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Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates. / Ph. D.

Le processus aléatoire des recombinaisons V(D)J permet d’obtenir un répertoire d’anticorps (Ac) ou immunoglobulines (Ig) hautement diversifié. En revanche, le caractère imprécis des jonctions V(D)J conduit à l’apparition de décalages du cadre de lecture dans deux tiers des cas. Ainsi, la plupart des cellules B hébergent des allèles d’Ig avec des réarrangements V(D)J non-productifs au sein de leur génome. Plusieurs études incluant celles menées au laboratoire ont montré que ces allèles non-productifs sont transcrits mais subissent une régulation post-transcriptionnelle impliquant le mécanisme de dégradation des ARNm appelé NMD « Nonsense-Mediated mRNA Decay ». Cette surveillance ARN diminue ainsi le taux d’ARNm codant pour des chaînes d’Ig tronquées. En revanche, l’impact de l’épissage alternatif des transcrits d’Ig non-productifs sur la production d’Ig aberrantes reste jusqu’ici peu exploré. L’étude de ce processus appelé NAS (« Nonsense-associated Altered Splicing »), et en particulier du phénomène de saut d’exon, présente un grand intérêt car cet épissage alternatif peut permettre la synthèse d’Ig tronquées présentant des délétions internes. Les projets développés lors de cette thèse ont révélé la toxicité des chaînes d’Ig dépourvues de domaine variable (V) dans les plasmocytes, et mis en évidence l’existence d’un nouveau point de contrôle au cours de la différenciation plasmocytaire. Ce phénomène nommé TIE-checkpoint (Truncated-Ig Exclusion) conduisant à l’élimination des plasmocytes exprimant des Ig tronquées, est la conséquence d’un saut d’exon lors de l’épissage des transcrits Ig non-productifs. Pour étudier les évènements de NAS lors de l’épissage des transcrits d’Ig dans les plasmocytes, il faut par conséquent limiter l’activation du TIE-checkpoint. A l’aide d’un modèle murin présentant un exon non-sens additionnel au locus IgH, nous avons pu analyser in vivo l’épissage alternatif par « saut d’exon » des transcrits d’Ig non-productifs. En effet, l’élimination de cet exon addtionnel aboutit à la synthèse d’une chaîne d’Ig normale et non à la production de chaînes tronquées. Cette étude a été menée dans des cellules B primaires et des plasmocytes. Les résultats obtenus ont révélé que l’hypertranscription des gènes d’Ig, qui accompagne la différenciation plasmocytaire, favorise l’épissage alternatif des transcrits d’Ig non-productifs, par un phénomène de saut d’exon. Nous avons également étudié les éventuelles connexions entre le mécanisme de NMD, impliqué dans la surveillance des ARNm, et l’UPR (« Unfolded Protein Response ») permettant de réguler l’homéostasie protéique dans les plasmocytes. De façon originale, nous avons identifié une boucle de régulation positive entre les processus de surveillance ARN (NMD) et protéique (UPR, autophagie, protéasome). La mise en évidence de cette coopération dans les plasmocytes constitue un exemple unique au vue de la littérature et, aurait pour effet de limiter la synthèse d’Ig tronquées tout en autorisant la synthèse massive d’Ig. Enfin, nous avons étudié le rôle de l’épissage des transcrits d’Ig non-codants (appelés transcrits I « germinaux ») au cours du processus de CSR « Class Switch Recombination ». Cette étude a apporté des précisions sur le rôle des sites donneurs d’épissage des exons I et révélé que la reconnaissance de ces sites d’épissage module l’intensité de la transcription de la région « switch » S adjacente, et par conséquent, son accessibilité à AID « Activation-Inducedcytidine Deaminase » lors de la CSR. / The random V(D)J recombination process contributes to the generation of a vast immunoglobulin (Ig) repertoire. However, imprecise V(D)J junctions lead to the appearance of frameshift mutations in two-third of the cases. Hence, numerous B-lineage cells retain non-productively V(D)J rearranged Ig alleles in their genome. Several studies including ours have shown that these non-productive alleles are transcribed but rapidly degraded by NMD « Nonsense-Mediated mRNA Decay », thus decreasing the level of mRNA encoding truncated Ig. However, less is known about the impact of alternative splicing on non-productive Ig transcripts, and especially « exon skipping », with regard to the production of truncated Ig with internal deletions. During my thesis, we have shown that truncated Ig chains lacking variable (V) domain exhibted toxic effects in plasma cells revealing a new « Truncated-Ig Exclusion » (TIE-) checkpoint during plasma cell differentiation. The TIE-checkpoint eliminates plasma cell-expressing truncated Ig, as a consequence of exon skipping during splicing of non-productive Igκ transcripts. However, the TIE checkpoint activation limits the analysis of NAS (« Nonsense associated Altered Splicing ») of Ig transcripts in plasma cells. Using a mouse model harboring an additional frameshift-inducing V exon at the IgH chain locus, we could analyze NAS of non-productive Ig transcripts in primary B cells and plasma cells. This study revealed that hypertranscription of Ig genes accompanying plasma cell differentiation favors alternative splicing of non-productive Ig transcripts. We also investigated potential connections between the NMD mechanism, involved in mRNA surveillance, and the UPR (« Unfolded Protein Response ») pathway that regulates protein homeostasis in plasma cells. Interestingly, we identified a positive regulatory loop between RNA (NMD) and protein (UPR, autophagy, proteasome) surveillance processes. In view of the literature, the occurrence of such cooperation is unique to plasma cells, and this should help to limit the expression of truncated Ig while allowing massive Ig synthesis. Finally, we studied other aspects of Ig RNA splicing, and investigated the role of splice donor site on non-coding « germline » I transcripts during CSR (« Class Switch Recombination »). Using dedicated mouse models, we found that the deletion of Iƴ1 splice donor site drastically decreased CSR to IgG1. Overall, this study demonstrated that the recognition of I exon donor splice site enhances transcription of « switch » regions S, facilitating their opening and the subsequent recruitment of AID « Activation-Induced cytidine Deaminase » during CSR.

Prevalence information on excessive gingival display in post-orthodontic patients is limited. By studying one aspect, namely the size relationship of the clinical crowns of teeth, in an orthodontic population, we can begin to quantify their need for periodontal plastic surgery. In this two part study, 200 plaster models were used as subjects, followed by a clinical exam of 31 of those subjects. These models represented patients before and directly after orthodontic therapy, and the Part 2 clinical exams were performed at least five years later. The lengths and widths of the six anterior teeth were measured and these values were compared to known ideals. This study revealed a significant increase in the length of the maxillary anterior teeth over the three examinations, yet these values were still approximately 1.5mm shorter than ideal. The mean tooth width-to-length ratio was 87-88% for maxillary central incisors, clearly below the accepted "ideal." As well, 61-71% of maxillary central incisors exceeded allowable tooth width-to-length ratios, and 61% of subjects displayed asymmetry in gingival architecture. Although this study only examined one aspect of excessive gingival display, it is the first study to show that in a predominantly young, post-orthodontic population, the prevalence of non-ideal width-to-length ratios is greater than 65%, and that the presence of asymmetry is greater than 60%. Therefore, close interaction between the periodontist and the orthodontist is necessary to diagnose these conditions in order to provide patients with all options for improving their smile.

Abstract FACTORS AFFECTING GINGIVAL EXCESS, ALTERED PASSIVE ERUPTION AND RECESSION IN THE MANDIBULAR ANTERIOR AND PREMOLAR SITES By William F Bohlen, D.M.D. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Dentistry at Virginia Commonwealth University. Virginia Commonwealth University, 2010 Major Director: Thomas Waldrop, DDS, MS Program director, Department of Periodontics, Virginia Commonwealth University AIM: The aim of this study was to determine the factors affecting gingival excess, altered passive eruption and recession. METHODS: 100 subjects were examined clinically and models of their mandible were fabricated. Demographic, periodontal and cast measurements were recorded for each subject. Measurements were made on casts with digital calipers and included clinical crown length, clinical crown width, papillary height and gingival width. The W:L ratio was calculated and the proportion compared to the maxillary arch ideal of .80. Values greater than .80 were used as a cutoff point for defining gingival excess. Measures of periodontal health were also examined and included probing depths, clinical attachment loss and bleeding on probing. Other patient variables examined were history of orthodontics, presence of occlusal and incisal wear, presence of parafunctional habits, subjective appearance of gummy smile and biotype. RESULTS: The mean W:L ratio was found to be 79.6 %. Tooth type (p<0.001), gender (p<0.0237) and biotype (p<0.0081) were found to significantly contribute to a W:L ratio >.80. There was a significant correlation between the subjective appearance of gingival excess and the W:L ratio, regardless of biotype. There was no association between recession and gingival excess. CONCLUSION: Subjectively, 17% of the study subjects had gingival excess. When the author (WB) made the determination that gingival excess was present, there was a significant increase in the W:L ratio for all teeth, regardless of biotype versus teeth without the presence of gingival excess. Proposed ideal W:L ratios for the mandibular anterior teeth from the second premolar to central incisor are listed in Table 11.

This theoretical study attempts to reflect the general problematic of modern society in complexity, the gradual constitution and development of estranged being as well as the contemporary effort to interconnect being as an integral whole. The study also contains an analysis of altered states of consciousness, which are understood as a certain means to view the world as integrated. We use the notion of consciousness to define the altered states of consciousness. The structure of the thesis is divided into five chapters. While the first chapter is focused on description and analysis of alienation and other negative aspects of modern society, the second chapter is based on this reflection of the crisis of western society. Its subject is the work of several scientists who contribute by their works to a qualitative change of the mechanistic and materialistic conception of the world and who promote holistic approach to reality. The key topic of the third chapter is phenomenon of consciousness and interdisciplinary theories of consciousness. We deal also with the qualitative altered states of consciousness, that offer some potential. The fourth chapter concentrates on the history and methods of research of altered states of consciousness and delineates their basic characteristic and their functions. The...

Classic psychedelic substances, such as lysergic acid diethylamide and the active compound in magic mushrooms, psilocybin, are being studied again in a renaissance of psychedelic research. Psychedelic substances have profound effects on perception, emotion, and cognition, as well as the capacity to induce mystical-type experiences and ego-dissolution. Recent clinical studies indicate that these substances have positive effects on patient populations and healthy participants, both acutely and long-term. Neuroimaging studies show that psychedelics alter neural integration, by the disintegration of normally stable resting state networks, and increasing network connectivity between normally anticorrelated networks. This thesis will review the phenomenological characteristics of the psychedelic-induced altered state of consciousness, the therapeutic potential of the psychedelic-induced altered state of consciousness, and neuroimaging studies on the psychedelic state. Two theoretical accounts are compared on the brain basis of psychedelic-induced altered state of consciousness. From the recent research on psychedelics a novel theory of conscious states has evolved, the entropic brain theory. This theory will be compared to the integrated information theory, a well-established theory of consciousness within cognitive neuroscience.

A temporalidade compõe os atos da pessoa no mundo. A subjetividade assume papel importante, a partir dela é que o tempo adquire sentido e significado. Desta forma, a percepção subjetiva do tempo torna-se essencial para a percepção da realidade, delineando uma distinção entre o decurso temporal dos eventos vividos ao longo da vida, o que implica que o processamento da informação temporal é indispensável ao cotidiano. A Nova Estética Experimental, que estuda estímulos musicais ou outros fenômenos estéticos por meio de experimentos, nos quais atributos musicais são manipulados, é um campo de pesquisa que se dedica a investigações sobre o tempo subjetivo e visa verificar quais são seus efeitos sobre algum aspecto do comportamento do ser. No contexto ritualístico, as músicas xamânicas e de meditação são utilizadas por participantes que realizam rituais em função da bebida ayahuasca. Os rituais xamânicos afetam estados de consciência, dependendo do uso da substância e das músicas. Alterações nos estados de consciência devem alterar o tempo subjetivo, em função destes elementos específicos do ritual que produzem estas alterações de estados de consciência. Os participantes dos rituais xamânicos relatam que suas experiências possuem caracteres diferentes ao serem expostos por músicas conhecidas ou desconhecidas durante os ritos. Questiona-se qual seria a influência da atividade musical em exposição ao gênero conhecido ou desconhecido e como essa relação se daria em condições nas quais os participantes apresentassem alterações nos estados da consciência em função da bebida ayahuasca em rituais xamânicos. Assim, a proposta deste trabalho foi examinar os efeitos do tempo subjetivo de participantes com estados alterados da consciência em função de uso da bebida ayahuasca em rituais xamânicos envolvendo música. Foram selecionados apenas participantes experientes, que tomaram ayahuasca mais de 60 vezes nos últimos três anos. Cada participante foi avaliado nas condições do ritual, com o registro de estimações subjetivas de tempo, que foram reproduzidos por estímulos musicais com durações de 20 segundos, contendo músicas conhecidas e músicas desconhecidas pelos participantes dos rituais. Foi preenchida uma Lista de Estados de Ânimo Presentes antes da ingestão da ayahuasca, no decurso temporal da ação da bebida e ao final do ritual. Após a coleta de dados, os participantes foram entrevistados com perguntas envolvendo os rituais xamânicos com ayahuasca. O registro de alteração de tempo subjetivo e a Lista de Estados de Ânimo Presentes permitiram a identificação de como os elementos do ritual afetam as alterações de estados de consciência, com ayahuasca em comparação ao uso de um veículo-controle desenvolvido para a presente pesquisa, no contexto do ritual. As estimações temporais realizadas pelos participantes apontaram que as exposições às musicas conhecidas ou desconhecidas durante os rituais xamânicos, em função da bebida ayahuasca, alteram a percepção subjetiva de tempo. Todos os participantes subestimaram o tempo de reprodução dos estímulos musicais, no entanto, os participantes ao serem expostos às músicas desconhecidas eliciaram uma maior subestimação quando comparados às exposições às músicas conhecidas / Temporality composes the acts of people in the world. Subjectivity assumes an important role, because it is through it that time acquires meaning. Thus, time subjective perception becomes essential for reality perception, outlining a distinction between time course of lived events during life, which implies that temporal information processing is imperative to daily life. The New Experimental Aesthetics, that studies musical stimuli or other aesthetic phenomena through experiments, in which musical attributes are manipulated, is a research field that dedicated itself to investigations about subjective time and aims to verify which are their effects on some aspect of human behavior. In the ritualistic context, shamanic and meditation songs are used by participants of rituals with ayahuasca brew. The shamanic rituals, affect the states of consciousness, depending on the music and substance use. Changes in states of consciousness must also change subjective time due to these rituals specific elements that generate changes in states of consciousness. The same report that their experiences have different characteristics when exposed to known or unknown music during the ayahuasqueiras ceremonies. Exposure to known or unknown music can change subjective time. We questioned what would be the influence of musical activity during exposure of a known or unknown musical genre and how this relation would appear in conditions where participants would present alterations in states of consciousness as consequences of ingestion of ayahuasca brew in shamanic rituals. Thus, the aim of this work was to evaluate the evaluate the effects on participants subjective time in altered states of consciousness as a consequence of ingestion of ayahuasca brew in shamanic rituals involving music. Only experienced participants were selected, people who have drank ayahuasca more than 60 times in the last three years. Each participant was evaluated during the ritual condition, with the register of subjective time estimations, that were reproduced for musical stimuli with durations of 20 seconds, containing known and unknown music for the ritual participants. It was filled a Lista de Estados de Ânimo Presentes (LEAP Present Mood List) before drinking ayahuasca, during the time course of effects and in the ritual ending. After data collection, participants were interviewed with questions about shamanic rituals with ayahuasca. The register of subjective time and the LEAP allowed to identificate how rituals elements affected changes in states of consciousness, with the use of ayahuasca in comparison with a vehicle developed for the present research in the ritual context. Time estimations made by participants pointed that exposure to known or unknown music during shamanic rituals, due to ayahuasca brew, chaged the subjective perception of time. All participants underestimated the reproduction time of musical stimuli. However, the participants that were exposed to unknown music presented a higher under estimation when compared to the exposures of known music

The thesis aims at investigating the connection between nucleus mechanical characteristics with pluripotency state and differentiation associated with altered cell gene expression levels. The project investigates the deformation characteristics of the cell nucleus during unconfined compression in a 3D cell-seeded agarose constructs. The studies report modification in the mechanical behaviour of the nucleus in different embryonic stem cell phenotypes based on various pluripotent states (naïve or primed states) or following triggering of early differentiation. A multi-scale model is also presented to simulate dynamic details of mechanical perturbation to cells during compression. The first chapter presents a review of the relevant literature to introduce current progress in the related research field and the second chapter describes the general methods used in the thesis including cell culture, agarose construct preparation, construct compression and microscopy recording. The third chapter presents findings of studies involving the application of compression to embryonic stem cells in naïve and primed sate within agarose scaffolds. A range of parameters relating to the relative cell/nucleus morphological modifications are recorded with analysis and discussion. Chapter four presents studies that investigate the early differentiation of embryonic stem cells from either the naïve and primed pluripotency, achieved by altering cell culture condition, and further reveals the nuclear mechanical characteristic changes. The fifth chapter describes a multi-scale model developed to simulating the 3D cell-seeded agarose compression reported in previous chapters. This model is also used to estimate cell mechanical parameters and show accurate deformation detail in different locations within the construct. A final discussion of the thesis is provided in chapter 6 with a plan for future work.