Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Jul 7.

No description available.

Tumour-associated macrophages (TAMs) have been shown to play an important role in tumour survival and progression. Thus, high numbers of macrophages in the tumour tissue are often associated with a poor prognosis. Identification of factors responsible for recruiting macrophages to the sites of different types of tumours might help to develop more effective cancer treatment. Burkitt's lymphoma (BL) is characterised by uncontrolled cell proliferation, high rate of spontaneous apoptosis and significant macrophage infiltration. Although BL cells undergo extensive apoptosis, in situ their corpses are cleared very effectively by macrophages infiltrating the tumour. It is now widely believed that dying cells are themselves able to release chemotactic molecules to ensure macrophage chemotaxis and subsequent clearance of their site of death. Previous work carried out in this laboratory identified fractalkine/CX3CL1 (FKN) released from dying BL cells to be an important player in macrophage chemotaxis to BL. Yet, these results have also indicated that FKN may not be the only chemokine involved in this process. Following from those observations, the first part of this work focused on examination of the potential role of monocyte chemoattractant protein-1 (MCP-1) in macrophage recruitment to BL. Despite the initial promising results, careful analysis of the data obtained by various techniques led to the conclusion that MCP-1 is, probably, not expressed by BL cells. Subsequently, effort was concentrated on understanding mechanisms regulating FKN processing during cell death. The studies performed before in this laboratory identified a new form of FKN to be present in apoptotic BL cells and showed that this is the form that is, most likely, responsible for mediating macrophage migration. Here, this apoptosis-related 60 kDa FKN was found to be a likely caspase-3 cleavage product. Moreover, it was demonstrated that FKN and active caspase-3 are released together in apoptotic BL cell-derived microparticles, suggesting that the proteolytic events could take place also extracellularly. In the final results chapter the differences between BL cell lines in the way they process FKN during cell death were revealed and a new cell death-associated 55 kDa FKN was observed. Through several lines of evidence, this new form was identified to be a possible product of calpain-mediated proteolysis. To conclude, this work provides the first evidence for a possible direct participation of the two major cell death executioner proteases – caspases and calpains, in production of ‘find me’ signals for macrophages and thus, ensuring effective clearance of dying cells. These results indicate that FKN cleavage and release might be of key importance during cell death. Moreover, the studies presented here contribute to better understanding of the process of FKN secretion.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Autophagy is the major cellular, conservative, lysosomal catabolic process to eliminate and recycle intracellular waste and organelles through autophagosomes. Enhancing autophagy to promote the clearance of toxic proteins is developing as a promising approach to treat proteinopathy disorders like Alzheimer's disease (AD). AD is the most common aging-associated neurodegenerative disease. It is characterized by the aggregation of aberrantly hyperphosphorylated tau (p-Tau) and excessively produced Amyloid-beta (Aβ) into neurofibrillary tangles (NFTs), and amyloid plaques (AP) respectively. Reprogramming autophagy lysosomal pathway (ALP) through autophagy master controller, transcription factor EB (TFEB), is developing as an attractive strategy to treat AD. It is already proven that TFEB overexpression can promote Aβ and p-Tau lysosomal clearance, attenuate NFT and AP deposition and restore the behavioural deficits in AD mice models. Previously Song et al., 2016 have identified a small molecule curcumin derivative C1. They reported that C1 could bind to recombinant TFEB and enhance ALP both in vitro and in vivo conditions independent of mTOR inhibition. In the current study, C1 is systematically evaluated for its bioavailability, anti-AD efficacy in vitro, and in vivo AD experimental models. To validate TFEB mediated anti-AD efficacy of C1 in vitro, we tested the C1 effect on amyloid precursor protein (APP) and p-Tau degradation in vitro neuronal AD cell culture models. In N2a cells overexpressed with APP (695) and EGFP-P301L tau plasmids, C1 induced APP, CTFβ, and Tau lysosomal degradation. To demonstrate the TFEB dependent autophagic clearance effects of C1, TFEB is silenced in N2a cells with lentiviral shRNA particles. Under TFEB silenced condition, C1 induced reduction of FL-APP, CTFβ, and Tau was compromised. Overall In vitro experiments show that C1 induced lysosomal digestion of FL-APP, CTFβ, and p-Tau in a TFEB dependent manner. To further demonstrate C1 brain bioavailability, C1 and curcumin comparative pharmacokinetic studies (Pk study) in both mice (time course Pk study) and rat (single time point analysis) are conducted. In Pk studies, both C1 and curcumin are dosed at 10 mg/kg to determine their concentration in the whole brain (mice), separate brain regions (rat), CSF (rat), and plasma. The WinNonlin analysis of C1 and curcumin mice Pk study data revealed that C1 is significantly more bioavailable than curcumin in both brain and plasma, which is also corroborated by the single time point analysis in rats. To illustrate C1 anti-AD activity in vivo, C1 is screened in homozygous P301S (Tau), heterozygous 5xFAD (Aβ), and homozygous 3xTg (both Aβ and Tau) AD transgenic mice models. These mice were started to treat with C1 before the onset of AD pathology until the AD pathological phenotype is expressed to cause impairment in mice behaviour. In mice behavioural examination, C1 treatment has significantly improved mice motor function (Rotarod-P301S), restored cognitive impairment related to the cortex (contextual fear conditioning-5xFAD), hippocampus (Morris water maze-3xTg) and improved cholinergic activation (open field-3xTg). In the brain biochemical examination, C1 activated the TFEB mediated ALP pathway to degrade FL-APP, CTFα/β, Aβ, and p-Tau and reduced the amyloid plaque load, extra neuronal-NFT positive cells. Notably, C1 treatment in 5xFAD mice has significantly restored hippocampal synaptic function. In summary, the current study validates C1 as an orally bio-available potent small molecule TFEB activator which restores mice cognitive impairment, altered behaviour, and synaptic plasticity by reducing Aβ and tau levels in AD experimental models. Overall, the TFEB activator C1 can be a promising drug to treat AD.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 142-166.

Equine hyperelastosis cutis (HC or HERDA), a connective tissue disorder in American Quarter Horses, results in hyperelastic skin with poor wound healing. Similar conditions are found in many species and all forms display decreased skin tensile strength. Fibroblasts produce collagen and elastin fibers, forming networks, providing the dermis with strength, and elasticity. This study aims to carry out a 3-part evaluation between horse skin fibroblast (cells from horses affected with HERDA, cells from horses that are carriers of HERDA (recessive HERDA gene), and cells from horses that are normal (neither affected or carriers of HERDA); Studies include: 1. Cell proliferation assay 2. Apoptosis analysis of fibroblasts 3. Mechanobiology of stretched fibroblast. Studies have shown cellular deformation to have an overall effect on mechanical properties of healthy and unhealthy tissues. This investigation provides a micromechanical evaluation of HC/HERDA in an effort to quantify the cellular level differences between each condition.

OmiHtrA2 is a highly conserved mammalian serine protease that belongs to the HtrA family of proteins. Omi shares homology with the bacterially expressed heat shock protease HtrA, which functions as a protease at higher temperatures and a chaperone at lower temperatures. Additionally, Omi shares sequence similarity with the mammalian homologs L56/HtrA1 and PRSP/HtrA3. Omi was first isolated as an interacting protein of Mxi2, an alternatively spliced form of the p38 stress-activated kinase, using a modified yeast two-hybrid system. Omi localizes in the mitochondria and in response to apoptotic stimuli the mature form of this protein translocates to the cytoplasm. In the cytoplasm Omi participates in both the caspase-dependent as well as caspase-independent apoptosis. Additionally, recent studies suggest that Omi may have another unique function, maintaining homeostasis within the mitochondria. In an effort to further elucidate the function of Omi, a yeast two-hybrid screening was performed to isolate novel interacting proteins. This screening identified a novel protein (HOPS), as a specific interactor of Omi. The predicted amino acid sequence of this protein does not provide any information about its potential function in mammalian cells. However, experiments show that HOPS is cleaved in vitro by Omi. Furthermore, in response to apoptotic stimuli, HOPS is also degraded in vivo. This study suggests that HOPS could be a physiological substrate of Omi that is cleaved and removed during apoptosis.

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.