University, Diss., 2006--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 2 uppsatser.

Univ., Diss., 2005--Frankfurt (Main) / Zsfassung in engl. und dt. Sprache

Type II diabetes is one of the leading cause of morbidity in the U.S. and other parts of the world. Insulin resistance which precedes Type II diabetes is a complex state of the body where the body fails to respond to insulin. Its complexity lies in its multifactorial origin that is to say various environmental and polygenic components come into play. Here we try to dissect one of these components - `Alox8' in transgenic mice and try to see if it affects blood glucose homeostasis. Comparison of glucose tolerance and insulin sensitivity among sixteen mice comprising of six wild type, five heterozygous and five knockout mice with respect to Alox8 gene showed that wild type mice had relatively more glucose tolerance than knockout mice and this corresponded with relatively more insulin sensitiveness of wild type mice with respect to the knock out. However, these findings were not significant statistically at p=0.05. In search of any relevant biological significance, periodic acid schiff staining of the liver sections from these mice in three independent repeated experiments revealed that the knockout phenotype led to accumulation of glycogen deposits as compared to the wild type mice, an indication of insulin resistance. Taken together, our data suggests that these findings when extrapolated to human which carries ALOX15B instead of mice orthologue Alox8, could lead to a benefit of administration of lower doses of insulin in the wild type phenotype as compared to its polymorphic alleles carrying individuals.

No description available.

Morphogenesis, or the development of tissues, structures, and organs, is at the heart of embryonic development. Morphogenesis is a complex, multi-tissue process that requires coordinated cellular communication, migration, and differentiation; due to this complexity, the mechanisms that underlie morphogenesis remain poorly understood. The sea urchin embryo is morphologically and genetically more simple than most other developmental model organisms, and is optically transparent, making it a highly tractable system in which to study morphogenetic processes. The sea urchin larval endoskeleton is a biomineral that is secreted by primary mesenchyme cells (PMCs). The PMCs ingress into the embryo and remain individual, mesenchymal cells that migrate into a stereotypic three-dimensional (3-D) pattern within the blastocoel, prefiguring the form of the ensuing skeleton, which they subsequently secrete. PMC positioning is directed by cues originating in the overlying ectoderm; however, the molecular identity of those cues has remained unknown. The work described in this dissertation combines systems-level approaches with in vivo 3-D spatial analysis to identify novel skeletal patterning genes and to define their functional roles in skeletal patterning. A transcriptomics-based screen identified numerous novel candidate skeletal patterning cues. Of those cues, two were further pursued for detailed functional studies. First, the sulfate transporter SLC26a2/7 (SLC) was found to promote ventral accumulation of sulfated proteoglycans that is both necessary and sufficient to attract PMCs to the ventral territory for ventral skeleton formation. Second, the enzyme 5-lipoxygenase (LOX) was found to be required for ventral and rotational skeletal patterning, and its product, 5(S)-HETE was found to be a chemoattractant for PMCs, thereby identifying a novel role for lipoxygenase enzymes in embryonic patterning and morphogenesis. Recent work from other groups has demonstrated that PMCs diversify their gene expression profiles during skeletal patterning, implying that PMC diversification is involved in skeletal patterning, likely as a response to locally distinct spatial cues. The studies herein identify Tbx2/3 and Pks2 as important PMC subset-specific genes whose spatial expression is modulated by SLC and LOX, respectively. Together, these results provide new mechanistic insights that define our molecular understanding of the regulation of sea urchin ventral skeletal patterning.

Ferroptosis is a recently characterized cell death pathway associated with the iron-dependent accumulation of lipid hydroperoxides in phospholipid bilayers. The origin of these hydroperoxides has been an ongoing topic of debate and many researchers argue for a lipoxygenase (LOX) enzyme-controlled mechanism of initiation, given their known role as dioxygenases of polyunsaturated fatty acids (PUFAs). In response to this, our lab investigated the induction and inhibition of ferroptosis in human embryonic kidney (HEK-293) cells transfected to overexpress the three most prevalent LOX isoforms, 5-LOX, p12-LOX, and 15-LOX-1. These studies did not support a role for LOX in the execution of ferroptosis; LOX inhibition was not associated with ferroptosis suppression and in fact, anti-ferroptotic activity was directly tied to purported LOX inhibitors’ ability to act as radical-trapping antioxidants (RTAs). We have investigated the effects of LOX inhibitors on ferroptosis in human fibrosarcoma (HT-1080) cells, the cell line in which ferroptosis was initially characterized, and mouse hippocampal neuronal (HT-22) cells, the cell line in which the closely related cell death modality oxytosis was characterized. In sum, our findings mirror those obtained in HEK-293 cells, and the effectiveness of an inhibitor is tied to its off-target RTA activity, not inhibition of LOX. Moreover, we observed suppression of ferroptosis via necrostatin-1 (Nec-1), a known receptor-interacting serine/threonine-protein kinase 1 (RIPK1) (and necroptosis) inhibitor. Herein, we show that Nec-1 is not an RTA and exerts its effects by a yet unknown mechanism which we investigate in a series of exploratory experiments. Conjugated fatty acids – particularly α-ESA – have recently been reported to induce ferroptosis by an unclear mechanism. Theorizing this phenomenon was tied to the autoxidation of α-ESA’s conjugated trienic unit, we aimed to investigate the kinetic and biological properties of natural α-ESA alongside a deuterated isotopologue. Herein, we report preliminary work to derive biologically relevant rate constants for addition and hydrogen-atom transfer (HAT) of α-ESA. Moreover, we report our progress towards the synthesis of a deuterated α-ESA which will facilitate future study alongside its natural counterpart.

No description available.

No description available.

5-Lipoxygenase (5LO) is a lipid-peroxidizing enzyme which inserts molecular oxygen into fatty acids leading to the biosynthesis of leukotrienes. This protein is widely expressed in the brain including the cortex and hippocampus regions, where its levels and activity increase in an age-dependent manner. Previous work has shown that 5LO modulates both amyloid beta (A) and tau pathology in Alzheimer's disease (AD) models. However, whether the effect of 5LO on tau is direct or indirect still remains unclear. Tau is a microtubule-associated protein usually found in the axons of neurons where it promotes assembly and stabilization of microtubules. In post-mortem brains of AD patients, tau is hyperphosphorylated and altered conformationally, followed by the formation of intracellular aggregates known as neurofibrillary tangles (NFTs), which are also the major pathological hallmark of another group of neurodegenerative diseases collectively referred to as tauopathies such as Pick's Disease, Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD) and Parkinsonism linked to chromosome 17. The central hypothesis of the thesis is that 5LO directly influences tau metabolism, the development of related neuropathology and behavioral phenotype. To prove this hypothesis, a comprehensive genetic and pharmacologic experimental approach, combining both in vivo and in vitro experiments, was implemented. We initially showed that human brains from patients with a confirmed diagnosis of PSP, had significantly higher levels of 5LO when compared with brains form healthy controls. Next, we assayed the levels of 5LO in brains from htau (transgenic tau mice) mice at 4 different age time-points and two regions (cortex and cerebellum). Interestingly, compared with wild type controls, cortices from htau mice had a non-significant increase in 5LO protein levels as early as 6 months of age, which became significant by 10 months of age in the cortex only. Taken together, the age-dependent and region-specificity of the 5LO up-regulation supports the hypothesis that this pathway may have a functional role in the development of the tauopathy phenotype. To prove it, we treated tau mice with a selective 5LO inhibitor, zileuton, and explored the effect on learning and memory. Treatment of the htau mice with zileuton restored their short term working memory and spatial memory deficits. Shortly after completion of the behavioral tests, mice were euthanized and brains harvested for biochemistry and immunohistochemistry analyses. In association with the changes in behavior, we observed that pharmacologic inhibition of 5LO had an influence on tau metabolism, more specifically a significant decrease in tau phosphorylation. In search for the molecular mechanism involved in this biological effect, we assayed different kinases and phosphatase which have been implicated in tau metabolism and showed the specific involvement of the cdk5 pathway. This observation was further confirmed by in vitro studies, in which by using primary neuronal cells we showed that zileuton decreased tau phosphorylation via a cdk-5-dependent mechanism. Since the development of tau pathology results in biochemical and functional manifestation of synaptic deficits, next we assessed levels of pre- and post-synaptic protein markers. Compared with wild type, htau mice had significant reduction in the levels of three distinct markers of synaptic integrity (that is synaptophysin, post-synaptic density protein-95 and microtubule associated protein-2). By contrast, the decrease was completely restored to wild type levels by zileuton treatment. To further support the involvement of this pathway in the improvement of the behavioral and cognitive deficits, we explored the effects of its pharmacological blockade on synaptic function by performing electrophysiological studies. As reported previously, there was a significant difference in Long Term Potentiation (LTP) between the wild type and htau mice, with the latter showing significant deficits. However, pharmacologic blockade of 5LO in the htau mice was adequate to restore the LTP responses to a level comparable to those measured in the wild type mice. In the genetic portion of the study, WT and htau pups were intracranially injected with both AAV2/1 control vector and AAV2/1 5LO vector. Compared with the htau control group, the htau mice injected with AAV2/1 5LO displayed a significant deficits in cognition and memory associated with a decline in their synaptic integrity. Also, genetic upregulation of 5LO yielded significant increases in tau phosphorylation associated with an increase in cdk-5 kinase activation both in vivo and in vitro. Taken together these results describe a pluripotent role for 5LO in the context of tauopathy by representing its direct functional role in modulating behavior along with tau phosphorylation, neuroinflammation and synaptic function in a relevant mouse model of the human disease. The demonstration of the pleiotropic role 5LO in tauopathy pathogenesis makes it not only a valid pharmacological target, as 5LO inhibitors are already FDA approved but, more importantly represents a unique therapeutic opportunity with true disease modifying potential for the treatment of these dementing disorders for which there is no cure. / Pharmacology