Les pâtes surgelées sont relativement stables et peuvent être fabriquées à l’échelle industrielle, distribuées et cuites à la demande au moment de la vente ou de la consommation (point chaud). La congélation des pâtes sucrées induit une baisse de volume et une augmentation du temps de fermentation, ces conséquences sont dues à deux facteurs : la baisse de la production de CO2 (viabilité des levures) et la faible capacité de rétention de gaz du réseau gluténique. La perte de la qualité des pâtes congelées est accélérée durant le stockage. Cette thèse porte sur l’étude de l’effet de la congélation et de la conservation sur les levures et les propriétés techno-fonctionnelles des pâtes sucrées type Kougelhopf. Ce travail vise à l’étude de l’impact de la vitesse de congélation sur les propriétés microbiologiques, rhéologiques, structurales et sensorielles de ces pâtes. Elles ont été congelées à différentes températures (-20 °C, -30 °C, -40 °C et une immersion dans l'azote liquide) puis conservées à -40 °C pendant 9 semaines. Les principaux résultats de cette étude ont permis de mettre en évidence le rôle de la vitesse de congélation et de la durée de conservation sur les propriétés intrinsèques des pâtes sucrées surgelées. Il en découle que l’activité fermentaire et l’intégrité du réseau du gluten sont tributaire de la vitesse de congélation. En effet, cette dernière contrôle la taille et la localisation des cristaux de glace d’où la recherche d’un compromis entre une vitesse de congélation ni trop rapide pour diminuer la viabilité des levures ni trop lente pour former de gros cristaux pouvant perforer le réseau de gluten de la pâte. Ce travail a démontré que le surdosage de levure reste valable uniquement pour les pâtes sucrées surgelées destinées à être conservées au-delà de 4 semaines. Ce surdosage améliore ainsi la qualité globale du Kougelhopf en compensant la perte de l'activité des levures pendant la congélation et le stockage / The frozen doughs are relatively stable and can be manufactured on an industrial scale, distributed and baked on demand at the point of sale or consumption (Bake-off). Freezing sweet dough induces a decrease in specific volume and an increase in fermentation time, these effects are due to two factors: lower production of CO2 (yeast viability) and losing capacity to retain gas (gluten network integrity). The loss of quality of frozen dough is accelerated during storage. This study focuses on the freezing and frozen storage effects on Kougelhopf sweet doughs. The aim of this work is to study the impact of freezing rate on microbiological, rheological, structural, and sensory properties of sweet doughs. The sweet doughs were frozen at different temperatures (-20°C, -30°C, -40°C and an immersion in liquid nitrogen) and stored at -40°C for 9 weeks. The main results obtained showed an impact of freezing rate and frozen storage duration on the frozen doughs intrinsic properties. This study shown the dependence of fermentation activity and integrity of the gluten network with freezing rate, which controls size and location of ice crystals resulting in research of a compromise between freezing rate nor too fast to reduce yeast viability, nor too slow to form large ice crystals that could perforate gluten network. Added the yeast amount is necessary only for frozen sweet doughs to be stored beyond 4 weeks, which improves the overall quality of Kougelhopf by compensating for yeast activity decrease during freezing and frozen storage

Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...

High throughput screening techniques that are routinely used in modern drug discovery processes result in a higher prevalence of poorly water-soluble drugs. Such drugs often have poor bioavailability issues due to their poor dissolution and/or permeability to achieve sufficient and consistent systemic exposure, resulting in sub-optimal therapeutic efficacies, particularly via oral administration. Alternative formulations and delivery routes are demanded to improve their bioavailability. Nanoparticulate formulations of poorly water-soluble drugs offer improved dissolution profiles. The physiology of the lung makes it an ideal target for non-invasive local and systemic drug delivery for poorly water-soluble drugs. In Chapter 2, a particle engineering process ultra-rapid freezing (URF) was utilized to produce nanostructured aggregates of itraconazole (ITZ), a BCS class II drug, for pulmonary delivery with approved biocompatible excipients. The obtained formulation, ITZ:mannitol:lecithin (1:0.5:0.2, w/w), i.e. URF-ITZ, was a solid solution with high surface area and ability to achieve high magnitude of supersaturation. An aqueous colloidal dispersion of URF-ITZ was suitable for nebulization, which demonstrated optimal aerodynamic properties for deep lung delivery and high lung and systemic ITZ levels when inhaled by mice. The significantly improved systemic bioavailability of inhaled URF-ITZ was mainly ascribed to the amorphous morphology that raised the drug solubility. The effect of supersaturation of amorphous URF-ITZ relative to nanocrystalline ITZ on bioavailability following inhalation was evaluated in Chapter 3. The nanoparticulate amorphous ITZ composition resulted in a significantly higher systemic bioavailability than for the nanocrystalline ITZ composition, as a result of the higher supersaturation that increased the permeation. In Chapter 4, pharmacokinetics of inhaled nebulized aerosols of solubilized ITZ in solution versus nanoparticulate URF-ITZ colloidal dispersion were investigated, under the hypothesis that solubilized ITZ can be absorbed faster through mucosal membrane than the nanoparticulate ITZ. Despite similar ITZ lung deposition, the inhaled solubilized ITZ demonstrated significantly faster systemic absorption across lung epithelium relative to nanoparticulate ITZ in mice, due in part to the elimination of the phase-to-phase transition of nanoparticulate ITZ. / text

Thesis (PhD (Food Sc))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Ostrich meat has become increasingly popular in South Africa and abroad, driven by the health conscious trend and ostrich meat’s natural low intramuscular fat, high polyunsaturated fatty acid and low cholesterol content. This increased demand led to the investigation of novel packaging regimes to improve its attractiveness and shelf-life. Different modified atmospheric packaging regimes were studied for fresh and frozenthawed ostrich M.iIliofibularis steaks stored at ±4°C for 10 days. Oxygen MAP (30 CO2:70 O2) was applied with great success to fresh steaks, and resulted in significantly improved colour stability, decreased drip loss and a 10-day shelf-life. Oxygen MAP of frozen-thawed ostrich steaks was not successful as the colour deteriorated within 3 days, coupled with high lipid and protein oxidation. The microbial shelf-life was not influenced by freezing and thawing. The use of nitrogen MAP (30 CO2:70 N2) as an alternative to vacuum packaging for fresh and frozen-thawed ostrich meat was inconclusive due to trace amounts of residual oxygen in the headspace accelerating myoglobin oxidation by depleting the metmyoglobin reducing activity. The differences in oxidative stability of the fresh and frozen-thawed ostrich meat led to the investigation of the source of these differences and a system that would allow control over the freezing and thawing practice. This was supported by the industry that is under increasing pressure to reduce the excessive (15-20%) thaw weight loss that is continually reported. Subsequently, a mathematical prediction model based on the control volume approach was developed that predicted the rate of freezing and thawing of intact whole vacuum-packed ostrich muscle. The model predicted with greater accuracy than existing models, and can be used successfully by the industry to optimally design, control and operate their systems. Furthermore research was conducted to investigate the effect of different freezing and thawing rates on the ice crystal formation and the quality of ostrich M. femorotibialis stored at ±4°C post freeze/thaw. Five characteristic freezing rates (FR (time from 0°C to -7°C): 1, 2, 4, 8, 24h) were paired with five characteristic thawing rates (TR (time form -7°C to 0°C): 1.5, 3, 6.5, 14, 21h) in a completely randomised block design. Results showed that thawing had no impact on any of the tested quality parameters, including thaw loss. Freezing rate however did influence the ice crystal formation and at a characteristic freezing rate of one hour (FR_1h) only intracellular ice crystals were observed throughout the M. femorotibialis leading to the lowest thaw loss (2.57%) and highest shear force. Freezing rate of 2h, 4h and 8h (FR_2h, FR_4h and FR_8h) were dominated by extracellular ice crystals. FR_2h and FR_8h showed major dehydration of the muscle fibres and excessive distortion of the muscle fibre matrix that led to significantly lower oxidative stability. FR_24h (approximately commercial rates) formed columns of ice from the surface to the centre of the meat resulting in the highest thaw loss (6.24%). FR_4h was judged to deliver the best quality product with moderate thaw (3.93%) and drip loss, low cooking loss, good colour stability and extremely low TBARS. FR_4h is an achievable rate of freezing for the industry and if implemented should decrease the thaw loss problem as well as increase revenue and throughput in the processing facility. / AFRIKAANSE OPSOMMING: ‘n Hedendaagse strewe na ‘n gesonde lewenstyl tesame met die natuurlike lae vetinhoud (d.i. hoë poli-onversadigde vetsure en lae cholesterolvlak) van volstruisvleis het gelei tot ‘n toename in die nasionale en internasionale aanvraag. Hierdie het ook die behoefte laat ontstaan om nuwe verpakkingstegnieke te ondersoek om sodoende die aanvaarbaarheid en rakleeftyd van die produk te verleng. Die invloed van verskeie gewysigde atmosferiese verpakkingsmetodes (GAV) op die kleurstabiliteit, drupverlies en rakleeftyd van vars en bevrore/ontdooide volstruisvleis (spesifiek M. Iliofibularis), gestoor by ±4°C vir 10 dae, is ondersoek. Die suurstof GAV (30 CO2:70 O2) van vars volstruisvleis het ‘n verbetering in kleurstabiliteit, verlaagde drupverlies en 'n verlengde mikrobiese rakleeftyd (10 dae) tot gevolg gehad. Die suurstof GAV van bevrore-ontdooide volstruisvleis het geen voordelige effek op kleurstabiliteit en rakleeftyd getoon nie. Die bevrore/ontdooide monsters het totaal verkleur na 3 dae en ‘n hoë vlak van lipied- en proteïenoksidasie is waargeneem. Die bevriesing en ontdooiing het geen invloed op rakleeftyd gehad nie. Die moontlikheid om stikstof GAV (30 CO2:70 N2) as 'n alternatief tot vakuumverpakking vir vars en bevrore/ontdooide volstruisvleis te gebruik, is beïnvloed deur die teenwoordigheid van oorblywende suurstof in die lugruimte van die GAV, met ‘n gevolglike toename in die tempo van mioglobien oksidasie as gevolg van ‘n afname in die metmioglobien reduksie aktiwiteit. Die verskil in die oksidatiewe stabiliteit van vars en bevrore/ontdooide volstruisvleis het gelei tot ‘n verdere ondersoek na die oorsaak van die verskille en die ontwikkeling van ‘n moontlike protokol om die oksidatiewe stabiliteit en dus rakleeftyd van volstruisvleis, te verbeter en bestuur. Die behoefte aan so ‘n protokol is beklemtoon deur die bedryf wat onder toenemende druk verkeer om buitensporige ontdooiingsverliese (15-20%) te verminder. ‘n Wiskundige voorspellingsmodel, gebaseer op die beheer-volume benadering, is ontwikkel om die tempo van bevriesing en ontdooiing van vakuumverpakte heel volstruisspiere te voorspel. Die model het tempo van bevriesing en ontdooiing met groter akkuraatheid as die bestaande modelle voorspel en kan suksesvol gebruik word in die bestuur van slagpale. Verder navorsing is onderneem om ondersoek in te stel na die effek van die tempo van bevriesing en ontdooiing op die kwaliteit van die bevrore-ontdooide volstruisvleis tydens berging by ±4°C. Die invloed van die kombinasie van vyf bevriesingstempo’s (FR (tydsverloop 0°C tot -7°C): 1, 2, 4, 8, 24 h) met vyf ontdooiingstempo’s (OT (tydsverloop -7°C to 0°C): 1.5, 3, 6.5, 14, 21 h) is ondersoek. Die resultate het getoon dat ontdooiingstempo geen beduidende invloed op enige van die kwaliteit kriteria, insluitend ontdooiingsverlies, gehad het nie. Bevriesingtempo het egter ‘n groot rol ten opsigte van yskristalvorming gespeel. Teen ‘n kenmerkende bevriesingstempo van een uur (FR_1h) was slegs intrasellulêre yskristalle waargeneem dwarsdeur die M. femorotibialis wat gelei het tot die laagste ontdooiingsverlies (2.57 %). Die ander bevriesingstempo’s, d.i. FR_2h, FR_4h en FR_8h, het gelei tot die vorming van meestal ekstrasellulêre yskristalle. Bevriesingstempo’s van FR_2h en FR_8h het beduidende dehidrasie van die spiervesels en vervorming van die spiervesel matriks tot gevolg gehad, wat tot verhoogde oksidasie gelei het. ‘n Bevriesingstempo van FR_24h (d.i. kommersieële tempo), het gelei tot die vorming van yskolomme van die oppervlak na die middelpunt van die spier, wat gevolglik die grootste ontdooiingsverlies (6.24%) tot gevolg gehad het. ‘n Bevriesingstempo van vier ure (FR_4h) is bestempel as die mees geskikte bevriesings tempo as gevolg van ‘n matige ontdooiingsverlies (3.93%), drupverlies, lae kookverlies, goeie kleur stabiliteit en uiters lae lipiedoksidasie te verseker. Die FR_4h bevriesingstempo is haalbare in die bedryf. Indien dit geïmplementeer word, sal dit waarskynlik die deurset van die slagpalesiklus asook die kostedoeltreffendheid van die volstruisbedryf vebeter, deur die vermindering van ontdooiingsverlies en verbetering van die algehele kwalitiet van die vleis.

Doctor of Philosophy / Department of Biology / Ruth Welti / The molecular mechanisms by which plants respond to environmental stresses to sustain growth and yield have great importance to agriculture. Lipid metabolites are a major element of plant stress responses. The model plant Arabidopsis thaliana is well-suited to study stress-driven compositional dynamics, metabolism, and functions of lipid metabolites. When Arabidopsis plants were subjected to wounding, infection by Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr), infection by Pseudomonas syringae pv. maculicola (Psm), and low temperature, and 86 oxidized and acylated lipids were analyzed using mass spectrometry, different sets of lipids were found to change in level in response to the various stresses. Analysis of plant species (wheat versus Arabidopsis), ecotypes (Arabidopsis Columbia 0 versus Arabidopsis C24), and stresses (wounding, bacterial infection, and freezing) showed that acylated monogalactosyldiacylglycerol was a major and diverse lipid class that differed in acyl composition among plant species when plants were subjected to different stresses. Mass spectrometry analysis provided evidence that oxophytodienoic acid, an oxidized fatty acid, is significantly more concentrated on the galactosyl ring of monogalactosyldiacylglycerol than on the glycerol backbone. A mass spectrometry method, measuring 272 lipid analytes with high precision in a relatively short time, was developed. Application of the method to plants subjected to wounding and freezing stress in large-scale experiments showed the method produces data suitable for lipid co-occurrence analysis, which identifies groups of lipid analytes produced by identical or inter-twined enzymatic pathways. The mass spectrometry method and lipid co-occurrence analysis were utilized to study the nature of lipid modifications and the roles of lipoxygenases and patatin-like acyl hydrolases in Arabidopsis during cold acclimation, freezing, and thawing.

La nucléation hétérogène de la glace est un processus, entre autres, qui concerne les phénomènes complexes de l'Atmosphère terrestre, cependant son impact est déterminant pour les propriétés et le temps de vie des nuages : ce processus affecte l'épaisseur optique et la durée de vie des nuages en phase mixte et froide et il est responsable d'une proportion significative des précipitations à l'échelle mondiale. La nucléation hétérogène de la glace est liée à la présence de particules d'aérosol spécifiques, nommées noyaux glaçogènes (acronyme INP depuis l’anglais), avec la propriété de réduire l’énergie de seuil exigée pour la formation des cristaux de glace, et dont le rôle est influent pour des températures de nuage >-38 °C. Dans les dernières décennies, des avancements significatifs ont été faits dans la description des noyaux glaçogènes, cependant leur compréhension reste incomplète et ceci influence les incertitudes sur les estimations de forçage radiatif dans les modèles climatiques. Il manque un grand nombre d’observations sur les distributions et propriété des 'INP' à l’échelle globale, spécialement en région éloignée. Dans la première partie de cette thèse, sont présentées des mesures de noyaux glaçogènes faites à l'Observatoire Climatique Italien "O. Vittori" sur la Montagne Cimone (2165 m au-dessus du niveau de la mer), pendant le printemps 2014 et l’automne 2015, sous les projets Bacchus et Air Sea Lab. Pour la première fois des mesures de INP hors ligne, reliés à un site de haute altitude du bassin méditerranéen sont présentées. Pendant la période du 19 au 29 mai 2014, une autre campagne a eu lieu en parallèle à la station San Pietro Capofiume, un site rural de basse altitude dans la Vallée du Po. Pendant quelques jours, les deux campagnes ont été concernées par un phénomène de Transport de Poussière Saharienne, qui a été enregistré simultanément dans la station de bas et de haute altitude. Nous avons examiné la concentration atmosphérique de INP activés à travers une congélation par condensation de vapeur (à -18°C et au-dessus de la pression de vapeur saturante). Dans la deuxième partie de cette thèse, sont présentées les observations qui ont été menées pendant la campagne arctique Parcs-Maca, dans la période de transition entre la nuit polaire et le jour polaire. Pour la première fois on reporte la caractérisation des propriétés glaçogènes et physiques/chimiques de l’aérosol marin primaire Arctique, dans une approche de génération contrôlée en laboratoire, qui a été combinée à une expérience de mesocosm. Le but de l'expérience de mesocosm était d'adopter une approche pluridisciplinaire afin d’étudier l'effet de la pollution marine sur les émissions marines. Nous avons trouvé une diminution modérée mais significative de la concentration de noyaux glaçogènes dans l'eau de mer polluée (par rapport à l'eau de mer du contrôle) pour des noyaux glaçogènes actives en congélation par immersion entre -8.5 et -19 °C. En ce qui concerne le spray marin, nos mesures indiquent une relation parmi les INP actifs à des températures chaudes (en congélation par immersion et au-dessus de -15 °C) et un enrichissement du Calcium détecté dans les filtres PM1 (suivi par un apparent épuisement du Chlorure). Sur la base de nos observations et des résultats publiés en littérature, quelque indication a été suggéré sur la nature de ces noyaux glaçogènes marins. En conclusion, les mesures effectuées dans cette thèse fournissent des nouvelles informations sur les concentrations de noyaux glaçogènes pour des particules d’aérosols en régions éloignées (un observatoire à haute altitude dans la région méditerranéenne centrale) et par rapport à une source spécifique (le spray marin Arctique). / Heterogeneous ice nucleation is one element inside the overall complexity of the Earth's atmosphere, however, it has a profound impact on our representation of cloud properties: this process affects the optical thickness and lifetime of mixed-phase clouds and cirrus clouds, and it is responsible for a significant proportion of precipitations formed globally. Heterogeneous ice nucleation is related to the presence of specific aerosol particles, named ice nuclei particles (INP), with the unique ability of lowering the energy barrier required for the formation of ice crystals, especially where cloud’s temperatures are >-38 °C. In the last decades, significant advancements have been made to the fundamental understanding of ice nucleation, however the lack of knowledge on the cloud ice phase still contributes to major uncertainties in climate model prediction of radiative forcing. This is partly due to limited observational data quantifying INP distributions and properties all over the world, especially in remote locations. In the first part of this thesis, field observations of ice nucleating particles have been performed at the Italian Climate Observatory “O. Vittori” on Mountain Cimone (2165 m above sea level), in the spring 2014 and autumn 2015, within the Bacchus and Air Sea Lab projects. For the first time we report the results of offline INP measurements, performed at a high altitude site within the Mediterranean basin. In the period 19-29 May 2014, a parallel campaign took place at the low-altitude station San Pietro Capofiume, a rural site in the Po Valley. The two campaigns were concerned, for a few days, by a Saharan Dust transport Event, which was recorded simultaneously at the high and the low-level station. We investigated the ambient number concentration of INP under condensation freezing activation mechanism (at -18 °C and above water saturation). In the second part of this thesis, we present the observations that were performed during the Arctic campaign Parcs-Maca, in the period of transition among the polar night and the polar day. We could characterise for the first time the ice nucleating and physical/chemical properties of the Arctic Primary Marine Aerosol, in a laboratory-controlled generation approach, that was combined to a mesocosm experiment. The aim of the mesocosm experiment was to adopt a multidisciplinary approach to study the effect of marine pollution on marine emissions. We found a moderate but significant decrease of the ice nuclei concentration in the polluted seawater (with respect to the control seawater) recorded in the freezing range between -8.5 and -19 °C and activated through immersion-freezing. Within the seaspray our measurements have indicated a relation among INP active at warm temperature (above -15 °C through immersion-freezing) and a calcium enrichment detected in PM1 filters (and followed by an apparent Chloride depletion). On the basis of our observations, and the results reported from other studies, a few suggestions on the nature of these marine ice nuclei have been suggested. In summary, the measurements made for this thesis provide new information on the concentrations of ice nuclei in ambient aerosol particles in remote regions (a high-altitude observatory in the central Mediterranean region) and in relation to a specific source (the Arctic sea spray).

Orientador: Maria Denise Lopes / Banca: Frederico Ozanan Papa / Banca: Jussara Tebet / Resumo: A análise dos efeitos da refrigeração sobre a congelabilidade da célula espermática da cauda do epidídimo de gatos domésticos foram avaliados nesse experimento. Após prévia refrigeração a 5O C por 24 horas alíquotas de sêmen foram submetidas a criopreservação e posteriormente descongeladas para análise morfofuncional. 15 animais foram submetidos a orquiectomia de conveniência e seus epidídimos foram manipulados para obtenção de amostras que foram suspensas em meio TE (TRIS/Equex/Gema de Ovo), analisadas, refrigeradas e congeladas. Amostras referentes ao grupo controle foram congeladas imediatamente após a colheita. A analise morfofuncional consistiu em motilidade, vigor, morfologia espermática e Integridade de membrana. Após a descongelação os resultados dos grupos controle e trabalho foram analisados estatisticamente e observou-se que não existe diferença estatística para nenhuma das variáveis analisadas. Embora tenha sido observada diferença estatística entre os momentos pós-colheita e pós-refrigeração por 24 horas para as variáveis motilidade, vigor, integridade de membrana e defeitos primários, nenhuma variável diferiu entre as amostras congeladas pós-colheita e as amostras congeladas pós-refrigeração prolongada. Tais resultados demonstram que um transporte refrigerado a 5O C por 24 horas não interfere sobre a congelabilidade de sêmen do epidídimo de gatos domésticos. Do ponto de vista da conservação tais resultados abrem portas para um melhor fluxo de gametas de espécies selvagens para centrais de biotecnologia permitindo um melhor aproveitamento e conservação deste material. / Abstract: The present study analyzed the effect of cooling on sperm cells obtained from the epididymis tail from domestic cats in respect to their capability for freezing. The treatment group had their samples submitted to freezing after being cooled at 5° C during 24 hours, and th~ control group samples were frozen immediately after collection, Samples from both groups were then thawed and submitted to mo rphofunctiona I analysis. Fifteen animais were submitted to a convenient routine orchiectomy and their epididymis were processed to obtain a semen samples which were suspended in TE medium (TRIS/Equexl egg yolk). The samples were then analyzed, coo,led and frozen. The morphofunctional analysis consisted of sperm motility, vigor, morphology and membrane integrity. After thawing, the semen samples from treatment and control groups were analyzed and no statistic difference was found among ali variables described above. / Mestre

Durante situações aversivas, como choque nas patas, imobilidade ou restrição dos movimentos, há um aumento nos níveis de acetilcolina no hipocampo de ratos. Além disso, o aumento desse neurotransmissor está envolvido com a modulação comportamental do MCC, principalmente em sua porção dorsal, HD. Diante disso, é proposto neste estudo que os receptores muscarínicos presentes no HD de ratos, modulam a expressão da REC, no modelo do MCC. Ratos wistar foram submetidos às sessões de condicionamento aversivo contextual, sendo divididos nos grupos: condicionado (6 choques de 3s; 1,5 mA) e não condicionado (sem choques) . Quarenta e oito horas após esta sessão foram avaliadas a comportamental (congelamento) e respostas autonômicas (PAM, FC e queda da TC). Além disso, foi verificada a distribuição de receptores M1 e M3 nas subáreas do HD e a quantificação dos mesmos em animais condicionados, não condicionados e naive, 48 após a sessão de condicionamento. A microinjeção bilateral de hemicolínio, inibidor da captação de colina, no HD promoveu uma redução do tempo de congelamento durante a reexposição ao contexto aversivo, caracterizando um efeito do tipo ansiolítico. Além disso, este tratamento inibiu o aumento da PAM, FC e a queda da TC induzidos pelo MCC. O mesmo ocorreu com a administração de atropina, antagonista não seletivo de receptores muscarínicos, de forma dose-dependente em todas as respostas observadas. Adicionalmente, a microinjeção de diferentes doses de J104129 fumarate bloquearam o tempo de congelamento (de forma dose-dependente, semelhante a atropina), a elevação da PAM, FC e a queda da TC durante a re-exposição ao contexto aversivo. Como o J104129 fumarate bloqueia receptores muscarínicos tanto M1 quanto M3, foi utilizado um antagonista de maior afinidade para receptores do tipo M1, pirenzepina. Todas as doses utilizadas de pirenzepina inibiram as respostas autonômicas, sem afetar o tempo de congelamento induzidos pelo MCC. A análise de imunofluorescência, por duplamarcação, mostrou que receptores M1 e M3 estão distribuídos nos mesmos compartimentos celulares nas subáreas do HD. A quantificação dos receptores 48 horas após o condicionamento evidencia apenas o aumento de receptores M3 no hipocampo de ratos, sem alteração na população de receptores M1. Com este conjunto de resultados podemos concluir que a ACh no HD é essencial para a expressão da REC. Especificamente, eles sugerem que os receptores muscarínicos do tipo M1 presentes nesta estrutura estão envolvidos com as respostas autonômicas e somente os receptores M3 participam das respostas comportamentais. / During aversive and stressful situations, such as footshock, stillness and restriction of movements, there is an increase in the levels of acetylcholine in rat hippocampus. Moreover, the increasing in the synaptic level of this neurotransmitter is involved with behavioral modulation of contextual fear conditioning (CFC), especially in the dorsal portion, DH. Therefore, this study investigated the involvement of muscarinic receptors in DH of rats in the expression of conditioned emotional response (CER) in the CFC. Moreover, we verified the expression of these receptors in the DH by double labeling immunofluorescence. Male Wistar rats were subjected to aversive contextual conditioning sessions and were divided into two groups: conditioned and unconditioned. Forty-eight hours after this session were evaluated autonomic (mean arterial pressure, MAP, heart rate, HR and tail temperature, TT) and behavioral (freezing) responses. Moreover, the distribution of M1/M3 receptors in subareas of DH was observed and the quantification of these receptors were performed 48 hours after the conditioning session in conditioned, unconditioned and naive animals. The bilateral microinjection of hemicholinium, inhibitor of choline reuptake, in DH, caused a decrease of freezing during re-exposure to the aversive context, featuring an anxiolytic-like effect. Furthermore, this treatment inhibited the increase in MAP, HR and TT drop by CFC. DH muscarinic receptors antagonism evoked by atropine, a non-selective muscarinic antagonist, reduced the freezing, in a dose-dependent manner. Similar reduction was observed in autonomic responses. The selective antagonism of M1/M3 receptors evoked by J104129 fumarate also reduced freezing, in a dose-dependent manner, when compared with control animals. The same effect was observed with autonomic responses in all the tested doses. As J104129 fumarate blocks both M1 muscarinic as M3 muscarinic, was used a higher-affinity M1 antagonist, pirenzepine. All doses inhibited cardiovascular responses and decrease the TT drop, without affecting the freezing induced by CFC. The immunofluorescence analysis revealed that M1 and M3 receptors are distributed in the same cellular compartments in DH. The quantification of receptors showed an increase of M3 receptors in rat hippocampus, while no change in the density of receptors M1 was detected. These findings support that cholinergic neurotransmission present in DH is involved with the expression of responses evoked by fear contextual conditioning, through muscarinic receptors activation. In particular, M3 muscarinic receptors modulate behavioral responses, M1 and M3 muscarinic receptors modulate the autonomic responses.

Introdução: O Estudo Longitudinal de Saúde do Adulto é um estudo de coorte multicêntrico com o objetivo de identificar os fatores de risco associados ao diabetes tipo 2 e à doença cardiovascular na população brasileira. Nosso principal objetivo é explicar a concepção e implementação das rotinas do laboratório central e biobanco do Estudo Longitudinal de Saúde do Adulto (ELSA-Brasil); destacando as forças e limitações do protocolo. O segundo objetivo é descrever os pré-testes usados para validar o protocolo de antes do início do estudo. Métodos: O estudo optou pela centralização dos exames em um laboratório central no Hospital Universitário da Universidade de São Paulo (USP), Com base em dados recentes que confirma a estabilidade da glicose em amostras congeladas, até os testes de glicose no sangue foram centralizadas. Porém o processamento das amostras foi realizado nos laboratórios locais o que reduziu os custos do transporte das amostras para o laboratório central. A estratégia implicou na necessidade de implementação de equipes nos laboratórios em cada Centro de Investigação para coletar e processar as amostras antes do transporte. O estudo incluiu exames para a avaliação do metabolismo da glicose, resistência à insulina, perfil lipídico, eletrólitos, ácido úrico, hormônios da tireóide, função hepática e contagem total de células do sangue. Além disso, as amostras de DNA, urina plasma, e de soro foram colhidas e Além desses exames, o estudo também extraiu DNA de leucócitos, colheu e estocou amostras de urina, plasma e soro. Para garantir a homogeneidade do protocolo todos os membros das equipes locais passaram por treinamento e certificação centralizado, com visitas cruzadas entre os centros durante o campo para controle de qualidade. Resultados: A opção por um laboratório central garantiu a uniformidade da metodologia utilizada para a realização dos exames, evitando as variações inevitáveis entre laboratórios. Durante 26 meses, cerca de 375.000 testes foram realizados no laboratório central. Não houve perda de amostras biológicas durante o estudo. A implementação do biobanco usando palhetas armazenados em repositórios de nitrogênio foi realizada sem problemas importantes desde 2008 até agora. Conclusão: O ELSA-Brasil mostrou a exequibilidade de exames multicêntricos no Brasil com todos os exames realizados em um laboratório central, de uma maneira custo-efetiva. A logística de armazenamento de amostras biológicas foi feito com custos aceitáveis e de qualidade, sendo um modelo para estudos futuros / Background: The Longitudinal Study of Adult Health is a multicenter cohort study aimed to identify risk factors associated with type 2 diabetes and cardiovascular disease in Brazilian population. Our main objective is to explain the conception and implementation of the routines of the central laboratory and biobank of the Longitudinal Study of Adult Health (ELSA-Brasil) highlighting the strength and the limitations of the protocol The second objective is to describe the pre-tests used to validate the protocol before the start of the study. Methods: The study made an option to centralize the exams in one central laboratory at the University Hospital, São Paulo University (\"USP\"). Based on recent data that confirms the stability of glucose in freeze samples, even blood glucose tests were centralized. However, biological samples were processed in the local laboratories, reducing the weight of the material to be transported, and diminishing costs of transportation to the central lab. Especially trained local teams collected and processed biological samples before transportation. The study included tests for glucose metabolism, insulin resistance, lipid profile, electrolytes, uric acid, thyroid hormones, hepatic function, and total blood cell count. In addition, DNA, urine, plasma and serum samples were collected and stored. In order to guarantee protocol homogeneity, all team members underwent centralized training and certification, and cross-visits in each research center were done. Results: The choice of a central laboratory assured uniformity of the methodology used for the exams, avoiding the variations between laboratories During 26 months, approximately 375,000 tests were done in the central- laboratory. There was no loss of biological samples during the study. The implementation of the biobank using straws stored in nitrogen repositories was performed without important problems since 2008 until now. Conclusion: The ELSA-Brazil showed the feasibility of a multicenter study in Brazil with all the analyses performed in a central laboratory, in a cost-effective way. The logistic of storage of biological samples was done with acceptable costs and quality being a model for future studies

Les automates cellulaires forment une famille bien connue de modèles dynamiques discrets, introduits par S.Ulam et J. von Neumann dans les années 40. Ils ont été étudiés avec succès sous différents points de vue: modélisation, dynamique, ou encore complexité algorithmique. Dans ce travail, nous adoptons ce dernier point de vue pour étudier la famille des automates cellulaires coagulants, ceux dont l’état d’une cellule nepeut évoluer qu’en suivant une relation d’ordre prédéfinie sur l’ensemble de ses états. Nous étudions la complexité algorithmique de ces automates cellulaires de deux points de vue : la capacité de certains automates coagulants à simuler tous les autres automates cellulaires coagulants, appelée universalité intrinsèque, et la complexité temporelle de prédiction de l’évolution d’une cellule à partir d’une configuration finie, appelée complexité de prédiction. Nous montrons que malgré les sévères restrictions apportées par l’ordre sur les états,les automates cellulaires coagulants peuvent toujours exhiber des comportements de grande complexité.D’une part, nous démontrons qu’en dimension deux et supérieure il existe un automate cellulaire coagulants intrinsèquement universel pour les automates cellulaires coagulants en codant leurs états par des blocs de cellules ; cet automate cellulaire effectue au plus deux changements d’états par cellule. Ce résultat est minimal en dimension deux et peut être amélioré en passant à au plus un changement en dimensions supérieures.D’autre part, nous étudions la complexité algorithmique du problème de prédiction pour la famille des automates cellulaires totalistiques à deux états et voisinage de von Neumann en dimension deux. Dans cette famille de 32 automates, nous exhibons deux automates de complexité maximale dans le cas d’une mise à jour synchrone des cellules et nous montrons que dans le cas asynchrone cette complexité n’est atteinte qu’à partir de la dimension trois. Pour presque tous les autres automates de cette famille, nous montrons que leur complexité de prédiction est plus faible (sous l’hypothèse P 6≠NP). / Cellular automata are a well know family of discrete dynamic systems, defined by S. Ulam and J. von Neumannin the 40s. The have been successfully studied from the point of view of modeling, dynamics and computational complexity. In this work, we adopt this last point of view to study the family of freezing cellular automata, those where the state of a cell can only evolve following an order relation on the set of states. We study the complexity of these cellular automata from two points of view, the ability of some freezing cellular automata to simulate every other freezing cellular automata, called intrinsic universality, and the time complexity to predict the evolution of a cell starting from a given finite configuration, called prediction complexity. We show that despite the severe restriction of the ordering of states, freezing cellular automata can still exhibit highly complex behaviors.On the one hand, we show that in two or more dimensions there exists an intrinsically universal freezing cellular automaton, able to simulate any other freezing cellular automaton by encoding its states into blocks of cells, where each cell can change at most twice. This result is minimal in dimension two and can be even simplified to one change per cell in higher dimensions.On the other hand, we extensively study the computational complexity of the prediction problem for totalistic freezing cellular automata with two states and von Neumann neighborhood in dimension two. In this family of 32 cellular automata, we find two automata with the maximum complexity for classical synchronous cellular automata, while in the case of asynchronous evolution, the maximum complexity can only be achived in dimension three. For most of the other automata of this family, we show that they have a lower complexity (assuming P 6≠NP).