The objective of my thesis is to explore the use of the conducting polymer, polypyrrole, in neural applications. In addition a supplementary aspect of dissertation will involves understanding the effects of external stimuli on nervous system cells, with the ultimate goal of designing therapeutic systems for nerve regeneration. In normal development and peripheral nervous system repair, nerves encounter naturally occurring chemical, physical, and electrical stimuli. Polypyrrole (PPy) has attracted much attention for use in numerous biomedical applications as it presents chemical, physical and electrical stimuli. In addition, PPy is particularly exciting because the extent by which chemical, physical, and electrical cues are presented to the injured nerve can be easily tailored. Thus, conducting polymers are excellent scaffolds for the exploration of how the cellular components of the nervous system (i.e., Schwann cells and neurons) interact with chemical, topographical, and electrical stimuli. This dissertation covers three main objectives and is supplemented by two additional topics. The two additional topics explore the effect stimuli present on the conducting polymer PPy have on neural interfaces. These fundamental studies use computational modeling to gain a better understanding of cellular motility on substrates containing different stimuli. Both topics are covered in the appendices of this dissertation. With regards to the three main objectives, I first characterized and optimized the electrochemical synthesis of the conducting polymer, PPy, for Schwann cell biocompatibility. Next, I investigated the effect the application of electrical cues through PPy has on Schwann cell migration. In addition to investigating the effect of the direct electrical current on Schwann cells I also considered the effect that electrical stimulation provided by PPy has on protein adsorption. Finally, I developed a hybrid PPy material that will provide advantageous properties for neural interfaces. Specifically, I describe the development of a polypyrrole:poly-(lactic-co-glycolic) acid blend for neural applications. In summary the three specific objectives covered in my thesis are: Specific Aim 1: Characterize and optimize the electrochemical synthesis of the conducting polymer, polypyrrole, for Schwann cell biocompatibility Specific Aim 2: Determine the effect of electrical stimulation on Schwann cell migration Specific Aim 3: Develop polypyrrole:poly-(lactic-co-glyolic) acid blends for neural engineering applications. / text

Fas Ligand (FasL) binds to the Fas receptor to induce apoptosis or activate other signaling pathways. FasL can also transduce "reverse signals" and thus participate in bidirectional signaling. The FasL intracellular domain contains consensus sequences for phosphorylation and a proline rich protein interaction domain. This latter region of FasL has previously been implicated in FasL reverse signaling and regulation of FasL surface expression. In this report, we sought to identify novel FasL interacting proteins to help understand signaling through and trafficking of this death factor. Using mass spectrometry, we identified sorting nexin 18, adaptin beta, Grb2, PACSIN2 and PACSIN3 as FasL interacting proteins. RNAi mediated knockdown of Grb2 significantly reduced the surface expression of FasL and increased its expression intracellularly. Our data show that Grb2 controls the subcellular localization of FasL. All other proteins identified in our screen could be classified as trafficking-associated proteins, highlighting the complex regulation of the surface expression of this death factor.

Hohenheim, Univ., Diss., 2007.

In the peripheral nervous system (PNS), Schwann cells ensheathe and myelinate axons to promote saltatory conduction of nerve impulses. Close interactions between Schwann cells and axons, and Schwann cells and the basal lamina are essential for the regulation of Schwann cell development and function. Myelinating Schwann cells are highly polarized radially and longitudinally for specifying distinct domains in the axon, which is required for fast action potential propagation. In addition, the Schwann cell cytoplasm is organized into discrete compartments, called Cajal bands, which contain different dystrophin-glycoprotein complexes that are believed to segregate the Schwann cell plasma membrane into appositions between the outer surface of the myelin sheath and the cytoplasmic face of the Schwann cell plasma membrane. Periaxin is expressed in myelinating Schwann cells, and homodimerizes at its PDZ domain to form a transmembrane complex with dystrophin-related protein 2 (DRP2) and dystroglycan. This PDG complex is concentrated at the appositions, and is essential for myelin sheath maintenance and stability in the mature PNS. In mice lacking Periaxin, an intact myelin sheath is formed but subsequently becomes unstable. Periaxin-null Schwann cells are also shorter, which has been proposed to result in a reduction in nerve conduction velocity. This thesis is a study of how Periaxin PDZ domain dimerization contributes to the regulation of PDG complex stability, apposition maintenance, Schwann cell internodal distance and myelin stability. I have studied the function of Periaxin by generating a conditional mutant mouse that lacks the PDZ domain, which is predicted to abrogate dimerization. In these mutants, DRP2 is severely depleted and appositions containing DRP2 fail to form. Mutant Schwann cells also have disrupted Cajal bands and shorter internodal lengths. In the mature peripheral nerves, mutant mice display a peripheral neuropathy characterized by hypermyelination with focally folded myelin. Nerve conduction velocity, motor coordination and sensory function were also studied in these mutant mice. Taken together, these data suggest that dimerization of the Periaxin PDZ domain is required for the stabilization of the PDG and appositions, and regulation of Schwann cell elongation and myelin maintenance. By analyzing a tamoxifen-inducible conditional mouse lacking Periaxin’s PDZ domain in mature myelinating Schwann cells, this work also shows that Periaxin dimerization is essential for maintaining Schwann cell compartmentalization and myelin stability in adult nerves. Finally, studies of single amino acid mutations of the Periaxin PDZ reveal that subtle changes in the structure of the PDZ domain can abrogate dimerization,and a possible mechanism for PDZ-PDZ homodimerization of Periaxin is proposed.

No description available.

No description available.

Würzburg, Univ., Diss., 2009. / Zsfassung in dt. Sprache.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-12-27T17:57:17Z No. of bitstreams: 1 luciana_s_rodrigues_ioc_bcm_0026_2010.pdf: 8319063 bytes, checksum: 23ce271789168f4d9723b9a7df6ffe11 (MD5) / Made available in DSpace on 2012-12-27T17:57:17Z (GMT). No. of bitstreams: 1 luciana_s_rodrigues_ioc_bcm_0026_2010.pdf: 8319063 bytes, checksum: 23ce271789168f4d9723b9a7df6ffe11 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A lesão neural é uma das principais consequências da hanseníase e responsável pela instal ação de deformidades e incapacidades físicas, al ém de contribuir para o esti gma da doença. O dano ao nervo é exacerbado com o desenvolvimento de episódi os reaci onais (Ti po I e Ti po II) e está correlaci onado à resposta imunol ógi ca desenvolvida pel o indivíduo, contra o Mycobacterium leprae – agente eti ol ógico da hanseníase que apresenta especi al tropismo por macrófagos e células de Schwann (CS) nos nervos periféri cos. Os fatores de crescimento semelhante à Insulina (IGFs) são hormôni os pept ídi cos implicados no metabolismo, indução de proliferação, inibição de apoptose e diferenciação de diferentes ti pos cel ulares. Evi dências da li teratura apontam também propri edades imunomoduladoras e anti -inflamatóri as do IGF-I. O objetivo do presente estudo vi sa a invest i gação da parti cipação do si stema IGF na infecção pel o M. leprae. Ini cialmente, verificamos o efei to anti -apoptóti co da bactéri a sobre CS humanas primárias e da linhagem ST88-14 cul t ivadas em condições livres de soro pel a inibição da at ivação de caspase-3. Demonstramos, ainda, através de ensai os de imunoci toquímica, que o bacil o é capaz de induzir a proliferação da CS, tal efei to provavelmente mediado pel a indução de IGF-I, confi rmado pel a técnica de RT-PCR quant i tativo e pel a detecção da proteína em sobrenadantes de cul tura através de ensai o imunoenzimát i co. Na segunda etapa do trabalho, avaliamos a part i cipação do IGF-I ci rculante na evol ução natural da hanseníase. Utilizando ELISA quimi oluminescente, quantificamos os níveis de IGF-I, da principal proteína ligadora de IGF (IGFBP-3) e TNF-a no soro de indivíduos sadi os e pacientes que desenvolveram ou não quadros reacionais ao l ongo do tratamento. No caso dos pacientes reaci onais, a dosagem de IGF-I, IGFBP-3 e TNF-a fo i realizada em duas etapas: i ) no momento do diagnóstico e ii ) durante o aparecimento da reação, antes do tratamento específico. Ini cialmente, numa comparação entre paci entes que não desenvolveram reação, verificamos que 81% e 72% dos paci entes lepromatosos (LL) apresentaram níveis de IGF-I e IGFBP-3, respectivamente, abaixo do normal por i dade, diferentemente dos paci entes com outras formas clínicas. Dentre os pacientes reaci onais, 93% e 86% do grupo BL também apresentou níveis de IGF-I e IGFBP-3, respectivamente, abaixo do normal por i dade, diferentemente do grupo BL não-reaci onal , que apresentou níveis de IGFs similares aos indivíduos sadi os. Durante o desenvolvimento dos epi sódi os reaci onais, houve uma queda dos níveis de IGF-I, IGFBP-3 e da rel ação IGF/TNF-a no grupo LL com reação ti po II. Já no grupo de paci entes BL, observamos um aumento dos níveis de IGF-I e IGFBP-3, como uma tentativa de alcançar os níveis normais. Nossos dados sugerem a xv parti cipação destes fatores de crescimento endócrinos na ínt ima relação entre bacil o e a CS, como uma estratégi a de obtenção e manutenção de um nicho favorável de mul t iplicação e, ainda, os revelam como potenci ais candidatos a bi omarcadores dos episódi os reaci onais na hanseníase. / Neural injury is a maj or consequence of l eprosy and responsible for the disabili t ies installat i on, beyond to contribute to the st i gma of the disease. The nerve damage is exacerbated by the devel opment or l eprosy react i ons (Type I and Type II) and is correl ated to the immune response against Mycobacterium leprae – et i ol ogi c agent of leprosy that has especial tropism for macrophages and Schwann cells (SC) in peri pheral nerves. Insulin-like growth factors (IGFs) are pept i de hormones involved in metabolism, proliferati on induct i on, apoptosis inhibi t i on and cell different i at i on. Evidence from the li t erature also indicate immunomodulatory and ant i -inflammatory properti es of IGF-I. The aim of this study is to investi gate the involvement of IGF system in the M. leprae infect i on. Ini t ially, we verified the ant i -apoptoti c effect of the bacteria on human primary CS and ST88-14 lineage growing in serum-f ree condi t i ons by inhibi t ing caspase-3 activat i on. It was also demonstrated by immunocytochemistry, that the bacill us is able to induce the SC proliferat i on, this effect i s probably mediated by induct i on of IGF-I, as verified by quant i tative RT-PCR and confirmed by protein detecti on supernatants using immunoenzimat i c assay (ELISA). On the second phase we evaluate the parti cipat i on of ci rculating IGFs in the natural course of leprosy. Through chemi ol uminescent ELISA, we quantified the IGF-I, the main IGF binding protein (IGFBP-3) and TNF-a serum levels in heal t hy individuals and pat i ents who devel oped or not reacti onal states during the treatment. In the case of react i onal pat i ents, the IGF-I, IGFBP-3 and TNF-a was performed in two steps: i ) at the diagnosis of leprosy and ii) during react i onal episode, pri or to specific treatment. Ini t i ally, a comparison of nonreact i onal pat i ents, we found that 81% and 72% of lepromatous l eprosy (LL) showed IGF-I and IGFBP-3 levels, respectively, bel ow normal for age, unlike pat i ents wi th other clinical forms. Among the reacti onal pat i ents, 93% and 86% of BL group also showed IGF-I and IGFBP-3 levels, respectively, bel ow normal for age, unlike the nonreact i onal BL group, wi ch showed similar levels of IGFs to heal thy individuals. During the devel opment of reacti onal episodes, there was a decrease in the levels of IGF-I, IGFBP-3 and the IGF/TNF-a rat i o in the LL group wi t h type II reacti on. In the BL group undergoing type II reacti on, the IGF-I and IGFBP-e levels increased, as an attempt to reach normal levels. Our data suggest the involvement of these growth factors in the relat i onship between bacilli and CS as a strategy for obtaining and maintaining a favorable niche for mul t iplicat i on, and also reveal the IGFs as potencial candidates for bi omarkers of react i onal episodes in leprosy.

Abstract The function of the vertebrate nervous system is based on the rapid and accurate transmission of electrical impulses. The myelin sheath is a lipid-rich membrane that envelops the axon, preventing the leakage of the nervous impulse to the environment. Myelin is formed when the plasma membrane of a myelinating glial cell differentiates and wraps around an axon. The compaction of myelin leads to the extrusion of most of the glial cell cytoplasm from the structure. Both the compact and noncompact regions of myelin carry distinct subsets of proteins. The myelin-associated glycoprotein (MAG) is present in noncompact myelin. It is a cell adhesion molecule expressed only by myelinating glial cells. Two isoforms of MAG, S- and L-MAG, exist, and these forms differ from each other only by their cytoplasmic domains. Until now, little information has been available on the differences between the MAG isoforms. This study was carried out in order to gain information on the cytoplasmic domains of S- and L-MAG. Significant differences were observed in the properties of the MAG cytoplasmic domains. An interaction between the L-MAG cytoplasmic domain and the S100b protein was characterised, and a role for this interaction was found in the regulation of L-MAG phosphorylation. Evidence was also obtained for the dimerisation of the L-MAG cytoplasmic domain. The S-MAG cytoplasmic domain bound zinc, which induced a change in the surface properties of the protein. The S-MAG cytoplasmic domain was also found to interact directly with tubulin, the core component of microtubules. In conclusion, this study has brought information on the functions of the MAG cytoplasmic domains. The results are complementary with ealier hypotheses on the roles of the MAG isoforms in myelinating glia. While the properties of L-MAG suggest a role as a signaling molecule, a dynamic structural role for S-MAG during myelin formation and maintenance can be envisaged.

The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. Historically, SMA has been characterised as a disease primarily affecting lower motor neurons. However, recent breakthroughs have revealed defects in other non-neuronal cells and tissues. In vivo analysis of peripheral nerve showed defects in Schwann cells, manifesting as abnormal myelination and delayed maturation of axo-glia interactions. The experiments in this thesis were designed to build on these observations and examine whether Schwann cell defects are intrinsic and occur as a primary result of low levels of SMN in that cell type, or rather represent a secondary consequence of pathology in neighbouring motor neurons. I initially developed a protocol to allow isolation of high-yields of purified, myelination-competent Schwann cells from ‘Taiwanese’ SMA mice. SMA-derived Schwann cells had significantly reduced SMN levels and failed to respond normally to differentiation cues. Increasing SMN levels restored myelin protein expression in Schwann cells from SMA mice. Perturbations in expression of key myelin proteins were likely due to failure of protein translation and/or stability rather than transcriptional defects. Co-cultures of healthy neurons with SMA Schwann cells revealed a significant reduction in myelination compared to cultures where wild-type Schwann cells were used. The presence of SMA Schwann cells also disrupted neurite stability. Perturbations in the expression of key extracellular matrix proteins, such as laminin α2, in SMA-derived Schwann cells suggests that Schwann cells were influencing neurite stability by modulating the composition of the extracellular matrix. Previous studies have demonstrated that low levels of SMN lead to disruption of ubiquitin homeostasis and decreased expression of ubiquitin-like modifier activating enzyme (UBA1) in the neuromuscular system, driving neuromuscular pathology via a beta-catenin dependent pathway. Label-free proteomics analysis of SMA and control Schwann cells identified 195 proteins with modified expression profiles. Bioinformatic analysis of these proteins using Ingenuity Pathway Analysis (IPA) software confirmed that major disruption of protein ubiquitination pathways was also present in Schwann cells from SMA mice. Immunolabeling and proteomics data both revealed that UBA1 levels were significantly reduced in SMA-derived Schwann cells. However, loss of UBA1 in Schwann cells did not lead to downstream modifications in beta-catenin pathways. Pharmacological inhibition of UBA1 in healthy Schwann cells was sufficient to induce defects in myelin protein expression, suggesting that UBA1 defects contribute directly to Schwann cell disruption in SMA. I conclude that low levels of SMN induce intrinsic defects in Schwann cells, mediated at least in part through disruption to ubiquitination pathways.