Conceitos prévios, ou alternativos, são ideias introjetadas no universo cognitivo dos estudantes que diferem daqueles credenciados pela ciência estabelecida. São bastante estudadas as consequências da presença deste tipo de conceitos para o aprendizado. Entre outras dificuldades provocadas pelos conceitos alternativos está a impossibilidade de utilizá-los para embasar novos conhecimentos. Nestas condições, incapazes de decodificar apropriadamente as novas informações apresentadas pelas disciplinas, os alunos são inconscientemente encaminhados para a memorização. A origem dos conceitos prévios é variada. Uma das causas já detectadas é a precariedade da transposição didática, feita pelos autores dos livros textos ou por docentes. Uma das moléculas fundamentais para os estudos bioquímicos é a adenosina trifosfato (ATP), que desempenha múltiplas funções. Uma das principais é participar de processos que requerem energia. A compreensão do papel desta molécula é fundamental para o entendimento dos processos dos quais ela participa. Um dos objetivos do presente trabalho foi investigar equívocos sobre o papel termodinâmico do ATP nos processos celulares. Os testes foram realizados com alunos do ensino médio (EM), graduação (G) e pós-graduação (PG). A existência de concepções alternativas foi verificada, assim como sua estabilidade nos diferentes níveis de escolaridade: um resultado mostra que 68% EM, 92% G e 91% PG afirmaram que a energia da hidrólise de ATP é responsável por conduzir os processos celulares. Os resultados gerais mostram que os estudantes carregam equívocos em conceitos termodinâmicos básicos, tais como transferência de energia e espontaneidade de reações químicas. Duas possíveis fontes de conceitos alternativos da termodinâmica do ATP são o professor e o livro didático. Nesse trabalho foi verificado que os livros de ensino médio e graduação podem contribuir para a instalação de conceitos alternativos referentes ao ATP. Nos livros analisados, principalmente os de ensino médio, foram encontrados passagens, analogias e esquemas que podem contribuir para isso. E por fim, uma interferência didática foi feita com o intuito de corrigir o entendimento dos alunos no que diz respeito ao papel do ATP nas reações químicas. Alunos monitorados por meio de pré e pós-testes apresentaram resultados animadores em relação à atenuação de conceitos relacionados à termodinâmica do ATP. Dos alunos que participaram da intervenção, mais de 80% responderam e justificaram corretamente os testes feitos pós-intervenção / Misconceptions, or alternative concepts, are introjected ideas in the students` cognitive universe that differ from those established by science. The consequences of alternative concepts for the learning process are widely studied. Among other difficulties caused by alternative concepts is the impossibility to use them to support new knowledge. Under these conditions, unable to properly decode the new information submitted by the disciplines, students are unconsciously driven to memorization. The origin of the alternative concepts is varied and an already detected reason is the precariousness of didactic transposition by textbooks writers and teachers. One of the fundamental biochemical molecules is ATP which play multiple roles. A key role is its participation in energy requiring processes. Understand this molecule role is fundamental to understand the processes which it takes part. The present work aims at the investigation of misconceptions on the ATP\'s thermodynamic role on cellular processes. Tests were carried out with high school students (HS), undergraduate (U) and graduate students involved in PhD programs (G). Misconceptions were observed as well as its stability along different levels of education: 68% (HS), 92% (U) and 91% (G) students stated that the energy from ATP hydrolysis is responsible for driving cellular energy-demanding processes. The overall results show that students carry misconceptions on basic thermodynamic concepts such as energy transfer and chemical reactions spontaneity. Misconceptions on ATP thermodynamics have the teacher and the textbook as possible sources. In this study it was found that books from all levels may contribute to insert or preserve misconceptions on ATP. From the analyzed books, especially for the high school, there were quotes, analogies and diagrams that can contribute to it. Finally, in order to correct the students\' understanding regarding the role of ATP in chemical reactions a didactic intervention was made. Students were tested through pre- and post-tests dealing with the subject in study. Of the students who participated in the intervention, over 80% correctly answered and explained the post-intervention test

Nitrogenase consists of two metalloproteins, the MoFe protein and the Fe protein. The MoFe protein is an α2β2 heterotetramer and the Fe protein is an α2 homodimer. The catalytic cycle of nitrogenase involves binding of the Fe protein to each αβ catalytic half of the MoFe protein, electron transfer followed by ATP hydrolysis, Pi release and eventually dissociation of the two proteins. This cycle has to be repeated eight consecutive times to reduce one molecule of N2. The two catalytic halves of the MoFe protein had been considered to be independent of each other. The research presented here showed that there is negative cooperativity associated between the two catalytic halves of the MoFe protein. The results suggested that only one half of the MoFe protein is operative during the first turnover of the enzyme. In order to understand the substrate reduction mechanism of nitrogenase, the study focused on two important enzymes of the biogeochemical nitrogen cycle: nitrite (NO2 -) and nitrate (NO3 -). Two intermediates of NO2 - reduction were trapped by a remodeled nitrogenase (α-70Ala/α-195Gln MoFe protein) and characterized by advanced spectroscopic studies. These intermediates were found to be identical to the intermediates trapped during reduction of diazene (N2H2) and hydrazine (N2H4). The pathway for reduction NO2 - to ammonia (NH3) was also proposed. NO3 - was established as a new substrate of nitrogenase. The advanced spectroscopic studies confirmed that the same two intermediates were trapped by the remodeled nitrogenase. Kinetic studies showed that two competing pathways lead to NO3 - reduction by nitrogenase, a primary 2 e- reduction pathway to form nitrite and a secondary 8 e- reduction pathway to form NH3. The pathways for reduction of NO3 - to NO2 - and NH3 were proposed.

Back pain is the second most common neurological ailment in the United States and the leading cause of pain and disability. More than 80% of the total US population experiences back-pain during their life time and the annual back pain related healthcare costs exceed 100 billion dollars. While the exact cause of low back pain (LBP) is still unknown, degeneration of the intervertebral disc (IVD) has been suggested as a primary contributor. IVD is the largest avascular tissue in the human body and it is composed of three integrated tissues (annulus fibrosus - AF, nucleus pulposus - NP and cartilaginous end plate - CEP). IVD functions as a shock-absorber during motion and provides flexibility to motion of the spine. Maintaining IVD tissue integrity is an energy demanding process. Studies have shown that mechanical loading affects cellular biosynthesis of IVD tissue and may also promote IVD degeneration. However the path to this effect is still unknown. We propose a link between mechanical loading and cell energy production which contributes to altered cellular biosynthesis. Thus, we investigated the effects of mechanical loading on IVD cell energy metabolism under various mechanical loading regimes. Porcine AF and NP cells were isolated and seeded in 2% agarose at a 5,000,000 cells/mL cell density. A custom made bioreactor was used to conduct compression experiments. The experiment groups were: 15% static compression; 30% static compression; 0.1, 1 and 2 Hz dynamic compression at 15% strain magnitude. Experiment duration was 4 hr. ATP concentration in cell-agarose construct and culture media were measured using Luciferin-luciferase method to evaluate ATP production and ATP release from cells respectively. Lactate concentration in media was measured using lactate dehydrogenase enzymatic assay. Nitrite (stable metabolite of nitric oxide - NO) concentration in media was measured by Greiss Assay. DNA content per sample was measured using fluorometric assay. DNA content per sample was used as an internal control; all compressed samples were then normalized to unstrained control group. ATP production of AF cells was up regulated by static and dynamic mechanical loading. Data suggests that AF cell response to mechanical loading is primarily loading amplitude dependent. NP cells exhibited an increased ATP production at 1 Hz dynamic loading but remained comparable to control samples at other tested conditions. AF cells produced an increase in NO production at 1-, 2 Hz dynamic loading. NO production of NP cells was up regulated by mechanical loading at all tested conditions. ATP release was up regulated at higher frequencies in AF cells. In addition to higher frequencies (1 Hz and 2 Hz) NP cell ATP release was also up regulated by 30% static compression. Thus, this study clearly illustrates that mechanical loading affects IVD cell energy production.

Pannexins represent a recently discovered second family of gap junction proteins in vertebrates. However, instead of forming intercellular gap junction channels like connexins, pannexins operate as unpaired pannexons, allowing the flux of molecules from the cytoplasm to the extracellular space and vice versa. Pannexins appear to play a vital role in the local control loop of blood perfusion and oxygen delivery. The properties of Panx1 channels indicate that this protein is the most probable candidate for an ATP release channel and is involved in the propagation of intercellular calcium waves. It is also proposed to mediate the large pore formation of the P2X7 receptor death complex. Prolonged activation of this receptor can lead to cell death. There must be some mechanisms to stop this ATP-induced ATP release and opening of the lethal pore. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly. ATP analogues, including BzATP, suramine, and BBG were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. The ATP inhibitory effect is largely decreased when R75 on the first extracellular loop of Pannexin1 is mutated to alanine, strongly indicating that the ATP regulates this channel through binding. To further investigate the structural property of the ATP binding, we did alanine scanning mutagenesis of the extracellular loops and found that mutations on W74, S237, S240, I247 and L266 on the extracellular loops severely impair the BzATP inhibitory effect indicating that they might be direct binding partners for the ligands. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 have largely decreased BzATP sensitivity. Mutations on other residues didn't change the BzATP sensitivity compared to the wild type except for some nonfunctional mutants. All these data demonstrate that some amino acid residues on the extracellular loop of Pannexin 1 mediate ATP sensitivity. However, how these residues form the ATP-binding pocket remains to be elucidated.

Parmi les différents types de canaux ioniques, les canaux potassium (K+) sont très largement exprimés au niveau des cellules eucaryotes. Ils se répartissent en plusieurs familles et sous-familles. Parmi celles-ci, les canaux K+ ATP-dépendants (KATP) représentent une classe tout à fait particulière. En effet, ils ont la particularité d’être sensibles à la concentration cytosolique d’ATP et permettent ainsi de coupler le potentiel membranaire de la cellule à son statut métabolique. Le canal KATP est un complexe hétéro-octamérique constitué de 2 sous-unités : une sous-unité Kir6.x (Kir6.1 ou Kir6.2) formant le pore du canal et appartenant à la famille des canaux potassiques de type « inward rectifier », et une sous-unité régulatrice SURx (SUR 1 ou SUR2A/B) faisant partie des protéines ABC (ATP-binding cassette). L’expression hétérologue des sous-unités Kir6.x et SURx suivant différentes combinaisons conduit à la formation de plusieurs types de canaux KATP possédant des propriétés électro-physiologiques et des sensibilités aux nucléotides et aux agents pharmacologiques distinctes. Les canaux KATP ont, à ce jour, été très peu étudiés dans la fonction de reproduction. Au cours de ce travail, nous avons notamment caractérisé la répartition tissulaire des différentes sous-unités Kir6.x et SURx constitutives des canaux KATP dans les différents segments de l’appareil reproducteur du rongeur mâle. Des marquages immuno-histochimiques ont permis de détecter les sous-unités Kir6.2 et SUR2 au niveau du testicule, de l’épididyme et du canal déférent. Au niveau du testicule, ces marquages sont localisés dans les cellules de Sertoli, ainsi que dans les spermatides et les spermatozoïdes présents au centre du tube séminifère. Les cellules de l’épithelium épididymaire sont également marquées ainsi que les spermatozoïdes présents dans la lumière. De plus, le marquage Kir6.2 et SUR2 est présent dans l’épithélium du canal déférent et se retrouve au niveau de l’épithélium de plusieurs glandes annexes (prostate, vésicule séminale, glande de coagulation). Aucun marquage n’est détecté pour les sous-unités Kir6.1 et SUR1. Une étude plus détaillée des marquages observés au niveau de l’épithélium épididymaire révèle une colocalisation des sous-unités Kir6.2 et SUR2 dans les cellules principales de l’épithélium épididymaire avec un marquage particulièrement intense au niveau de l’appareil de Golgi. La présence de ces deux sous-unités au sein de l’épididyme a été confirmée par immunoblots. La présence d’ARNm codant pour Kir6.2 et SUR2 a été détectée par RT-PCR à partir d’extraits d’ARN d’épididymes adultes et pré-pubères de souris. Nous avons également démontré, par immunofluorescence indirecte, une association Kir6.2 / SUR2 dans des échantillons d’épididymes humains, bovins, canins et félins. Les marquages ont été répétés sur des étalements de spermatozoïdes provenant de rongeurs mais aussi d’autres espèces (humains, canins et équins). Les sous-unités Kir6.2, SUR2 mais également Kir6.1 ont été observées. Leur présence a été confirmée par immunoblotting. L’ARNm codant pour ces différentes sous-unités, mais pas pour la sous-unité SUR1, a été détecté par RT-PCR. Nous avons tenté ensuite de mieux cerner le rôle physiologique potentiel des canaux KATP au niveau des spermatozoïdes de souris. Comme les canaux KATP participent indirectement au contrôle de l’activité des canaux calciques potentiel-dépendants et à l’influx de calcium, nous avons mesuré la concentration de calcium intracellulaire de spermatozoïdes isolés et incubés dans différentes conditions expérimentales. Nous avons pu démontrer que différents agents pharmacologiques connus pour inhiber l’activité des canaux KATP étaient susceptibles de provoquer une augmentation rapide et soutenue de la concentration cytosolique en Ca2+ libre de spermatozoïdes isolés et périfusés. L’absence de calcium extracellulaire abolit l’effet des inhibiteurs des canaux KATP, ce qui confirme le rôle de ces canaux ioniques dans le contrôle de l’influx de calcium au niveau des spermatozoïdes. Nous avons ensuite évalué l’implication des canaux KATP dans la réaction acrosomiale, processus physiologique calcium-dépendant indispensable à la fécondation de l’ovocyte. Pour ce faire, nous avons initialement validé une technique de détection et de quantification de la réaction acrosomiale pour le spermatozoïde de souris. Tous les agents pharmacologiques que nous avions précédemment testés et qui augmentaient la concentration cytosolique en Ca2+ libre, induisent également une réaction acrosomiale. Dans le but de mieux cerner le rôle que pourraient jouer les canaux KATP dans les phénomènes sécrétoires participant à la fonction de reproduction, nous nous sommes intéressés au placenta humain dont le processus hormonal sécrétoire (hCG et hPL) est un processus Ca2+-dépendant bien caractérisé. Nous avons mis en évidence, par immunohistochimie, la présence des sous-unités Kir6.2 et SUR2 dans le syncytiotrophoblaste de placentas humains à terme. La présence de ces deux sous-unités a été confirmée par immunoblots et RT-PCR. La sécrétion d’hCG et d’HPL a été étudiée en réponse à divers agents pharmacologiques modulant l’activité des canaux KATP. Dans un modèle expérimental d’explants placentaires incubés, l’addition d’inhibiteurs et/ou d’activateurs des canaux KATP n’affecte pas, de manière significative, la sécrétion d’hCG et d’hPL. En conclusion, nos travaux démontrent, pour la première fois, l’existence de différentes sous-unités formant les canaux KATP au niveau de plusieurs structures de l’appareil reproducteur. Dans le cas des spermatozoïdes, ces canaux semblent être fonctionnels et sont impliqués dans le contrôle de l’entrée de calcium.

The mammalian brain contains a regulatory center in the diencephalic region known as the hypothalamus that plays a critical role in physiological homeostasis, and contains a variety of centers for behavioral drives, such as hunger and thirst. Located deep within the hypothalamus is the suprachiasmatic nucleus (SCN), or the master biological clock, that organizes rhythmic physiology and behavior, such that critical events take place at the most appropriate time of the day or night and in the most appropriate temporal, phase relationships. Cell-to-cell communication is essential for conveying inputs to and outputs from the SCN. The goal of the present study was to use an immortalized neural cell line (SCN2.2), derived from the presumptive anlage of the rat suprachiasmatic nucleus, as an in vitro model system to study intercellular communication among SCN cells. I tested whether the pineal neurohormone melatonin could modulate cell-to-cell signaling, via both dye coupling (gap junctional communication) and calcium waves (ATP-dependent gliotransmission). I also tested whether extracellular ATP could influence the spread of calcium waves in SCN2.2 cells. Lastly, the ability of extracellular ATP to modulate SCN physiological responses to melatonin in SCN2.2 cells was examined. I show that melatonin at a physiological concentration (nM) reduced dye coupling (gap junctional communication) in SCN2.2 cells, as determined by a scrape loading procedure employing the fluorescent dye lucifer yellow. Melatonin caused a significant reduction in the spread of calcium waves in cycling SCN2.2 cultures as determined by ratiometric calcium imaging with Fura-2 AM, a calcium sensitive indicator dye. This reduction was greatest when an endogenous circadian rhythm in extracellular ATP accumulation, determined by luciferase assay, was at its trough or lowest extracellular concentration. In addition, melatonin and ATP interacted in the regulation of gliotransmission (calcium waves), and this interaction was also specific to particular phases of the endogenous SCN physiological rhythmicity. Thus, I have established that a complex interaction exists between established melatonin signaling pathways and this newly discovered ATP accumulation rhythm, with the mechanisms underlying this relationship linked to endogenous cycling of SCN cellular physiology.

The research project, The Enzymatic Synthesis of CoA Derivatives Using a New ATP Regeneration System, describes the multiple lab trials conducted to develop an ATP regeneration system using various concentrations of specific substrates and the two enzymes MatB and PrpE. Reactions were combined using different concentrations of ADP, the addition or removal of ADK (adenylate kinase), and the substitution of either MatB or PrpE. Further reactions were combined using trans-crotonyl, trifluoroacetic acid, butyric acid, acetic acid, and creatine phosphokinase. This report also includes the methods used and analysis of the different chromatographs of each sample tested. / text

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