Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases.

Chemnitz, Techn. Univ., Diplomarb., 2008.

Made available in DSpace on 2014-06-11T19:23:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-18Bitstream added on 2014-06-13T20:29:52Z : No. of bitstreams: 1 000721435.pdf: 591654 bytes, checksum: 7b7b1e9b77dc7c172c5d9fbb988ca07b (MD5) / toxina Épsilon (ETX) produzida pelo Clostridium perfringens tipos B e D é uma das mais potentes toxinas clostridiais superada apenas pelas neurotoxinas botulínica e tetânica. É responsável por quadros fatais de enterotoxemia em ovinos, caprinos e ocasionalmente em outros animais, caracterizados por edema em vários órgãos e aumento da permeabilidade vascular. Nos estudos “in vitro”, a linhagem de células “Madin-Darby canine kidney” (MDCK) é susceptível à ação da ETX, que se heptameriza nas membranas celulares formando um poro complexo que evolui para a lise celular. No presente estudo, foram avaliadas a morfologia e a viabilidade celular, a despolarização da membrana mitocondrial e a expressão de mediadores de morte celular programada (Bax e Bcl-2), após a exposição das células MDCK, com a ETX, a cada 24 horas, durante intervalo de 1 a 5 horas. Verificou-se o aparecimento de vacúolos no interior do citoplasma celular associados à perda de viabilidade celular, que evoluíram de forma progressiva, nos períodos de 1 a 5 horas pós-exposição. Foram realizadas análises por citometria de fluxo acústica para obtenção de uma visão mais aprofundada da patogenia causada pela ETX. Utilizando a citometria de fluxo acústica, considerada altamente sensível, as células MDCK expostas à ação da ETX, nos períodos de 1 a 5 horas, revelaram uma diminuição do potencial de membrana mitocondrial, seguido da expressão das proteínas Bax (25,48 %) e Bcl-2 (45,45 %) na fase de formação do pré-poro (1 hora pós exposição). Estes resultados, juntamente com alta citotoxicidade e visualização de vacúolos celulares, demonstra que a análise por citometria de fluxo acústico representa potencialmente uma ferramenta eficaz para estudar a patogênese da ETX / Epsilon toxin (ETX) produced by Clostridium perfringens types B and D is one of the most powerful clostridial toxins surpassed only by neurotoxins botulinum and tetanus. Studies blame the ETX by developing a fatal enterotoxemia in sheep, goats and occasionally in other animals, characterized by edema in multiple organs and increased vascular permeability. In in vitro toxicoinfection studies the Madin-Darby canine kidney (MDCK) cell line is susceptible to the action of ETX, which forms a heptamer in the cell membranes forming a pore complex that progresses to cell lysis. In the present study, we assessed cell viability and morphology, mitochondrial membrane depolarization and expression of programmed cell death mediators (Bax and Bcl-2) after exposure of MDCK cells with the ETX every 24 hours for range of 1 to 5 hours. Our results shows the appearance of vacuoles within the cytoplasm associated with loss of cell viability, which evolved gradually, in periods 1-5 hours after exposure. Analyzes were performed by acoustic flow cytometry to obtain further insight into the pathogenesis caused by ETX. Using acoustic flow cytometry, considered highly sensitive, MDCK cells exposed to the action of ETX, during 1 to 5 hours showed a decrease in mitochondrial membrane potential followed by the expression of Bax (25.48%) and Bcl-2 (45.45%) proteins at the pre-pore stage (1 hour post exposure). These results along with high cytotoxicity and visualization of cellular vacuoles, demonstrates that the flow cytometry analysis acoustic represents a potentially powerful tool for studying the pathogenesis of ETX

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Nonlinear optics represents a significant area of research and technology concerned with the modification of material optical properties using light. The interaction between light and such materials gives rise to a multitude of nonlinear optical effects, including second har-monic generation, third harmonic generation, high harmonic generation, and sum frequency generation. This thesis focuses on a specific and relevant nonlinear phenomenon within this field, namely the nonlinear Kerr effect, which involves the modification of a material’s re-fractive index through the exposure to an intense beam of light. The nonlinear Kerr effect holds promise for various applications, such as self-phase modulation in laser technology and the utilization of optical solitons in telecommunications. However, the limited availability of materials with sufficiently strong Kerr effects often restricts the practical application of this effect across different industries. Concurrently, optical time-varying systems play crucial roles in modern technologies, in-cluding optical modulators, LiDAR systems, and adaptive cameras. These systems involve the dynamic modification of optical properties. To achieve ultra-fast modulation of light properties, it is beneficial to explore materials with ultra-fast modulation speeds of the op-tical refractive index for integration into time-varying systems. While electro-optical effects represent the most common methods for achieving high-speed modulation of the effective refractive index, the utilization of all-optical methods, such as the nonlinear Kerr effect, presents an alternative approach. Nevertheless, the absence of simultaneous high speed and large nonlinear Kerr response in the majority of well-established materials restricts the utilization of the Kerr effect in time-varying systems.This thesis focuses on the study of a group of materials known as epsilon-near-zero (ENZ) materials, where the real part of the permittivity vanishes at a specific wavelength referred to as the ENZ wavelength. Specifically, indium-tin-oxide (ITO), a transparent conducting oxide, is investigated, with its ENZ wavelength falling within the infrared region of the elec-tromagnetic spectrum. ITO has been shown to possess a record-breaking large nonlinear Kerr effect with sub-picosecond response times, making it an excellent candidate for all-optical time-varying systems. The primary objective of this research is to investigate the applications of this large, fast nonlinear response and, where possible, enhance its effective-ness. One notable application of rapid and substantial modifications in the refractive index of a material is adiabatic wavelength conversion of light. In one project, a thin layer of ITO is subjected to a pump-probe setup, where an intense pump beam of light triggers the nonlinear response of ITO, causing the refractive index to rapidly change while a probe beam passes through the modulated system. Consequently, the wavelength of the probe beam undergoes conversion. Furthermore, it has been demonstrated that the nonlinear response of ITO can be sig-nificantly enhanced in the presence of a plasmonic metasurface. Metasurfaces consist of two-dimensional arrays of sub-wavelength scattering objects capable of manipulating the vectorial properties of light. In another project, we design a gradient metasurface composed of gold placed over ITO, enabling the diffraction of incident light into various diffraction orders depending on the ratio between the wavelength of light and the periodicity of the metasurface. This unique property is utilized to dynamically steer the diffraction orders of the probe beam, achieving wavelength conversion by exciting the nonlinear response of the ITO substrate with a second pump beam. Additionally, we investigate the interaction of resonance modes in an amorphous silicon metasurface, known as Mie modes, with an inherently dark mode in a thin layer of ITO known as the ENZ mode. Through experimental and analytical approaches, we demonstrate that two fundamental Mie modes, electric dipole resonance and magnetic dipole resonance, can strongly couple with the ENZ mode. This strong coupling creates a highly complex system with a large and rapid nonlinear response, enabling the manipulation of light on sub-picosecond timescales. In our final main project, we delve into investigating the nonlinear response of ITO nanoparticles. To accomplish this, we put forth a numerical recursive approach that allows us to incorporate the significant nonlinear Kerr effect of ITO into inherently linear simulation environments. Subsequently, we employ this proposed method to extract the scattering pattern of sub-wavelength antennas fabricated from ITO in both linear and nonlinear optical regimes. Our objective is to explore the potential applications of ITO nanoantennas in various fields. Moreover, this thesis encompasses other projects related to ENZ materials. We investi-gate the nonlinear response of an artificially created ENZ medium by stacking subsequent layers of materials with negative and positive permittivities within the visible range of the electromagnetic spectrum. Additionally, we explore the nonlinear response of nanoparticles made of ITO. Lastly, we present our investigations into the strong coupling of the ENZ mode in a thin layer of ITO with surface plasmon polaritons in a layer of gold in contact with ITO.

<p> An area of current research interest involves the diacylglycerol kinase (DGK) family. Diacylglycerol kinases (DGKs) are a group of enzymes that phosphorylate diacylglycerol (DAG), a second messenger involved in cell signaling. The product of this reaction, phosphatidic acid (PA), also has signaling roles. An interesting isoform is DGKε, that although it has no identifiable regulatory domains other than the C1 domains. In addition, the catalytic domain is homologous to that of other DGK isoforms; however, DGKε exhibits an unusual specificity toward acyl chains of DAG, selectively phosphorylating an arachidonoyl-DAG substituted at the sn-2 position. Recently, researchers have identified an N-terminal hydrophobic domain of about 19 amino-acids in human DGKε. The present study attempted to identify the function of the N-terminal putative transmembrane domain of human DGKε and its relationship to the activity and substrate specificity of this enzyme by designing a truncated form of DGKε lacking the putative transmembrane domain.</p> <p> We have shown that the putative transmembrane domain of DGKε is not required for enzyme activity or for substrate specificity. In a mixed micellar assay the enzyme-catalyzed reaction followed surface dilution kinetics with respect to diacylglycerol and followed Michaelis-Menten kinetics with respect to ATP. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increased the catalytic rate constant for all three substrates used in this study. It appears unlikely that the putative transmembrane domain, a segment unique to DGKε, has no functional role. It is possible that the hydrophobic segment may have a role in enzyme regulation by associating the enzyme in oligomers that are inactive in quiescent cells and get activated upon dissociation into monomers by increased levels of DAG in the membrane. We have shown that the presence of higher molecular species in the gel is not dependent on the presence or absence of the putative transmembrane domain. The only difference between the full-length and truncated enzyme is the monomer to dimer ratio. It appears likely that another segment of DGKε besides the putative transmembrane domain may be involved in oligomerization and that oligomerization is either transient or very weak. The absence of the hydrophobic domain of DGKε seems to cause no drastic changes either in the activity, the substrate specificity, or the state of oligomerization of the enzyme.</p> <p> Therefore, the next question is whether the hydrophobic domain of DGKε inserts itself in the membrane as a transmembrane helix or it only helps associate the enzyme to the surface of the membrane. We studied the topology of theN-terminal domain of DGKε in intact and permeabilized cells by indirect immunofluorescent microscopy. The results show that the N-terminal domain of the protein is present in the cytosol. The data supports a model in which the hydrophobic domain of DGKε forms a hydrophobic loop that attaches to the inner layer of the plasma membrane or that the hydrophobic domain attaches to the inner leaflet through its nonpolar surface of a horizontal helix. The first hypothesis is supported by the presence of a Pro residue in the middle of the hydrophobic domain. This Pro would introduce a kink in the helix creating a loop, but the absence of one or more glycine residues proximal to proline may hinder the formation of the loop. The second hypothesis is sustained by the presence of a polar surface on one side of the helical wheel. This orientation indicates the presence of a slightly horizontal helix attached to the surface of the inner layer of the plasma membrane.</p> <p> Regardless of the orientation of the helix, the weak association of the enzyme with the membrane is supported by previous data on the ease of extractability of the enzyme with high salts and on the Triton X-114 phase partitioning.</p> / Thesis / Master of Science (MSc)

O poli(&epsilon;-caprolactona) (PCL) é um polímero biocompatível e biodegradável, aprovado pelo Food and Drug Administration (FDA) para ser usado como biomaterial. Diversos estudos utilizando sua forma linear ou ramificada têm demonstrado resultados promissores para seu uso no desenvolvimento de dispositivos médicos e em aplicações na área farmacêutica. O objetivo deste trabalho foi utilizar o PCL com estrutura estrelada (PCLE) para obter microesferas biorreabsorvíveis. Primeiramente realizou-se a avaliação das propriedades físico-químicas do PCLE por meio da cromatografia de permeação em gel (GPC), ressonância magnética de prótons (1H-RMN) e carbono (13C-RMN), calorimetria exploratória diferencial (DSC) e espectrometria por infravermelho com transformada de Fourier (FT-IR). A avaliação toxicológica do PCLE foi obtida por meio do ensaio de citotoxicidade utilizando células CHO-K1 e o corante vital 5-(3-carboximethoxifenil)-2-(4,5-dimetiltiazolil)-3-(4-sulfofenil) tetrazolium e do acoplador de elétrons fenazine metilssulfato (MTS/PMS). O ensaio de biodegradação foi conduzido em pH 7,4 na presença de lipase a 37 ºC. Após essas análises o PCLE foi utilizado para preparação de esferas por meio de emulsão complexa A/O/A. O PCLE foi caracterizado como um polímero de baixa massa molar, com dispersão de tamanho unimodal e cerca de 68,8 % de suas moléculas apresentaram estrutura estrelada com três braços. Em relação às propriedades térmicas o PCLE apresentou temperatura de fusão de 57,3 ºC e temperatura de transição vítrea de -54,3 ºC. A avaliação da citotoxicidade mostrou que o extrato de PCLE é compatível com o metabolismo celular. As microesferas obtidas a partir do PCLE, por emulsão A/O/A apresentaram polidispersão de tamanho. / The poly(&epsilon;-caprolactone) (PCL) is a biocompatible and biodegradable polymer which has been approved by Food and Drug Administration (FDA). Many studies that are using its linear or branched form have showed promising results for medical devices and controlled drug delivery applications. The aim of this research was the use of star-shaped PCL (PCLE) to prepare bioreabsorbable microspheres. At first, the physical-chemical properties were characterized by Gel Permeation Chromatography (GPC), Protons Resonance Magnetic Nuclear (1H-RMN), Carbon Resonance Magnetic Nuclear (13C-RMN), Differential Scanning Calorimetry (DSC) and Fourier Transformed Infrared Spectroscopy (FT-IR). The toxicological property was investigated by colorimetric assay using CHO-K1 cells and the vital dye (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) and the electron acceptor phenazine methosulfate (PMS). The biodegradation behavior was evaluated in the presence of lipase at 37 ºC and pH 7.4. The microspheres were prepared by complex emulsion W/O/W. The PCLE was characterized as low molecular weight polymer with monomodal distribution and about 68,8 % of the molecules were three-arm branched. The melting and glass transition temperatures were 57.3 ºC and -54.3 ºC, respectively. The cytotoxicity evaluation showed that PCLE extract was cell compatible. The obtained microspheres showed diameter polydispersity.

O poli(&epsilon;-caprolactona) (PCL) é um polímero biocompatível e biodegradável, aprovado pelo Food and Drug Administration (FDA) para ser usado como biomaterial. Diversos estudos utilizando sua forma linear ou ramificada têm demonstrado resultados promissores para seu uso no desenvolvimento de dispositivos médicos e em aplicações na área farmacêutica. O objetivo deste trabalho foi utilizar o PCL com estrutura estrelada (PCLE) para obter microesferas biorreabsorvíveis. Primeiramente realizou-se a avaliação das propriedades físico-químicas do PCLE por meio da cromatografia de permeação em gel (GPC), ressonância magnética de prótons (1H-RMN) e carbono (13C-RMN), calorimetria exploratória diferencial (DSC) e espectrometria por infravermelho com transformada de Fourier (FT-IR). A avaliação toxicológica do PCLE foi obtida por meio do ensaio de citotoxicidade utilizando células CHO-K1 e o corante vital 5-(3-carboximethoxifenil)-2-(4,5-dimetiltiazolil)-3-(4-sulfofenil) tetrazolium e do acoplador de elétrons fenazine metilssulfato (MTS/PMS). O ensaio de biodegradação foi conduzido em pH 7,4 na presença de lipase a 37 ºC. Após essas análises o PCLE foi utilizado para preparação de esferas por meio de emulsão complexa A/O/A. O PCLE foi caracterizado como um polímero de baixa massa molar, com dispersão de tamanho unimodal e cerca de 68,8 % de suas moléculas apresentaram estrutura estrelada com três braços. Em relação às propriedades térmicas o PCLE apresentou temperatura de fusão de 57,3 ºC e temperatura de transição vítrea de -54,3 ºC. A avaliação da citotoxicidade mostrou que o extrato de PCLE é compatível com o metabolismo celular. As microesferas obtidas a partir do PCLE, por emulsão A/O/A apresentaram polidispersão de tamanho. / The poly(&epsilon;-caprolactone) (PCL) is a biocompatible and biodegradable polymer which has been approved by Food and Drug Administration (FDA). Many studies that are using its linear or branched form have showed promising results for medical devices and controlled drug delivery applications. The aim of this research was the use of star-shaped PCL (PCLE) to prepare bioreabsorbable microspheres. At first, the physical-chemical properties were characterized by Gel Permeation Chromatography (GPC), Protons Resonance Magnetic Nuclear (1H-RMN), Carbon Resonance Magnetic Nuclear (13C-RMN), Differential Scanning Calorimetry (DSC) and Fourier Transformed Infrared Spectroscopy (FT-IR). The toxicological property was investigated by colorimetric assay using CHO-K1 cells and the vital dye (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) and the electron acceptor phenazine methosulfate (PMS). The biodegradation behavior was evaluated in the presence of lipase at 37 ºC and pH 7.4. The microspheres were prepared by complex emulsion W/O/W. The PCLE was characterized as low molecular weight polymer with monomodal distribution and about 68,8 % of the molecules were three-arm branched. The melting and glass transition temperatures were 57.3 ºC and -54.3 ºC, respectively. The cytotoxicity evaluation showed that PCLE extract was cell compatible. The obtained microspheres showed diameter polydispersity.

Neste trabalho estudamos problemas de evolução da forma \'d \' úpsilond\' SUP. \' úpsilon\' t\'\' + A (t,\'úpsilon\' )\' úpsilon\' = f(t,\'úpsilon\' ) \'úpsilon\'(0) = \' \' úpsilon\' IND. 0\' \', em um espaço de Banach X onde A(t, \'úpsilon\' ) : D \'está contido em\' X \'SETA \' X é um operador linear fechado e setorial para cada (t, \' úpsilon\' ). Quando o operador A(t, \' úpsilon\' ) é independente de \' úpsilon\' , isto é, A(t, \' úpsilon\') = A(t), mostramos um resultado de exitência, unicidade, continuidade relativamente a dados iniciais e continuação para o caso em que a não linearidade f tem crescimento crítico. Se A(t, \'úpsilon\' ) depende do tempo e do estado, então mostramos um resultado de existência, unicidade com f tendo crescimento sub-crítico semelhante aos resultados encontrados em [7, 33] / In this work we study initial value problems of the form \' d \'úpsilon\' SUP. dt + A (t, \'úpsilon\')\'úpsilon\' = f (t, \'úpsilon\' ) \' úpsilon\' (0) = \' úpsilon IND.0\', in a Banach space X where A(t,\' úpsilon\' ) : D \' this contained \' X \' ARROW\' X is an unbounded closed linear operator which is sectorial for each (t,\' úpsilon\' ). When the operator family A(t, \' úpsilon\' ) is independent of \' úpsilon\' , that is, A(t, \' úpsilon\' ) = A(t), we show a result on local well posedness and continuation with the nonlinearity f growing critically. If A(t,\' úpsilon\' ) depends on the time t and on the state \' úpsilon\' we show a local well posedness and continuation result that is similar to the result found in [7, 33]