Sedan implementeringen av digitala verktyg i läroplanen har en polarisering uppstått kring användandet av digitala verktyg med yngre barn där forskning visar både positiva och negativa aspekter. Massmedia lyfter fram att det råder delade åsikter kring användandet av digitala verktyg bland förskollärare. Syftet med denna studie är därför att undersöka förskollärares perspektiv på användandet av digitala verktyg i sin undervisning med yngre barn. Vi undersöker förskollärares attityder till att använda digitala verktyg tillsammans med yngre barn vilket kan synliggöras genom diskurser. I studien lyfter förskollärarna att de främst använder de digitala verktygen, projektor och lärplatta. Föreliggande studie har utförts med grund i en kvalitativ forskningsmetod med individuella semistrukturerade intervjuer. Den teoretiska grunden samt analysmetod som tillämpats är inspirerad av Faircloughs kritiska diskursanalys. Transkriberingen har analyserats utefter Faircloughs tredimensionella modell som ingår i den kritiska diskursanalysen.  Resultatet av studien påvisade att förskollärarnas perspektiv på digitala verktyg med yngre barn handlar om att vara uppmärksam om hur och med vilket innehåll verktygen bör och ska användas. Uppmärksamheten tolkas ur förskollärarnas konstruktion av två integrerade diskurser: medvetenhets- och vaksamhetsdiskurs, vilket kan spåras till både förskolans styrdokument och vetenskapliga texter. Studien synliggjorde förskollärarnas attityd och säkerhet i talet i relation till användandet av digitala verktyg med yngre barn utifrån hur de talade och deras konstruerade diskurser.

Facial expressions convey non-verbal cues. Humans use facial expressions to show emotions, which play an important role in interpersonal relations and can be of use in many applications involving psychology, human-computer interaction, health care, e-commerce, and many others. Although humans recognize facial expressions in a scene with little or no effort, reliable expression recognition by machine is still a challenging problem. Automatic facial expression recognition (FER) has several related problems: face detection, face representation, extraction of the facial expression information, and classification of expressions, particularly under conditions of input data variability such as illumination and pose variation. A system that performs these operations accurately and in realtime would be a major step forward in achieving a human-like interaction between the man and machine. This document introduces novel approaches for the automatic recognition of the basic facial expressions, namely, happiness, surprise, sadness, fear, disgust, anger, and neutral using relatively low-resolution noisy sensor such as the Microsoft Kinect. Such sensors are capable of fast data collection, but the low-resolution noisy data present unique challenges when identifying subtle changes in appearance. This dissertation will present the work that has been done to address these challenges and the corresponding results. The lack of Kinect-based FER datasets motivated this work to build two Kinect-based RGBD+time FER datasets that include facial expressions of adults and children. To the best of our knowledge, they are the first FER-oriented datasets that include children. Availability of children data is important for research focused on children (e.g., psychology studies on facial expressions of children with autism), and also allows researchers to do deeper studies on automatic FER by analyzing possible differences between data coming from adults and children. The key contributions of this dissertation are both empirical and theoretical. The empirical contributions include the design and successful test of three FER systems that outperform existing FER systems either when tested on public datasets or in realtime. One proposed approach automatically tunes itself to the given 3D data by identifying the best distance metric that maximizes the system accuracy. Compared to traditional approaches where a fixed distance metric is employed for all classes, the presented adaptive approach had better recognition accuracy especially in non-frontal poses. Another proposed system combines high dimensional feature vectors extracted from 2D and 3D modalities via a novel fusion technique. This system achieved 80% accuracy which outperforms the state of the art on the public VT-KFER dataset by more than 13%. The third proposed system has been designed and successfully tested to recognize the six basic expressions plus neutral in realtime using only 3D data captured by the Kinect. When tested on a public FER dataset, it achieved 67% (7% higher than other 3D-based FER systems) in multi-class mode and 89% (i.e., 9% higher than the state of the art) in binary mode. When the system was tested in realtime on 20 children, it achieved over 73% on a reduced set of expressions. To the best of our knowledge, this is the first known system that has been tested on relatively large dataset of children in realtime. The theoretical contributions include 1) the development of a novel feature selection approach that ranks the features based on their class separability, and 2) the development of the Dual Kernel Discriminant Analysis (DKDA) feature fusion algorithm. This later approach addresses the problem of fusing high dimensional noisy data that are highly nonlinear distributed. / PHD / One of the most expressive way humans display emotions is through facial expressions. The recognition of facial expressions is considered one of the primary tools used to understand the feelings and intentions of others. Humans detect and interpret faces and facial expressions in a scene with little or no effort, in a way that it has been argued that it may be universal. However, developing an automated system that accurately accomplishes facial expression recognition is more challenging and is still an open problem. It is not difficult to understand why facial expression recognition is a challenging problem. Human faces are capable of expressing a wide array of emotions. Recognition of even a small set of expressions, say happiness, surprise, anger, disgust, fear, and sadness, is a difficult problem due to the wide variations of the same expression among different people. In working toward automatic Facial Expression Recognition (FER), psychologists and engineers alike have tried to analyze and characterize facial expressions in an attempt to understand and categorize these expressions. Several researchers have considered the development of systems that can perform FER automatically whether using 2D images or videos. However, these systems inherently impose constraints on illumination, image resolution, and head orientation. Some of these constraints can be relaxed through the use of three-dimensional (3D) sensing systems. Among existing 3D sensing systems, the Microsoft Kinect system is notable because it is low in cost. It is also a relatively fast sensor, and it has been proven to be effective in real-time applications. However, Kinect imposes significant limitations to build effective FER systems. This is mainly because of its relatively low resolution, compared to other 3D sensing techniques and the noisy data it produces. Therefore, very few researchers have considered the Kinect for the purpose of FER. This dissertation considers new, comprehensive systems for automatic facial expression recognition that can accommodate the low-resolution data from the Kinect sensor. Moreover, through collaboration with some Psychology researchers, we built the first facial expression recognition dataset that include spontaneous and acted facial expressions recorded for 32 subjects including children. With the availability of children data, deeper studies focused focused on children can be conducted (e.g., psychology studies on facial expressions of children with autism).

A Periodontite Agressiva (PA), que atualmente pertence ao grupo das Periodontites estágios 3 e 4, distingue-se dos demais tipos de doença periodontal por seu início precoce, agregação familiar dos casos e por afetar pacientes sistemicamente saudáveis. Além disso, pode ser subclassificada em duas formas, localizada (PAL) e generalizada (PAG), em função de sua extensão. Muitas vezes, os depósitos de biofilme bacteriano são desproporcionais à quantidade de destruição óssea e perda de inserção que o paciente apresenta, independente da subclassificação. O microrganismo mais relacionado à etiopatogênese da doença é o Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), incluindo os seus principais sorotipos a, b e c, amplamente estudados. Associado a estas condições, A. actinomycetemcomitans apresenta alguns fatores de virulência como uma leucotoxina, principalmente ligada ao sorotipo b - clone JP2 (que é altamente leucotóxico) e proteínas de membrana externa (OMPs), especialmente Omp29. A resposta de imunoglobulina G (IgG) sérica contra este patógeno foi anteriormente associada à ambas as formas de PA, porém, são escassos os estudos que avaliaram longitudinalmente a resposta sérica frente a variáveis como estas. Dessa maneira, o objetivo desse estudo foi avaliar a resposta sérica, de 27 pacientes com PA e 10 pacientes periodontalmente saudáveis, contra Omp29 e sorotipos de A. actinomycetemcomitans, através de um ensaio ELISA, correlacionando com o número de cópias de JP2 (obtidos por qPCR em tempo real) e parâmetros clínicos, a partir de dados anteriormente coletados por nosso grupo. Todos os dados foram obtidos antes do início do tratamento e um ano após seu término. O tratamento consistiu de orientações de higiene bucal, tratamento mecânico e antibioticoterapia. Os dados resultantes do estudo mostraram que em ambas as formas de PA houve uma redução significativa na profundidade clínica de sondagem (PCS)(p<0,001), nível clínico de inserção (NCI)(p<0,001) e na resposta sérica contra Omp29 e sorotipo c de A. actinomycetemcomitans(p>0,005). Após 1 ano, os valores de densidade óptica (D.O.) normalizados para Omp29 e sorotipos de A. actinomycetemcomitans, bem como o número de cópias do clone JP2 tornaram-se similares aos níveis encontrados nos controles. A redução no número de cópias do clone JP2 foi correlacionada com redução da PCS em PAL(r=0.80,p=0.0042) e valores de D.O. normalizados de Omp29 em PAG(r=0.66,p=0.005). O estudo concluiu que o tratamento periodontal foi eficaz em alterar a resposta sérica contra Omp29 e sorotipos de A. actinomycetemcomitans, além de reduzir o número de cópias do clone JP2 e melhorar os parâmetros clínicos. / Aggressive Periodontitis (AP), which currently belongs to the group of Periodontites stages 3 and 4, is distinguished from other types of periodontal disease due to its early onset, familial aggregation of cases and to affect systemically healthy patients. In addition, it can be sub classified into two forms, localized (PAL) and generalized (PAG), depending on its extent. Often, bacterial biofilm deposits are disproportionate to the amount of bone destruction and loss of insertion that the patient presents, regardless of sub classification. The most important microorganism related to the etiopathogenesis of the disease is Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), including its main serotypes a, b and c, widely studied. Associated with these conditions, A. actinomycetemcomitans presents some virulence factors such as leukotoxin, mainly linked to serotype b - clone JP2 (which is highly leukotoxic) and outer membrane proteins (Omp\'s), especially Omp29. Serum immunoglobulin G (IgG) response against this pathogen was previously associated with both forms of BP; however, there are few studies that longitudinally evaluated the serum response to variables such as these. Thus, the objective of this study was to evaluate the serum response of 27 patients with AP and 10 periodontally healthy patients against Omp29 and A. actinomycetemcomitans serotypes by an ELISA, correlating with the number of copies of JP2 (obtained by qPCR in real time) and clinical parameters, from data previously collected by our group. All data were obtained prior to initiation of treatment and one year after its completion. The treatment consisted of oral hygiene guidelines, mechanical treatment and antibiotic therapy. Data from the study showed that in both forms of BP there was a significant reduction in clinical depth of sampling (PCS) (p<0,001),, clinical level of insertion (NCI)(p<0,001) and serum response against Omp29 and serotype c of A. actinomycetemcomitans(p>0,005). After 1 year, normalized optical density (O.D.) values for Omp29 and A. actinomycetemcomitans serotypes, as well as the number of copies of clone JP2 became similar to the levels found in the controls. The reduction in copy number of clone JP2 was correlated with reduction of PCS in PAL(r=0.80,p=0.0042) and O.D. normalized from Omp29 to PAG(r=0.66,p=0.005). The study concluded that periodontal treatment was effective in altering the serum response against Omp29 and A. actinomycetemcomitans serotypes, in addition to reducing the number of copies of clone JP2 and improving clinical parameters.

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana. / Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis.

Bien que les mécanismes moléculaires précis à l’origine de la neurodégénérescence dopaminergique ne soient pas encore totalement connus, un ensemble de preuves épidémiologiques, cliniques et expérimentales indiquent que la neuroinflammation peut avoir un rôle important dans la pathogenèse de la MP. L’étude des liens spatio-temporels entre la neuroinflammation et la neurodégénérescence au cours de la MP pourrait améliorer la compréhension du mécanisme physiopathologique et aussi l'accessibilité à un diagnostic précoce et/ou à de nouvelles approches thérapeutiques anti-inflammatoires. Le développement actuel des méthodes non invasives d'imagerie moléculaire permettant la surveillance directe du processus de neuroinflammation devrait être utile à cet effet. La cible moléculaire de choix dans ce domaine est la protéine de 18 kDa translocateur (TSPO), biomarqueur sensible associée à la neuroinflammation, qui est surexprimé dans les microglies activées. Dans le travail présenté ici nous avons réalisé l'évaluation longitudinale des deux mécanismes physiopathologiques en parallèle avec les modifications de la fonction dopaminergique à plusieurs points au cours du temps après lésion à la 6-OHDA chez le rat, modèle qui imite un stade précoce de la MP. Après l'administration unilatérale, intra-striatale de la 6-OHDA, nous avons quantifié l'évolution temporelle de la TSPO, de l’immunoréactivité TH et du DAT dans le striatum et la SNC de 3 à 56 jours post-lésion (jpl). L’augmentation de la liaison des ligands de la TSPO utilisés, c-à-d [3H]-PK11195 et [125I]-CLINDE, a été observée dans le striatum lésé à 3, 7 et 14 jpl, suivie d'un retour progressif à un niveau basal à 56 jpl. Le profil de liaison dans la SNC a montré une augmentation progressive de la fixation qui débute à 3 jpl, avec un pic à 14 jpl, et diminue progressivement jusqu'à ce que 56 jpl. Dans ce modèle de rongeur de la MP, les processus neuroinflammatoire et neurodégénératif surviennent de façon concomitante. La présence transitoire de l'activation microgliale pourrait être impliquée dans l’apparition et l'installation durable de la perte neuronale dopaminergique. Cette étude confirme donc le lien entre la neuroinflammation et de la neurodégénérescence et met aussi l'accent sur l'intérêt du CLINDE comme traceur potentiel de la neuroinflammation in-vivo en fournissant des informations précieuses pour le diagnostic précoce et le suivi longitudinal de la progression de la maladie, avec des applications potentielles chez l'homme. En effet, la détection précoce de la neuroinflammation, de façon antérieure à une perte neuronale cliniquement significative, pourrait devenir un enjeu majeur dans la prise en charge pré-symptomatique de la MP. Dans ce sens, nous mettons en évidence l’existence d'une fenêtre thérapeutique, survenant juste après la lésion, qui peut être proposé pour l'introduction de traitements anti-inflammatoires qui viseraient à ralentir le processus neurodégénératif. La poursuite de l’exploration des relations entre la neuroinflammation et la neurodégénéréscence in-vivo dans le même modèle animal avec la méthode d’imagerie micro-TEP, transposable à l’homme, en utilisant en parallèle le [18F]-DPA714 pour la TSPO et le [18F]-LBT999 pour le DAT est en cours. / Although the precise molecular mechanisms causing the dopaminergic neurodegeneration are still not totally understood, a body of epidemiological, clinical and experimental evidence indicates that neuroinflammation may have an important role in the pathogenesis of PD. Study of spatio-temporal links between neuroinflammation and neurodegeneration during the course of PD would improve understanding of the physiopathological mechanism and also accessibility to early diagnosis and/or new antiinflammatory therapeutic approaches. The current development of non-invasive molecular imaging methods allowing direct monitoring of the neuroinflammation process should be valuable for this purpose. The molecular target of choice in this field is the 18 kDa translocator protein (TSPO), a sensitive biomarker associated with neuroinflammation, which is over-expressed in activated microglia. In the study presented here we achieved the longitudinal evaluation of both physiopayhological mechanisms in parallel with the modifications of dopaminergic function at several time-points after 6-OHDA lesion in the rat that mimics an early stage of PD. After unilateral intra-striatal 6-OHDA administration, we quantified the temporal evolution of the TSPO, TH immunoreactivity and DAT in the striatum and the SNc from 3 to 56 days post-lesion (dpl). Increased binding of TSPO ligands used, i.e. [3H]PK11195 and [125I]CLINDE, was observed in the lesioned striatum at 3, 7 and 14 dpl, followed by a progressive return to the basal level at 56 dpl. The binding profile in the SNc showed progressive binding beginning at 3 dpl, peaking at 14 dpl, and progressively decreasing until 56 dpl. In this rodent model of PD, the neuroinflammatory and neurodegenerative processes occurred concomitantly. The transitory occurrence of microglial activation could be involved in the advent and the lasting installation of dopaminergic neuron loss. This study supports the link between neuroinflammation and neurodegeneration and emphasizes the interest of CLINDE as potent in vivo tracer of neuroinflammation by providing valuable information for early diagnosis and longitudinal follow-up of disease progression, with potential applications to human patients. Indeed, early detection of neuroinflammation, prior to a clinically significant loss of neurons, could become a major issue in the management of pre-symptomatic PD. To support this idea, we demonstrate the existence of a therapeutic window, occurring just after the lesion, which may be proposed for the introduction of anti-inflammatory treatments that aimed to slow the neurodegenerative process. Further exploration of the relationship between neuroinflammation and neurodegeneration in vivo in the same animal model with the method of micro-PET imaging, transposable to humans, using in parallel the [18F]-DPA714 for TSPO and [18F]-LBT999 for DAT is pending.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana. / Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis.

Orientadores: Liana Maria Cardoso Verinaud, Luciana Campos Paulino / Dissertação (mestrado) - Universidade Estadual de Campoinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T22:23:58Z (GMT). No. of bitstreams: 1 DiGangi_Rosaria_M.pdf: 3869352 bytes, checksum: 6c11f8a4be727a58eb3974501efd287e (MD5) Previous issue date: 2012 / Resumo: A paracoccidioidomicose (PCM), uma micose sistêmica, cujo agente etiológico é o fungo Paracoccidioides brasiliensis, acomete primariamente os pulmões e pode causar lesões secundárias na pele e mucosas, que são extremamente dolorosas e de difícil tratamento por métodos convencionais com drogas anti-fúngicas. Estudos anteriores, conduzidos em nosso laboratório, usando modelos experimentais para PCM, mostraram a capacidade do laser HeNe em aumentar a produção de citocinas pró-inflamatórias e a síntese de fibras colágenas, reticulina e de elementos de matriz extracelular, como laminina e fibronectina. Estes eventos levaram a uma cicatrização tecidual mais rápida e eficaz das lesões. Além disso, as células fúngicas recuperados a partir das lesões após tratamento com laser mostrou nenhuma capacidade de crescimento, quando cultivadas in vitro. Neste trabalho foi avaliada a ação direta do laser HeNe sobre o fungo com o objetivo de fornecer suporte para o uso desta terapia durante a infecção PCM. Os resultados mostraram que o tratamento com laser de HeNe reduziu a capacidade de crescimento e a viabilidade de células fúngicas em 90% e 30%, respectivamente. Ademais, as células tratadas apresentaram núcleo mais eletrondenso, sugerindo que a irradiação com laser induz alterações ultra-estruturais que podem afetar a viabilidade e/ou a virulência do fungo. Entretanto, a patogenicidade do fungo não foi afetada, uma vez que nenhuma diferença foi observada no pulmão de animais infectados com o fungo tratado e não tratado. Além dos efeitos sobre a resposta inflamatória, acreditamos que o laser de HeNe modifica, ou mesmo destrói, estruturas que são usadas pelo patógeno como mecanismos de resistência. Certamente, esta atividade fungistática/fungicida do laser também desempenha um papel importante na aceleração da cicatrização de feridas e, deste modo, a terapia com o laser de HeNe pode ser considerada como um tratamento adjuvante na cura de lesões da pele observados durante PCM / Abstract: Paracoccidioidomycosis (PMC), caused by the fungus Paracoccidioides brasiliensis (Pb), mainly affects the lungs and also causes painful lesions in the skin and mucous membranes that can remain active for months even after the beginning of antifungal treatment. In this work, we evaluated the effects of laser treatment on fungal cells, aiming to provide support for the use of this therapy during PCM. Pb yeast cells were exposed to laser for three consecutive days and then analyzed for growth inhibition, viability, 43-kDa glycoprotein (gp43) expression, infectivity, and ultra-structural alterations. HeNe laser treatment reduced the growth capacity and the viability of fungal cells, and increased gp43 expression. Laser-treated cells also showed pronounced structural alterations, since they were collapsed and presented deep folds. Additionally, remarkable changes were observed in the nucleus that presented a very electrondense form. The pathogenicity of P. brasiliensisafter laser treatment was also evaluated,and no differences were observed in pulmonary lesions comparing animals infected with laser-treated and non-treated yeast cells. Possibly HeNe laser is able to modify fungal structures involved in resistance and virulence mechanisms and therefore can be considered as an adjunctive therapy in the treatment of skin lesions observed during PCM / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Prions são definidos como partículas protéicas infecciosas compostas, quase que exclusivamente, por uma proteína conhecida como prion scrapie (PrPsc). O envolvimento dessas partículas na etiologia de doenças neurodegenerativas, tanto em homens como em animais, já está bem determinado. Acredita-se que o PrPsc seja sintetizado através de modificações pós-traducionais que ocorreriam na isoforma celular da proteína prion (PrPc), uma glicoproteína expressa constitutivamente na superfície de vários tipos celulares, principalmente de neurônios, ancorada na membrana plasmática por glicosil-fosfatidil inositol (GPI). Apesar de ser uma molécula conservada em várias espécies, a função de PrPc ainda permanece desconhecida. Interessados nos possíveis papéis fisiológicos desempenhados pelo PrPc, decidimos investigar certas interações que o PrPc poderia realizar com outras moléculas, na tentativa de se encontrar pistas sobre a função dessa proteína em células normais. Assim, nosso grupo identificou e vem caracterizando duas interações nas quais o PrPc está envolvido: com o seu receptor de 60-66 kDa e com a principal proteína não colagênica da matriz extracelular, a laminina (LN). Grande parte da caracterização dessas duas interações vem sendo desenvolvida graças ao uso de PrPc recombinante produzido em sistema heterólogo (em E.coli). O trabalho em questão trata principalmente da produção de PrPc recombinantes em sistema heterólogo e a utilização destes como importantes ferramentas para a melhor caracterização das interações identificadas. Alguns trabalhos na literatura vinham sugerindo a existência de um receptor para prions. Através da teoria da hidropaticidade complementar dos aminoácidos, nosso grupo foi capaz de identificar uma proteína de 60-66 kDa como sendo esse provável receptor. Ensaios de ligação in vitro utilizando PrPc recombinante, ou PrPc nativo (ancorado na superfície de células) confirmaram que a forma desse receptor presente na membrana (de 66 kDa) é capaz de se ligar ao PrPc. Com a ajuda de PrPc recombinante também foi possível verificar uma ligação específica, saturável e de alta afinidade (Kd da ordem de 10-8 M) entre PrPc e a LN. Através de ensaios de competição, utilizando peptídeos sintéticos correspondentes a domínios da LN já bem caracterizados e de funções estabelecidas, fomos capazes de mapear o sítio dessa molécula que se liga ao PrPc. A sequência identificada (RNIAEIIKDI) encontra-se na região C-terminal da cadeia &#947;1 da LN e, como demonstrado na literatura, esse domínio é responsável por estimular tanto a adesão celular, quanto o crescimento de neuritos em neurônios de cerebelo em cultura primária. De fato, resultados obtidos pelo nosso grupo indicam que a interação PrPc/LN participa no processo de neuritogênese. Experimentos de esquiva inibitória, realizados em colaboração com o grupo do Prof. Dr. Ivan Izquierdo (UFRGS) indicaram que a ligação PrPc/LN também desempenha um importante papel nos processos de memória e aprendizado. / Prions are defined as proteinaceous infectious particles that mediate the pathogenesis of certain neurodegenerative diseases, in humans and in animals. The prion particle is composed largely, if not entirely, by PrPsc (prion scrapie), a posttranslationaly modified isoform of the cellular host-encoded prion protein (PrPc). PrPc is a glycosylphosphatidylinositol anchored protein that is constitutively expressed by several cell types, mainly on neuronal cell surface. However, the physiological role of this conserved protein remains unclear. ínterested in this normal function of PrPc, our group decided to investigate some interactions that PrPc could be done with other molecules in order to find some clues about it. We have been identified and characterized two interactions that PrPc is involved: with its putative 60-66 kDa receptor and with laminin. In order to better characterize these interactions it was necessary to produce purified PrPc. In this work we will report the expression and the purification of mouse PrPc protein in heterologous system and its use as important tools to investigate the PrPc interactions. A specific cell surface receptor for PrPc has been predicted. Using the concept of complementary hydropathy, our group has identified a 60-66 kDa membrane protein in mouse brain, which seems to be a putative PrPc receptor. ln vitro binding assays using recombinant and native PrPc were able to confirm that the membrane receptor (66 kDa) binds PrPc. Recombinant PrPc was also useful to demonstrate a specific and high affinity (Kd around 10-8M) interaction between PrPc and laminin. In an attempt to map the PrPc binding site in this molecule, laminin peptides with established physiological functions were used in cornpetition binding assays. A peptide derived frorn C-terminal &#947;-1 chain of mouse laminin, RNIAEIIKDI, was the only one that was able to block the binding of laminin to PrPc, suggesting that this region comprises the PrPc binding site. It was reported in the literature that this peptide simulates the neurite outgrowth and cellular adhesion. In collaboration with Dr. Izquierdo\'s group (UFRGS) we demonstrated that the interaction characterized between this laminin\'s domain and PrPc is involved in the neuritogenesis process, as well as in learning and memory.

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