-/- hearts at E17.5 is evident in the pattern of gene expression. GR-/- fetuses do not display the normal gestational changes between E14.5 and E17.5 that are seen in control mice, including in genes involved in the maturation of cardiac structure (eg myosin heavy chain-α, MyHC-α), function (atrial natriuretic peptide, ANP), energy metabolism (eg hexokinase-1, PPARγ coactivator-1α, PGC-1α) and calcium handling (ryanodine receptor, RyR; sarcoplasmic reticulum Ca2+-ATPase, SERCA2a). However, there are no genotype or gestational alterations in mRNA encoding the mineralocorticoid receptor, which is also a receptor for glucocorticoids in the heart. The normal gestational changes in the levels of modified histone H3 associated with the promoters of some of the genes (MyHC-α, ANP, PGC-1α) are not seen in hearts of GR-/- fetuses. This cardiac phenotype was not secondary to adrenal catecholamine insufficiency reported in other GR-/- models, as peripheral tissue levels of adrenaline were not different between genotypes. In order to test the hypothesis that the effects of glucocorticoids on the heart are mediated via GR in cardiomyocytes and to further elucidate the direct effects of GR deficiency specifically within the heart, mice with conditional deletion of GR selectively in cardiac and vascular smooth muscle cells were generated ("SMGRKO" mice). These show ~65% reduction in cardiac GR mRNA and protein levels. Circulating levels of corticosterone do not differ between genotypes at E17.5. SMGRKO fetuses at E17.5 display a phenotype strikingly similar to that of global GR-/-, namely edema, impaired cardiac function, impaired cellular architecture within the ventricle and alterations in the gene expression, implying that the GR-deficient phenotype is largely due to the direct actions of GR within the heart and not secondary to effects on other systems (eg kidney or liver). In order to investigate the pathways by which GR stimulates cardiomyocyte maturation, an in vitro model of murine primary fetal (E15.5-E16.5) cardiomyocytes was developed. Cultures contain >98% of troponin Tpositive cells which beat spontaneously. Treatment of cardiomyocytes with either synthetic (dexamethasone) or physiological (corticosterone) glucocorticoid induces time- and dose-dependent changes in gene expression, consistent with glucocorticoid-dependent changes seen in vivo in the late gestation heart. The effects of glucocorticoids on gene expression were abolished by either siRNA mediated knock-down of GR or RU486 antagonism of GR, but were unaffected by a mineralocorticoid receptor (MR) antagonist. Moreover, cycloheximide pretreatment (to block protein synthesis) suggested PGC-1α as a direct genomic target of GR. RNAseq transcriptome analysis performed on cardiomyocytes treated with dexamethasone and cycloheximide for 2h identified >600 genes as possible rapid and direct glucocorticoid response targets. Among them are genes involved in energy metabolism, calcium handling and sarcomere assembly. Glucocorticoid treatment of fetal cardiomyocytes also induces striking structural changes – formation of stress troponin T-associated actin fibers and sarcomere assembly. Spontaneous contractile activity is improved by glucocorticoid treatment, with a decrease in both contraction and relaxation time (without a change in frequency) and an improvement in the relaxation kinetics. In summary, glucocorticoid signalling in cardiomyocytes is required for the functional, structural and transcriptional maturation of the fetal heart in late gestation in vivo. Glucocorticoid treatment of primary murine fetal cardiomyocytes replicated the contractile, transcriptional and structural changes seen in vivo and was dependent on GR. Thus, GR is essential in cardiomyocytes for the structural and biochemical changes that underlie the maturation of heart function around the time of birth and an inadequate glucocorticoid environment could potentially lead to detrimental and permanent changes in postnatal cardiac function. Since prenatal glucocorticoids are routinely used clinically, it is important to consider any possible effects they might have on the heart development and its function later in life.

La première étape de la réponse immune est réalisée par des cellules "sentinelles": les cellules dendritiques (DC). Elles ont à la fois un rôle de surveillance de l’organisme et une capacité unique à alerter les lymphocytes T naïfs. Leur efficacité à présenter des antigènes rencontrés en périphérie à des lymphocytes résidant dans les organes lymphoïdes résulte d’une spécialisation de fonction au cours du temps. A l’état immature, elles capturent et apprêtent les antigènes protéiques au niveau de divers organes, mais ont une faible capacité stimulatrice. Par contre, à l’état mature, elles perdent la capacité de capturer des antigènes, acquièrent celle de sensibiliser des lymphocytes T et migrent vers les organes lymphoïdes. Cependant, des DC immatures sont présentes dans les organes lymphoïdes en contact avec les cellules T laissant supposer qu’elles pourraient jouer d’autres rôles que celui de sentinelles. L’immunisation de souris par injection de DC immatures ou matures nous a permis de mettre en évidence un rôle potentiel des DC immatures. Ces dernières induisent en effet une prolifération des cellules T CD4 et leur différenciation en cellules de mémoire en absence de réponse primaire effectrice (absence d’IFN-) suite à une production d'IL-10. La différenciation de cellules de mémoire par des DC immatures pourrait être un mécanisme permettant d’alerter le système immunitaire lors d’infections limitées ou dans un contexte peu activateur (en présence d'IL-10) ne nécessitant pas l’induction de réponse immune immédiate. De plus, les DC spléniques immatures et matures semblent migrer des sites d’immunisation vers la zone des cellules T des ganglions drainants dans les mêmes proportions. La migration est un mécanisme impliquant les DC injectées mais aussi une activité physiologique des souris immunisées: aucune migration n’est observée chez des souris anesthésiées. En outre, les résultats obtenus au cours de cette étude suggèrent qu’un transfert de membranes pourrait avoir lieu entre les DC injectées et celles de l’organisme receveur. Ce mécanisme permettrait d’amplifier le signal antigénique exposé par les DC migrant de la périphérie.

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In Nature, antibodies are capable of recognizing a huge variety of different molecular structures on the surface of antigens. The primary factor that defines the structural diversity of the antibody antigen combining site is the length variation of the complementarity determining region (CDR) loops. Following antigen stimulation, further diversification through the process called somatic hypermutation (SHM) leads to antibodies with improved affinity and specificity. Sequence diversification by SHM is mainly achieved by introduction of point substitutions and a small percentage of insertions/deletions (indels). Although the percentage of indels in affinity matured antibodies is low, probably due to the low rate incorporation of in-frame indels throughout the course of the SHM diversification process, it is likely that the antibody fold can accommodate higher diversity of affinity-enhancing indels. By in vitro evolution, other researchers have sampled either only restricted diversity of indels or extended diversity of insertions only in specific positions chosen based on structural information and natural length variation. The aim of this thesis was to study the impact of random and high diversity indels on antibody affinity by in vitro evolution. New approaches for construction of libraries with in-frame amino acid indels were applied to enable sampling of indels of different lengths across the entire antibody variable domains. I followed two different approaches for construction of indel libraries. Firstly, a recently developed random approach allowed the construction of libraries with random insertions and deletions. Secondly, a semi-random approach was developed to build libraries with different lengths of insertions that could be widely applied in future in vitro antibody affinity maturation campaigns. Libraries constructed by either of these approaches yielded variants with insertions with improved affinity. Overall, this thesis demonstrates that insertions besides offering alternative routes to affinity maturation can also be combined with point substitutions to take advantage of additive effects on function.

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