No description available.

No description available.

No description available.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Zugl.: Göttingen, Univ., Diss., 2008

No description available.

Development of the visual system involves the precise orchestration of neural connections between the retina of the eye, the thalamus (dorsal lateral geniculate nucleus; dLGN) and the superior colliculus (SC). During early development, receptor molecules on the growth cones of retinal ganglion cell (RGC) axons sense molecular guidance cues in the extra cellular matrix (ECM) that define their route and branching behaviour within the visual system. Heparan sulphate proteoglycans (HSPGs) are ECM molecules composed of a core protein and a variable number of disaccharide residues that have been implicated in mediating axon guidance. HSPGs are modified by a number of enzymes that contribute to their structural diversity. Based on this structural diversity; the “heparan sulphate code” hypothesis of Bulow and Hobert (2004) postulated that different HSPG modifications confer different axon navigation responses as the growth cones traverse the local environment. To investigate the roles played by specific modifications of HSPG molecules in the guidance of axons, we examined two lines of mutant mice harbouring mutations in the genes encoding HSPG modifying enzymes, Heparan sulphate-6-O-sulphotransferase-1 (Hs6st1) and Heparan sulphate-2-O-sulphotransferase (Hs2st). These two mutant lines were generated through the use of gene trapping. Previous observations of RGC axon development in the two mutant lines revealed distinct axon guidance errors at the optic chiasm. Loss of Hs6st1 sulphation resulted in RGC axons navigating ectopically into the contralateral eye. Loss of Hs2st sulphation resulted in RGC axons navigating outside the normal boundary of the optic chiasm. Early observations suggested that both Hs2st sulphation and Hs6st1 sulphation have distinct, non-overlapping actions and thus, influence different axon guidance signalling pathways at the optic chiasm. Based on our findings and previous work describing the expression patterns and functions of the chemo-repellent axon guidance molecules, Slit1 and Slit2 at the optic chiasm and their Robo2 in the retina, we formulated the hypothesis of an HSPG sulphation code where Hs2st sulphation is specifically required for Slit1-Robo2 signalling and Hs6st1 sulphation is specifically required for Slit2-Robo2 signalling at the optic chiasm. To further our understanding of the roles Hs2st sulphation and Hs6st1 sulphation have on axon guidance, we looked at a number of key choice points that navigating axons encounter and are known to be influenced by Slit signalling. Further observations of RGC axons at the optic chiasm of Hs2st-/- mutants and Hs6st1-/- mutants showed distinct axon guidance phenotypes, both resulting in statistically significant increases in the width of the optic chiasm at the midline. While Hs6st1 sulphation had no effect on RGC axon navigation within the eye (possibly due to 6-O-sulphation compensation by Hs6st3); the loss of Hs6st1 sulphation at the dLGN resulted in a significant increase in the defasciculation of the optic tract. Observations of other axonal tracts influenced by Slit signalling revealed the importance of Hs2st and Hs6st1 sulphation in aiding callosal axons to successfully traverse the midline in corpus callosum development. Observations of the thalamocortical (TCA)/corticothalamic (CTA) tracts revealed that neither Hs2st sulphation nor Hs6st1 sulphation was required for the development of the mouse TCA tract (the latter may be explained by 6-O-sulphation compensation by Hs6st2). To test whether Hs2st and Hs6st1 enzymes have redundant functions in optic chiasm development, we attempted to create Hs2st-/-/Hs6st1-/- double mutants. A PCR genotyping strategy was developed for the identification of Hs6st1 animals and showed that Hs6st1-/- mutants had high postnatal lethality with only 3% of the offspring surviving to weaning while Hs2st-/-/Hs6st1-/- double mutants all died very early during embryonic development. Observations of Hs2st-/-/Hs6st1+/- mutants and Hs2st+/-/Hs6st1-/- mutants that lacked three of the four Hst alleles showed no differences when compared to single Hst knockouts. Finally, we showed that altered Slit expression at the optic chiasm and Robo expression in the retina could not explain the mutant phenotypes observed in Hs2st-/- mutants and Hs6st1-/- mutants, and therefore we hypothesized that Hs2st sulphation and Hs6st1 sulphation regulate distinct aspects of Slit-Robo signalling at the surface of the navigating axon growth cone.

Heparanase (Hpa1) is an enzyme overexpressed in nearly all cancers, typically at the tumour growth front. It cleaves proteoglycan heparan sulfate (HS) chains to release growth factors necessary for tumour growth. While some carbohydrate-based mimetic inhibitors have progressed to advanced clinical trials, new inhibitors and tools to further investigate heparanase are of continued interest. This thesis proposes a HS mimetic trisaccharide sequence that can bind Hpa1 and is suitable both for biological evaluation and inhibitor development. Synthetic work was then undertaken toward the progression of this moiety. Exploring building blocks applicable to the trisaccharide, conformationally-locked glucose derivatives were developed. This included the introduction of a conformational switch that resulted in the isolation of constrained half-chair conformers. The synthetic work toward trisaccharide formation also evaluated the utility of 1,2-cyclohexane-diacetal as a protecting group with glucuronic acid. The disarming qualities of these moieties were assessed, leading to the development of alternate routes. A more linear approach resulted in the formation of important disaccharide building blocks that contribute toward the synthesis of the core trisaccharide, including isolated 1,2-orthoesters. Further development of the chemistry established herein should allow for the formation of the desired core trisaccharide, while contributions have additionally been made toward its tool functionalisation and use in multivalent schemes.

Heparan sulphate proteoglycans (HSPGs) are a family of molecules that are found on the surface of cells or in the extracellular matrix, where they are involved in regulating key signalling events required for normal mammalian brain development. It is thought that specificity of HSPGs for particular signalling processes is encoded by their heparan sulphate (HS) sugar side chains, which can be modified post-translationally to yield huge variation in HS structure. Different sulphation patterns are generated by the action of the heparan sulphate sulfotransferases (HSTs) and sulfatase enzymes, which add or remove sulphate groups to specific positions on residues of the HS side chains. Depending on the expression of these enzymes and the resulting heparan sulphate ‘code’, it is proposed that cells are then able to regulate signals they receive and send in the ligand rich extracellular environment of the developing forebrain. Hs6st1 and Hs2st catalyse 6-O and 2-O HS sulphation, respectively. Following loss of either of these two HSTs, commissural tracts including the corpus callosum fail to develop normally during late mouse embryogenesis. The telencephalic midline environment is perturbed, with a striking mis-positioning of glial cell populations that normally act to guide axons towards the contralateral hemisphere. Too many radial glial cells at the glial wedge (GW) migrate towards the indusium griseum (IG) in mutant embryos. The running hypothesis to explain this phenotype is a change in critical signalling pathways required to set up the correct midline glia environment, such as Fgf8/ERK signalling which has already been identified as up-regulated at the Hs6st1-/- corticoseptal boundary (CSB). In order to further study what changes are occurring at the developing midline of HST-/- embryos compared to WT, we took a hypothesis free approach using RNA-sequencing analysis. RNA extracted from dissected midline regions of WT, Hs2st-/- and Hs6st1-/- mouse embryos at E16.5 was sent for sequencing, and a list of differentially expressed genes obtained. Overall we find few differentially expressed genes at the Hs6st1-/- midline compared to WT. At the Hs2st- /- midline there are a larger number of differentially expressed genes. Following validation studies, we find a significant and specific increase in Fgf17 protein distribution at the CSB of Hs2st-/- embryos compared to WT at E14.5. The results suggest the hypothesis that Hs2st’s normal role is to regulate Fgf17 protein distribution to limit exposure of GW radial glia cells to this translocation signal. When 2-O HS sulphation is lost then in Hs2st-/- embryos, ectopic Fgf17 signalling induces aberrant glia migration which ultimately prevents callosal axons from crossing the telencephalic midline to form the corpus callosum. To test this hypothesis, we used ex vivo slice culture experiments and showed ectopic Fgf17 protein expression is sufficient to trigger precocious translocation of midline glia in WT CSB, phenocopying the glia behaviour of Hs2st-/- embryos. Also consistent with the hypothesis, the Hs2st-/- glia phenotype can be rescued by addition of an FgfR1 inhibitor which reduces number of translocated glia cells. From these results we find for the first time that 2-O sulphated HS plays a remarkably specific role in regulating Fgf17-mediated translocation of midline glia cells at the developing mammalian telencephalic midline.

<p>This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions.</p><p>The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals.</p><p>The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project.</p><p>The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.</p>