The synapse, the minimal element required for interneuronal communication in the nervous sytems, is a structure with a great deal of plasticity, capable of undergoing changes that alter transmission strength, and even forming new connections. This property has great implications for a number of processes, including circuit formation and learning and memory. However, the proteins behind this synaptic plasticity are still not fully understood. To uncover and characterize the proteins that regulate the plastic nature of the synapse, I turned to the Drosophilalarval neuromuscular junction (NMJ), a powerful and accessible model system. I began by examining synaptic cell adhesion, as Cell Adhesion Molecules (CAMs) have long been implicated in synaptic outgrowth as well as learning and memory. CAMs have traditionally been thought of as molecules that mediate cell adhesion between the pre- and postsynaptic membrane. However, through the course of the studies presented here I demonstrate a CAM function that goes beyond simple cell adhesion, acting as a receptor that transduces adhesive signals to the intracellular space. In particular, I have demonstrated a role for the Drosophila CAM, Fasciclin II(FasII), in a signaling complex involving the Amyloid Precursor Protein-Like (APPL) and the Drosophila homolog of X11/MINT/Lin-10 (dX11). Further results show that deletion of either APPL or dX11 inhibits the FasII mediated outgrowth. These studies show that during NMJ expansion the transinteraction between FasII molecules in the pre- and postsynaptic membrane results in the recruitment of APPL and dX11 to the presynaptic cell surface, and the initiation of a signaling cascade that leads to bouton outgrowth. The next question addressed here was regarding the cytoskeletal changes that must occur during synapse remodeling. In particular I centered on the evolutionarily conserved cell polarity complex aPKC-Par3-Par6, which is know to regulate axon growth, the cell cytoskeleton during polarized cell division, and learning and memory. To understand the role of the cytoskeleton during NMJ expansion, I examined the organization of microtubules and actin during this process. Further, I identified atypical protein kinase C (aPKC) as a regulator of microtubule dynamics. I found that aPKC is required for regulating the degree of stabilization of synaptic microtubules. This stabilization requires the Microtubule Associated Protein-1B (MAP1B) homolog Futsch, which I demonstrated was required for aPKC to associate with and stabilize the microtubule cytoskeleton. The process of synaptic expansion not only requires modifications to the presynapse, but to the postsynapse as well. Previous work demonstrates that levels of the scaffolding proteins DrosophilaMembrane Associated Guanlyate Kinase (MAGUK) protein Discs-large (DLG), as well as the vertebrate homolog Postsynaptic Density-95 (PSD-95), which are concentrated at synapses, determine the size of postsynaptic membranes. To identify the underlying mechanisms of the regulation of postsynaptic size, we performed a yeast two hybrid screen, searching for DLG interacting proteins. We found a novel interaction between DLG, and a t-SNARE, GUK-interacting Syntaxin (Gtaxin; GTX), and went on to demonstrate that this interaction is required for proper postsynaptic membrane addition. Strong hypomorphic mutations in either dlg or gtx show a dramatic reduction in postsynaptic expansion. Overexpression of DLG produces an increase of synaptic GTX, as well as an increase in postsynaptic size, and an increased formation of GTX positive SNARE complexes. Taken together, these observations suggest that the MAGUK DLG regulates postsynaptic membrane addition by modulating the formation of a SNARE complex of the t-SNARE Gtaxin, and by targeting GTX to sites of postsynaptic membrane addition. In summary, the studies performed in this thesis probe a trans-synaptic adhesion based signaling complex required for presynaptic expansion, a specific pathway for dynamic microtubule stabilization required for pre- and postsynaptic expansion, and how a scaffolding protein regulates postsynaptic membrane expansion. These processes are all interconnected to maintain the efficacy of the synapse. The studies conducted revealed important information about how these processes are accomplished, and constitute an important step to elucidate the mechanisms by which synapse plasticity occurs at the level of single synaptic terminals.

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A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems. Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma, being an embryonal tumor, show loss of function of genes controlling differentiation and apoptosis. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments. A common model for human neuroblastoma is the SH-SY5Y cell line resembling immature neuroblasts that can be differentiated in vitro with several agents including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the vitamin A-derivative all-trans retinoic acid. Here, the effect of ellagic acid on proliferation, cell detachment and apoptosis in non-differentiated and in vitro-differentiated SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Apoptosis was partly mediated by the intrinsic pathway. 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid both induced morphological differentiation, while only the latter induced G0/G1-arrest. Single-cell analysis revealed that 12-O-tetradecanoylphorbol-13-acetate-treated cells continued cycling during neuritogenesis while these two read-outs were mutually exclusive in all-trans retinoic acid-treated cells. 12-O-tetradecanoylphorbol-13-acetate- and especially all-trans retinoic acid-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis. / <p>Artikel 4 ("Altered sensitivity...") ingick som manuskript i avhandlingen, nu publicerad.</p>

Talin is a multifunctional cytoskeletal protein that plays a critical role in linking the actin cytoskeleton to the integrin family of transmembrane cell adhesion receptors. Two distinct integrin binding sites have been identified in talin, one present in the globular head domain (IBS1) and involved in integrin activation, and a second (IBS2), that has been delineated to a 130 residue fragment of the talin rod domain, but whose functional role is still elusive (Tremuth et al.,2004). The objective of the present study was to define the minimal structure of talin IBS2 and to investigate its functional role in the integrin-cytoskeleton connection. In the first part of this study, we used a combination of three different experimental approaches to define the minimal structure of talin IBS2: 1) an in silico bioinformatics approach to analyse sequence conservation of talin IBS2, 2) an in vivo cell biology approach to study the subcellular localization of recombinant talin fragments covering IBS2 in CHOáIIbâ3 cells, and 3) an in vitro biochemical approach consisting in protein overlay, pull down and Surface Plasmon Resonance (SPR) assays, to study the direct interaction between talin IBS2 and the integrin â3 subunit. We delineated IBS2 to a single amphipathic á-helical repeat of 23 residues within the talin rod domain. We further provided evidence that a two amino acid mutation(L2094I2095/AA) was sufficient to inactivate the IBS2 site, due to a disruption of the á helix structure, as demonstrated by infrared spectroscopy. In addition, we identified 2 lysine residues (K2085, K2089) exposed on the solvent face of á helix 50, which are directly involved in the talin IBS2-integrin interaction. In the second part of this study, we investigated the functional role of talin IBS2 in spreading defective talin (-/-) cells and showed that in contrast to full-length wild type talin, an IBS2 LI/AA mutant talin was unable to fully rescue the spread phenotype of these cells. These results provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the actin cytoskeleton.

The neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily and it mediates many adhesive interactions during brain development. Mutations in the L1 gene are associated with a spectrum of X-linked neurological disorders known as CRASH or L1 syndrome. The objective of this thesis was to use the zebrafish model to investigate the molecular mechanisms of L1 functions and the pathological effects of its mutations. Zebrafish has two L1 homologs, L1.1 and L1.2. Inhibition of L1.1 expression by antisense morpholino oligonucleotides resulted in phenotypes that showed resemblances to L1 patients. However, knockdown of L1.2 expression did not result in notable neural defects. Furthermore, analysis of the expression pattern of L1.1 has led to the discovery of a novel soluble L1.1 isoform, L1.1s. L1.1s is an alternatively spliced form of L1.1, consisting of the first four Ig-like domains and thus a soluble secreted protein. L1.1 morphants exhibited disorganized brain structures with many having an enlarged fourth/hindbrain ventricle. Further characterization revealed aberrations in ventricular polarity, cell patterning and proliferation and helped differentiate the functions of L1.1 and L1.1s. While L1.1 plays a pivotal role in axonal outgrowth and guidance, L1.1s is crucial to brain ventricle formation. Significantly, L1.1s mRNA rescued many anomalies in the morphant brain, but not the trunk phenotypes. Receptor analysis confirmed that L1.1 undergoes heterophilic interactions with neuropilin-1a (Nrp1a). Peptide inhibition studies demonstrated further the involvement of L1.1s in neuroepithelial cell migration during ventricle formation. In the spinal cord, spinal primary motoneurons expressed exclusively the full-length L1.1, and abnormalities in axonal projections of morphants could be rescued only by L1.1 mRNA. Further studies showed that a novel interaction between the Ig3 domain of L1.1 and Unplugged, the zebrafish muscle specific kinase (MuSK), is crucial to motor axonal growth. Together, these results demonstrate that the different parts of L1.1 contribute to the diverse functions of L1.1 in neural development.

Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

Cellular adhesion molecules (CAMs) and selectins are cell-surface proteins involved in the binding of cells to the vascular endothelium. Elevated levels of sCAMs and soluble E-selectin (sE-selectin) have been reported in patients with periodontitis. The aim of this study was to determine if periodontal scaling and root planing would influence the serum concentration of sICAM-1, sVCAM-1, and sE-selectin. Twenty-one subjects with chronic periodontitis received scaling and root planing in conjunction with blood serum sample analysis using enzyme-linked immunosorbent assay (ELISA), to determine if periodontal instrumentation results in changes in serum concentrations of sICAM-1, sVCAM-1, and sE-selectin. No change was observed in serum concentration of sICAM-1 or sVCAM-1. However, in a subset of 17 patients a statistically significant change in serum sE-selectin was observed (P < 0.05). This suggests that there is a decrease in endothelial activation following periodontal treatment.

The process of electrospinning has given the field of tissue engineering insight into many aspects of tissue engineered scaffolds, including how factors such as fiber diameter and porosity are affected by polymer concentration. However, the affects of fiber alignment upon the material properties of electrospun scaffolds remains unclear. The purpose of this study is to determine how the material properties of electrospun gelatin scaffolds are affected by changes in fiber alignment and starting gelatin concentration. Gelatin scaffolds, with starting concentrations of 80, 100, and 130mg/m1, were electrospun onto a target mandrel rotating at various speeds. Samples of each scaffold were taken parallel and perpendicular to the axis of mandrel rotation. Fast Fourier Transform (FFT) analysis was performed on these samples, to determine how fiber alignment is affected by starting polymer concentration and the rotational speed of the target mandrel. Mechanical tests were aiso performed on these samples. Results were analyzed by Three-way ANOVA. It was determined that starting gelatin concentration, mandrel speed, and direction of fiber alignment interact together to produce effects on the mechanical properties of electrospun gelatin scaffolds.

Cryopreservation in suspension is commonplace for a variety of cell types. However, cryopreservation of adherent cells has achieved limited success. This research aimed to cryopreserve adherent nerve cell networks in vitro in a manner that preserved network morphology and physiology. Successful implementation would enable long term storage of adherent neuronal networks on microelectrode arrays and on-demand access for use in pharmacological and toxicological testing. Based upon morphological assessments, excellent post-thaw preservation was obtained and post-thaw cultures survived in a transitional medium for up to 3.5 hours. However, transitions to native culture medium post-thaw presented difficulties, ultimately resulting in necrosis. A discussion of methods to supplement the current research and increase post-thaw viability is included in the thesis.