No description available.

Polysialylated NCAM (polySia-NCAM) is re-expressed in a number of tumours, including small cell lung carcinoma and neuroblastoma and is strongly associated with aggressive, invasive and metastatic tumours in the clinic. SiRNA knockdown of the polysialyltransferases (polySTs), the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration. PolySia-NCAM is thus a highly attractive novel therapeutic target. A library of potential polyST inhibitors has been synthesised, using substrate-based design and computational chemistry. Compounds synthesised include N-acylmannosamine analogues, thio-linked CMP-sialic acid analogues, N-acyl modified sialic acids and compounds incorporating elements of both approaches. Novel methodology development in the synthesis of many of the compounds is described, notably a novel route to N-acyl sialosides. In addition, compounds identified from in silico screening were considered. Routes to synthesis and isolation of analogues of biologically active compounds are described. Using an enzyme assay, compounds were evaluated for their ability to reduce polySia synthesis through polyST inhibition. Effects of agents on polySia expression in cells, and the ability of compounds to reduce cell migration in vitro was studied using a wound healing ‘scratch assay’. The data from these experiments revealed a number of potent modulators of polySia assembly and their efficacy in reducing cell migration, as well as the limits of the biosynthetic pathway to accept unnatural sialic acid precursors. This is the first example of polyST inhibition modulating tumour cell migration, and points to the potential of the polysialyltransferases as a therapeutic target in metastatic tumours.

The alpha-, beta-, and gamma-Protocadherins (gamma-Pcdhs) are cadherin superfamily adhesion molecules encoded by clustered gene families. The 22 gamma-Pcdhs are combinatorially expressed in the central nervous system (CNS) by neurons and astrocytes, and play critical roles in synaptogenesis, dendrite arborization, and the survival of subsets of neurons. The gamma-Pcdhs promiscuously form cis-multimers that interact strictly homophilically in trans (Molumby et al., 2016; Schreiner and Weiner, 2010); the alpha- and beta-Pcdhs were subsequently shown to interact in a similar homophilic manner (Rubinstein et al., 2015; Thu et al., 2014). The Pcdh gene clusters thus have the potential to generate millions of distinct adhesive interfaces, providing CNS cells with molecular identities that shape neuronal morphology. We demonstrated previously that, in mice lacking the gamma-Pcdhs in the cerebral cortex, pyramidal neurons exhibit severely reduced dendrite arborization (Garrett et al., 2012a). This, combined with many studies of gamma-Pcdh interactions in vitro, suggests that homophilic, adhesive gamma-Pcdh interactions between neurons, and between neurons and glia, provide a positive signal for dendrite growth. However, in retinal starburst amacrine cells and cerebellar Purkinje cells, loss of the gamma-Pcdhs resulted in aberrant dendrite fasciculation and self-crossing (Lefebvre et al., 2012), suggesting that these molecules can mediate repulsive self-avoidance between a neuron’s own dendrites. In Chapter I of this thesis I utilized transgenic mice to manipulate expression in vivo, to show that the complexity of a cortical neuron’s dendritic arbor is determined by homophilic gamma-Pcdh isoform matching with other cells. Expression of the same single isoform in a neuron can result in either exuberant, or minimal, dendrite complexity depending on whether surrounding cells express the same isoform. Additionally, loss of gamma-Pcdh in astrocytes, or induced astrocyte-neuron mis-matching, reduces dendrite complexity cell non-autonomously. This indicates a neuron’s pattern of connectivity is indeed regulated by specific interactions between cells that are distinct from the repulsive self-avoidance seen in isoneuronal processes of planar cell types. In addition to modulating dendrite branch development, the gamma-Pcdhs have been shown to regulate the progression of spinal cord synaptogenesis (Garrett and Weiner, 2009). A role for these molecules in cortical dendritic spines and synapses, however, had yet not been examined. In Chapter II of this thesis, I provide evidence that the gamma-Pcdhs negatively regulate synapse formation and spine morphogenesis in forebrain neurons. Mice lacking all gamma-Pcdhs in the cortex exhibit significantly increased spine and synapse density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 gamma-Pcdh isoforms. To explain this functional result, we present in vitro evidence to show that gamma-Pcdhs physically and functionally interact with the synaptic cell adhesion molecule neuroligin-1. This work suggests a potential new mechanism by which gamma-Pcdhs regulate the “choice” between dendrite arbor growth and formation and/or stabilization of dendritic spines and synapses in the developing brain. Given that disruptions in the pattern and density of dendritic arbors and spines are a hallmark of neurodevelopmental disorders such as autism and Down, Rett, and fragile X syndromes, my work may provide the basic science foundation for future therapeutic approaches focused on Pcdhs and their associated signaling pathways.

Skeletal muscle (SkM) possesses an impressive ability to regenerate in response to injury or chronic disease. This regenerative capacity is attributed to its resident mononuclear myogenic progenitors. Previous studies have identified several types of myogenic progenitors within SkM, some of which are isolated by fluorescence activated cell sorting (FACS) using cell surface markers. Studies in our laboratory have identified melanoma cell adhesion molecule (MCAM) as a cell surface marker expressed by myogenic progenitors in human fetal SkM. However, the relationship between MCAM expression and the degree of myogenic commitment of distinct MCAM+ populations has not been elucidated. In the present study, subpopulations of MCAM+ cells were purified by FACS on the basis of Hoechst 33342 dye uptake. Specifically, MCAM+ side population (SP) was isolated by Hoechst exclusion and MCAM+ main population (MP) on Hoechst incorporation. Sorted populations were first optimized for growth in vitro since SkM SP cells are difficult to maintain in culture. In particular, Invitrogen’s StemPro® MSC SFM medium was found to support propagation of human fetal SkM SP cells with minimal differentiation. Following this optimization, sorted populations were assessed for expression of myogenic markers before and after propagation and then for fusion potential in vitro and engraftment potential in vivo. The MCAM+ subpopulations were found to express myogenic markers to a significantly greater extent than MCAM- subpopulations. Furthermore, the MCAM+ subpopulations fused robustly into myotubes in vitro whereas the MCAM- subpopulations did not. Interestingly, the MCAM+ SP population exhibited the highest fusion potential in vitro and was the only MCAM+ subpopulation to engraft into dystrophic muscle in vivo following propagation. These results indicate that MCAM is associated with myogenicity and can be used to prospectively isolate a pure myogenic fraction from human fetal SkM tissue. Moreover, the MCAM+ SP retain its myogenic potential to a greater extent than MCAM+ MP after propagation. This suggests that the MCAM+ SP fraction contains a higher percentage of early myogenic progenitors compared to the MCAM+ MP fraction. Additional studies on MCAM-expressing populations in human fetal SkM may elucidate a potent population for use in cell-based therapeutic strategies for treating muscle diseases.

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.

The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. / This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in women and fourth in men globally. While expansions in preventative measures have increased the detection of CRC at the early stages of disease, only 40% of CRC patients are diagnosed when the disease is at a local stage. Moreover, many anti-cancer drugs fail to significantly improve the life expectancy of patients due to innate and acquired resistance, underscoring a need for better diagnostic and therapeutic strategies for CRC. Immunoglobulin-containing and proline-rich receptor-1 (IGPR-1) is a novel cell adhesion molecule (CAM) that was recently identified in our laboratory. IGPR-1 is expressed in epithelial and endothelial cells and promotes cell-cell adhesion. Expression of IGPR-1 in endothelial cells regulates angiogenesis; however, its role in epithelial cells, particularly cancer cells with an epithelial origin, remains unknown. The overall goal of this study was to investigate the possible function of IGPR-1 in CRC tumor cell growth and response to chemotherapeutic agents. Specifically, we aimed to test the hypothesis that increased expression of IGPR-1 in CRC tumor cells promotes cell survival and contributes to the resistance of tumor cells to doxorubicin. Human CRC tumor cell lines, HCT116 and HT29, were transduced via a retroviral system to express IGPR-1 or empty retroviral vector pQCXIP. The effect of overexpression of IGPR-1 in HCT116 and HT29 cells was measured by MTT assay in non-adherent 24-well plates. In addition, cells were viewed under a light microscope, and images were taken to assess multicellular aggregation. Results demonstrated that expression of IGPR-1 in HCT116 and HT29 tumor cells promoted CRC tumor cell growth, increased multicellular aggregation, and stimulated resistance to the conventional chemotherapeutic agent doxorubicin in non-adherent cell culture conditions in vitro. Intriguingly, treatment of cells with doxorubicin promoted phosphorylation of IGPR-1 at serine 220 (Ser220), suggesting a critical role for phosphorylation of IGPR-1 in the development of resistance to chemotherapeutics. In addition, non-adherent cell culture conditions promoted activation of the key pro-apoptotic kinase, p38 MAPK in CRC tumor cells. Ectopic expression of IGPR-1 reversed this activation. This data suggests that IGPR-1, by suppressing p38 activity, in part, promotes tumor cell survival and increases the resistance of tumor cells to the killing effects of doxorubicin. Our findings are the first to demonstrate that IGPR-1 promotes CRC tumor cell growth and increases the resistance of CRC tumor cells to the cytotoxic effects of chemotherapeutic agents. The data suggests that IGPR-1 plays an important role in CRC by inhibiting the cellular apoptotic response and promoting chemotherapeutic resistance. Finally, IGPR-1 phosphorylation at Ser220 in response to doxorubicin may account for the IGPR-1-mediated development of resistance to doxorubicin in CRC.

Polysialylated NCAM (polySia-NCAM) is re-expressed in a number of tumours, including small cell lung carcinoma and neuroblastoma and is strongly associated with aggressive, invasive and metastatic tumours in the clinic. SiRNA knockdown of the polysialyltransferases (polySTs), the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration. PolySia-NCAM is thus a highly attractive novel therapeutic target. A library of potential polyST inhibitors has been synthesised, using substrate-based design and computational chemistry. Compounds synthesised include N-acylmannosamine analogues, thio-linked CMP-sialic acid analogues, N-acyl modified sialic acids and compounds incorporating elements of both approaches. Novel methodology development in the synthesis of many of the compounds is described, notably a novel route to N-acyl sialosides. In addition, compounds identified from in silico screening were considered. Routes to synthesis and isolation of analogues of biologically active compounds are described. Using an enzyme assay, compounds were evaluated for their ability to reduce polySia synthesis through polyST inhibition. Effects of agents on polySia expression in cells, and the ability of compounds to reduce cell migration in vitro was studied using a wound healing ‘scratch assay’. The data from these experiments revealed a number of potent modulators of polySia assembly and their efficacy in reducing cell migration, as well as the limits of the biosynthetic pathway to accept unnatural sialic acid precursors. This is the first example of polyST inhibition modulating tumour cell migration, and points to the potential of the polysialyltransferases as a therapeutic target in metastatic tumours. / EPSRC and BACR / The full text will be available at the end of the extended embargo: 5th March 2027

Neuroblastoma is a highly metastatic and invasive tumour with poor prognosis. Despite recent advances in the treatment of neuroblastoma, mortality is still high due to uncontrolled metastatic disease, and novel therapeutic approaches for the treatment of neuroblastoma are therefore desperately needed. A potential novel approach for therapy of neuroblastoma relates to the polysialic acid decoration of the neural cell adhesion molecule (PSANCAM). PSA-NCAM is selectively re-expressed in a number of tumours including neuroblastoma, where it is thought to modulate tumour dissemination. Expression is strongly associated with poor clinical prognosis and an aggressive tumour phenotype. Inhibition of the enzymes responsible for synthesis of PSA, the polysialyltransferases (polySTs) presents a novel and selective therapeutic opportunity. The aims of the studies described in this thesis are to evaluate PSANCAM expression and function in neuroblastoma, and to develop and utilise cell-based models to pharmacologically investigate novel polyST inhibitors. PSA-NCAM was seen to be highly expressed in neuroblastoma clinical specimens and associated with phenotypes of tumour aggressiveness. A screening panel consisting of cell lines with a range of PSA-NCAM expression types was established and utilised to develop assays for pharmacologically assessing novel polyST inhibitors. Using cytidine monophosphate (CMP), a naturally-occurring inhibitor of polySTs, the robustness of the assays was confirmed before progression to evaluate novel molecules. From 16 compounds identified in an in vitro screen of polyST inhibition, three promising polyST inhibitors were identified. These promising polyST inhibitors modulated PSA-NCAM expression on the tumour cell surface and led to a significant reduction in cell migration. Therefore the work presented in this thesis suggests that targeting polySTs is a promising novel therapeutic strategy for neuroblastoma and further research in this area is warranted. / Mu'tah University and Jordan Armed Forces, Hashemite Kingdom of Jordan. / The full text will be available at the end of the extended embargo period: 5th March 2027