During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. Cellular events that occur at the time of and immediately after an insult are poorly understood. Immediately following traumatic brain injury (TBI), the neuronal plasma membrane may become disrupted and potentiate detrimental pathways by allowing extracellular contents to gain access to the cytosol. In the current study, neuronal plasma membrane disruption was assessed in vivo following moderate unilateral controlled cortical impact in rats using a normally cell-impermeant fluorescent compound as a plasma membrane permeability marker. This fluorescent dye was injected into the cerebrospinal fluid and was allowed to diffuse into the brain. TBI caused a widespread acute disruption of neuronal membranes which was significantly different compared to uninjured brains. Affected cells were present in cortex and hippocampal regions. These findings were complemented by an in vitro model of TBI where membrane disruption was quantified and its mechanisms elucidated. Permeability marker(s) were added to neuronal cultures before the insult as indicators for increases in plasma membrane permeability. The percentage of cells containing the permeability marker was dependent on the molecular mass, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were also positively correlated with the rate of insult. Membrane disruption was transient, evidenced by a robust resealing within the first minute after the insult. In addition, membrane resealing was found to be dependent on extracellular Ca2+, as chelation of the ion abolished a significant amount of resealing. We have also investigated the effects of mechanically-induced plasma membrane disruptions on neuronal network electrical activity. We have developed a multielectrode array system that allows the study of electrical activity before, during, and after a traumatic insult to neurons. Endogenous electrical activity of neuronal cultures presented a heterogeneous response following mechanical insult. Moreover, spontaneous firing dysfunction induced by injury outlasted the presence of membrane disruptions. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.

Cell adhesion plays an important role in inflammation and immunological responses. Adhesion molecules (e.g., selectins and integrins) are key modulators in mediating these cellular responses, such as leukocyte trafficking under shear stress. In this thesis, we use a bio-membrane force probe (BFP) to study force regulation on kinetics and conformations of selectin and LFA-1 integrin. A new BFP was built up, and a new assay, using thermal fluctuation of the BFP, was developed and used to monitoring selectins and their ligands association and dissociations. The new BFP was also used to investigate the force and force history dependence of selectin-ligand interactions. We found tri-phasic transition of force-dependent off-rates and force-history dependence of selectin/ligaind interactions. The BFP was also used to characterize force-dependent lifetimes of the LFA-1-ICAM-1 interaction. We found that LFA-1/ICAM-1 bonds behaved as catch bond and that LFA-1-ICAM-1's catch bonds were abolished blocking the downward movement of αA domain α7 helix. Finally, the BFP was applied to dynamically probe the global conformational changes of LFA-1 and to characterize force-regulated transitions among different conformational states on a living cell. We observed dynamic transitions of LFA-1 between extended and bent conformations on living cells. The observed average distance change of LFA-1's extensions was about 18nm, while that of the bending was only about 14nm. We also found that forces could facilitate extension but they slow down the bending of LFA-1. The observed transition time of extension was less than 0.1s, while that of contraction was longer than 0.2s. Our observations here are the first in-situ evidence to demonstrate how integrins dynamically transit different conformations and how force regulates these transitions.

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. <p>In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. <p>A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

A queimadura de 3º grau é um dos maiores traumas a que um ser humano pode ser submetido e em geral são as lesões mais frequentes na população mundial, tratando-se de um importante problema de saúde pública. Pensando neste problema, observou-se que a utilização da membrana amniótica (MA) pode ser o melhor tratamento que esses pacientes possam receber. Ela atua em benefício da epitelização, facilita a migração e a adesão das células epiteliais basais, a matriz estromal possui inibidores de proteases que previne a apoptose e restaura o fenótipo epitelial, antibacteriana do córion e âmnion, além de proteger a ferida e atuar na redução da dor. A descelularização, trata-se da retirada de todas as células e núcleo da MA utilizada, como forma de se evitar qualquer tipo de histoincompatibilidade. O presente trabalho teve como objetivo avaliar a eficácia do uso da membrana amniótica descelularizada (MAD) no tratamento de queimaduras de 3o grau em humanos, testada em ratos da linhagem Wistar. Trata-se de uma pesquisa descritiva, a qual investiga fundamentalmente a especificação e a disposição de dados acerca dos resultados obtidos a partir de um processo regenerativo de queimaduras utilizando-se a MAD. A amostra foi constituída de constituída por 20 ratos machos adultos da linhagem Wistar, dividida em 2 grupos (n=10): grupo controle (GC)–sem MA e grupo transplantado–com MA (TMAD). Os dois grupos foram submetidos a um processo padronizado de queimadura térmica, sendo retiradas duas amostras de tecido para análise, no 14º dia e no 30º dia. As amostras foram analisadas com o emprego de técnicas anatomopatológicas onde foram montadas em lâminas fragmentos de MA corados com solução Hematoxilina Eosina (HE) e, para análise histomorfométrica,as lâminas foram coradas com picro sirius red sob luz polarizada para verificação da descelularização e quantificação de colágeno do tipo I, II e III. As imagens foram quantificadas utilizando o programa Image Pro Plus®, versão 5.0 para Windows®. Os dados estatísticos foram analisados utilizando o programa computacional SSPS v.21.0. Observou-se um aumento significativo da quantificação de colágenos do tipo III no 14º dia no grupo TMAD em relação ao GC, do colágeno do tipo II no 14º e 30º dia comparado ao GC e do colágeno tipo I no 14º e 30º dia, mostrando que a utilização da MA foi eficaz no grupo TMAD. Concluiu-se que a MAD, aplicada topicamente, demonstrou eficácia no processo cicatricial em queimaduras de 3º grau. Esperava-se que a mesma promovesse a aceleração de cicatrização dos ferimentos, mas, o que se observou ao término do trabalho é que a MAD não só promoveu a cicatrização como foi mais eficiente no processo de regeneração dos tecidos lesados. / 3rd degree of burns are one of the greatest injury that a human being can be submitted and generally are the most frequent injuries in the world population, in the case of a major public health problem. Thinking this problem, it has been observed that the use of amniotic membrane (AM) may be the best treatment these patients can receive. It acts on behalf of epithelialization, facilitates migration and adhesion of basal epithelial cells, stromal matrix has protease inhibitors which prevent apoptosis and restores epithelial, antibacterial phenotype of the chorion and amnion, and protect the wound and act on reducing pain. The decellularization, it is the withdrawal of all cells and core AM used as a way to avoid any kind of histoincompatibility. This study aimed to evaluate the efficacy of the decellularized amniotic membrane (DAM) in the treatment of third degree burns in humans, tested in Wistar rats. It is a descriptive research which investigates mainly the specification and the arrangement of data on the results obtained from a regenerative process burns using DAM. The sample consisted of consisted of 20 Wistar adult male rats, divided into 2 groups (n = 10): control group (CG) - without MA, and group transplanted - with AM (TDAM). The two groups were submitted to a standard process of thermal burn, two samples being removed tissue for analysis on day 14 and day 30. Samples were analyzed with the use of pathological techniques which were mounted on slides DNA fragments stained with solution hematoxylin eosin (HE) for histomorphometric analysis, the slides were stained with picrosirius red under polarized light for verification of decellularization and quantifying collagen type I, II and III. Images were quantified using Image Pro Plus ®, version 5.0 for Windows. Statistical data were analyzed using the computer program SPSS v.21.0. There was a significant increase in type III collagen quantification on the 14th day in TDAM group compared to CG, type II collagen in the 14th and 30th day compared to the CG and type I collagen in the 14th and 30th day, showing that the use of MA were effective in TDAM group. It was concluded that the DAM, applied topically, has shown efficacy in the healing process in 3rd degree burns. It was expected that it would promote accelerated wound healing but which was observed at the end of work that MAD is not only promoted as the healing was more efficient the regeneration process of the damaged tissues.

Orientador: Eliana Aparecida de Resende Duek / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-08-09T22:07:51Z (GMT). No. of bitstreams: 1 Barauna_GrazielledosSantos_M.pdf: 1217156 bytes, checksum: ded7e97c7818fe0c3d87cb97d8c8f8aa (MD5) Previous issue date: 2007 / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

Les protéines associées aux membranes sont d'une importance cruciale pour la cellule. Cependant, en raison d'une plus grande difficulté de manipulation, les données biochimiques les concernant sont très lacunaires, notamment au point de vue de la formation de complexes entre ces protéines.<p><p>L'objectif global de notre travail consiste à combler ces lacunes et à préciser les interactions entre protéines membranaires chez la levure Saccharomyces cerevisiae et plus précisément, entre les transporteurs. Nous avons commencé notre travail par l'étude d'un jeu de données d'interactions à grande échelle entre toutes les perméases détectées par une méthode de double hybride spécialement adaptée aux protéines insolubles (split ubiquitin). Premièrement, la qualité des données a été estimée en étudiant le comportement global des données et des témoins négatifs et positifs. Les données ont ensuite été standardisées et filtrées de façon à ne conserver que les plus significatives. Ces interactions ont ensuite été étudiées en les modélisant dans un réseau d'interactions que nous avons étudié par des techniques issues de la théorie des graphes. Après une évaluation systématique de différentes méthodes de clustering, nous avons notamment recherché au sein du réseau des groupes de protéines densément interconnectées et de fonctions similaires qui correspondraient éventuellement à des complexes protéiques. Les résultats révélés par l'étude du réseau expérimental se sont révélés assez décevants. En effet, même si nous avons pu retrouver certaines interactions déjà décrites, un bon nombre des interactions filtrées semblait n'avoir aucune réalité biologique et nous n'avons pu retrouver que très peu de modules de protéines de fonction semblable hautement inter-connectées. Parmi ceux-ci, il est apparu que les transporteurs d'acides aminés semblaient interagir entre eux.<p><p>L'approche expérimentale n'ayant eu que peu de succès, nous l'avons contournée en utilisant des méthodes de génomique comparative d'inférence d'interactions fonctionnelles. Dans un premier temps, malgré une évaluation rigoureuse, l'étude des profils phylogénétiques (la prédiction d'interactions fonctionnelles en étudiant la corrééélation des profils de présence - absence des gènes dans un ensemble de génomes), n'a produit que des résultats mitigés car les perméases semblent très peu conservées dès lors que l'on considère d'autres organismes que les \ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The multidrug transporter LmrP, member of the major facilitator superfamily (MFS), confers L. lactis and recombinant E. coli cells resistance to an array of cytotoxic compounds including antibiotics. LmrP mediates drug extrusion from the plasma membrane by an electrogenic proton/drug exchange reaction, whereby a positively charged substrate may move towards the external medium in exchange for two or more protons moving towards the cytoplasm. Recent studies have suggested that MFS transporters require phosphatidylethanolamine (PE) for function and proper topology. However, the specificity of the PE requirement, as well as the contribution of the electrochemical gradient (the driving force of the substrate transport) to this lipid requirement was not addressed. Here we report a new approach for addressing PE specific requirement for the function and the structure of membranes transporters. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by phosphatidylcholine (PC) ( including energy transfer between the protein tryptophan residues and the lipid headgroups). We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.

Indiana University-Purdue University Indianapolis (IUPUI) / Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distr ibuted to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. T his research employs model membranes of well - defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega - 3 (n - 3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids . T he m olecular organization of 1 -[ 2 H 31 ]palmitoyl -2- eicosapentaenoylphosphatidylcholine (PEPC - d 31 ) and 1 -[ 2 H 31 ]palmitoyl -2- docosahexaenoylphosphatidylcholine (PDPC -d 31 ) in membran es with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid - state 2 H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n - 3 PUFAs found in fish oil, while PEPC -d 31 and PDPC -d 31 are phospholipids containing the respective PUFAs at the sn - 2 position and a perdeuterated palmitic acid a t the sn - 1 position . Analysis of s pectra recorded as a function of temperature indicate s that in both cases, formation of PUFA - rich (less ordered) and SM - rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infil trate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%) . The implication is that n - 3 PUFA s can incorporate into lipid rafts, which are domains enriched in SM and chol in the plasma membrane, and potentially disrupt the activity of signaling proteins that reside therein. DHA, furthermore, may be the more potent component of fish oil. PUFA - chol interactions were also examined through affinity measurements. A novel method utilizing electron paramagnetic resonance (EPR) was develope d, to monitor the partitioning of a spin - labeled analog of chol , 3β - doxyl - 5α - cholestane (chlstn), between large unilamellar vesicles (LUVs) and met hyl - β - cyclodextrin (mβCD). The EPR spectra for chlstn in the two environments are distinguishable due to the substantial differences in tumbling rates , allowing the population distribution ratio to be determined by spectral simulation. Advantages of this approach include speed of implementation and a vo idance of potential artifact s associated with physical separation of LUV and mβCD . Additionally, in a check of the method, t he relative partition coefficients between lipids measured for the spin label analog agree with values obtained for chol by isothermal titration calorimetry (ITC). Results from LUV with different composition confirmed a hierarchy of decreased sterol affinity for phospholipids with increasing acyl chain unsaturation , PDPC possessing half the affinity of the corresponding monounsaturated phospholipid. Taken together, the results of these studies on model membranes demonstrate the potential for PUFA - driven alteration of the architecture of biomembranes, a mechanism through which human health may be impacted.