Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Drosophila TGFbeta-Activating-Kinase 1 (dTAK1) is an essential component of both the Immune Deficiency (IMD) innate immune and TNF/Eiger apoptotic cascades. The IMD and JNK pathways bifurcate at the level of dTAK1. Hence, elucidating the regulatory mechanism of dTAK1 is pertinent to understanding the regulation of both innate immunity and apoptosis. In this study, Trabid was identified as a novel negative regulator of the Drosophila IMD pathway. Trabid interacted with dTAK1 and decreased K63-linked ubiquitination, thereby reducing immune signalling. Three tandem Npl4 Zinc Fingers (NZF) and C518 were required for Trabid activity. Lysines 142 & 156 were identified as the K63 Ub acceptor sites of dTAK1, required for K63-linked ubiquitination and signalling. Also, results show Lys 156 functioned as the K48 Ub acceptor site. Further, the ZF domain of TAK1-associated Binding Protein 2 (dTAB2) was important in modulating dTAK1 K63-linked ubiquitination and thereby the immune signal. These results indicate an elaborate and multi-tiered mechanism for regulating dTAK1 activity and modulating the immune signal. Further, Ariadne-2 (Ari-2) was identified as a novel component of the Drosophila TNF/Eiger pathway which functioned at the level of dTAK1. Results indicate that Ari-2 is essential for normal development and longevity. It enhances the apoptotic signal when concomitantly over-expressed with Eiger. Further, Ari-2 interacts with dTAK1, dTAB2 and dTRAF2 which are all implicated in TNF/Eiger signalling. Thus, evidence supports the hypothesis that Ari-2 functions as an adaptor, involved in assembling a distinct signalling complex which transduces the apoptotic signal without activating immunity.

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The study of the mechanism of traumatic brain injury (TBI) processes at the cellular level is vital to obtain characterization of nerve cell damage after mechanical deformation. This understanding is needed to find feasible therapeutic targets for mechanically damaged neurons. To study the cellular level of TBI damage, development of a new in vitro cellular model of TBI might be done to simulate in vivo cellular TBI. In this research, two studies were performed: (1) the design and construction of an in vitro cell stretching device to mechanically injure cells and (2) the characterization of the molecular and cellular level of the TBI mechanism. The cell stretching device design allows for the precise control of cell strain and duration of stretching cells such that TBI can be mimicked. Analysis of the cellular and molecular level mechanisms of TBI in the proposed in vitro model might help in the design of therapeutic strategies for the treatment of TBI. Our proposed mechanism of injury due to TBI is as follows: after the cell is stretched, a cellular signaling molecule is released to activate the cellular signaling pathway. The activated cell signal may activate kinases which phosphorylate proteins and initiate new protein synthesis. Newly phosphorylated and synthesized proteins may activate the apoptotic process. Using a variety of pharmacological agents, one could block steps in the hypothesized mechanism and examine the effect of those agents on downstream cellular processes and cell apoptosis. For example, the inhibitions of calcium transport, protein synthesis, and caspases were performed to examine the initial activation of the signaling pathway and the role of both in the apoptosis process. Proteomics of TBI may help the understanding of the mechanism of TBI related protein expression. This work will contribute to the discovery of new therapeutic targets and better treatments for TBI.

Few weeks after birth, a switch in peptide elongation factor 1As from EF-1alpha/EF1A-1 to S1/EF1A-2 occurs in brain neurons, heart and skeletal muscles of mammalians. In order to elucidate the reason behind this switch, I studied the expression of both homologous proteins during muscle differentiation and apoptosis and, documented the relation between peptide elongation factors and caspase-3 activation. I found that during in vitro muscle differentiation of L6 myoblasts, a switch in peptide elongation factors 1A occurs as physiologically observed in skeletal muscles. While EF-1alpha/EF1A-1 is expressed in replicating myoblasts, S1/EF1A-2 is solely found in differentiated myotubes where it replaces EF-1alpha/EFIA-1 as the major elongation factor. Similarly, upon serum deprivation-induced apoptosis, a reversion in peptide elongation factors 1A is observed: EF-1alpha/EF1A-1 replaces S1/EF1A-2 and becomes the major form of elongation factor 1A present in dying myotubes. This switch correlates in myotubes with the activation of caspase-3 protein, a cysteine protease involved in apoptosis. When L6 myotubes constitutively express S1/EF1A-2 as caused by adenoviral gene transfer, they become resistant to serum deprivation-induced apoptosis. In contrast, when L6 myotubes are transfected with EF-1alpha/EF1A-1 gene, they die more rapidly from serum deprivation-induced apoptosis than control cells. Transfection using anti-sense EF-1alpha/EF1A-1 gene protects myotubes from apoptotic cell death. Thus, both elongation factor 1As exert opposing effect on muscle survival: while EF-1alpha/EF1A-1 accelerates apoptotic cell death, S1/EF1A-2 protects muscles against apoptosis. / I found that skeletal muscles are the only tissues where, despite the constitutive expression of caspase-3 mRNA, the protein can be absent. Furthermore, I found that while immediately after birth, caspase-3 protein is present in skeletal muscles, a few weeks afterwards, the protein cannot be detected by Western blotting. In skeletal muscle, this change correlates with the observed switch in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 and suggests that caspase-3 is translationally regulated in skeletal muscles. The laboratory previously reported that while EF-1alpha/EF1A-1 protein reappears; S1/EF1A-2 protein becomes absent from regenerating muscles. However, once tissue regeneration is completed, the situation returns to normal as EF-1alpha/EF1A-1 disappears and S1/EF1A-2 reappears to become the only type 1A elongation factor expressed in muscle. / In conclusion, I found that the developmental switch observed in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 partly serves to protect muscle cells from apoptosis. Thus, I am the first to identify a noncanonical function for S1/EF1A-2. (Abstract shortened by UMI.)

Six long chain fatty acid esters of quercetin-3-O-glucoside (Q3G) acylated enzymatically were used for determining their antiproliferative action in comparison to precursor compounds (quercetin, Q3G and six fatty acids namely, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, eicosapentaenoic and docosahexanoic acids) using HepG2 cells. Long chain fatty acid esters of Q3G showed significant inhibition of cell proliferation (approximately 85% to 90%) compared to the precursor compounds and two prescribed anticancer-drugs (Sorafenib and Cisplatin) after 6 hrs and 24 hrs by inducing cell cycle arrest, apoptosis and DNA topoisomerase II inhibition. Among the six fatty acid esters of Q3G, oleic acid ester (OA-Q3G) displayed the greatest anti-proliferation action and upon further investigation showed significant regulation of expression of genes involved in cell cycle, growth, survival and apoptosis at gene and protein level. Overall, results of the study suggest strong potential of these novel compounds in treatment of liver cancer.

<p>The process of cytotoxic T lymphocyte (CTL) killing involves the recognition and destruction of foreign antigens by cytotoxic T cells and is of crucial importance to the defence of the organism against viral infections. Defects in this process can lead to various autoimmune diseases and cancer. The aim of this study was to identify more genes involved in the cell death pathway and to link CTL killing, apoptosis and cancer.</p>

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Ląstelės metabolizme svarbų vaidmenį vaidina oksidacijos – redukcijos reakcijos, kuriose deguonis yra elektronų akceptorius. Įvairių cheminių reakcijų metu gali susidaryti ir aktyviosios deguonies formos – superoksido anijonas, vandenilio peroksidas, hidroksilo radikalas, singletinis deguonis, kurios pažeidžia įvairias biomolekules. Augalai nuo seniausių laikų yra vartojami įvairioms ligoms gydyti. Flavonoidai – tai augalinės kilmės junginiai, randami vaisiuose, daržovėse arbatose, kurie pasižymi antioksidacinėmis savybėmis. Sintetiniai antioksidantai polifenoliai labai plačiai naudojami maisto pramonėje, kaip įvairūs priedai ir konservantai. Vienas jų - kvercetinas. Kvercetino antioksidacinis poveikis priklauso nuo to, kad jis reaguoja su laisvaisiais radikalais, sudarydamas fenoksradikalus, kurie yra ne tokie aktyvūs. Tačiau aukštos kvercetino koncentracijos yra citotoksiškos, o citotoksiškumo mechanizmai, nors ir labai plačiai tyrinėjami visame pasaulyje, lieka neaiškūs. Mūsų darbo tikslas ir buvo ištirti kvercetino ir kitų flavonoidų poveikį Hep 22a linijos ląstelėms. Hep 22a ląstelių linija pasirinkta neatsitiktinai. Tai pelių hepatomos ląstelių kultūra, kuri pasižymi navikinėms ląstelėms būdingomis savybėmis – neribota proliferacija ir ląstelių migracija. Vyrauja epitelinio tipo ląstelės, kurias persėjus po oda susidaro navikai. Ląstelės gerai auga tiek in vivo, tiek in vitro. Ląstelės pailgos, prisitvirtinusios prie substrato, sudaro monosluoksnį. Tirtų flavonoidų... [toliau žr. visą tekstą] / ABSTRACT Flavonoids are widely distributed in edible plants, and considered to be dietary antioxidants. Flavonoids can protect cell from „oxidative stress“, but the same flavonoids compound could behave in two ways as an both antioxidant and prooxidant, depending on the concentration used and free radical source. Among the flavonoids, quercetin is one of the most widely studied flavonoid and has biological, pharmocological, and medicinal properties. Besides the chemopreventive effects, other biological functions of quercetin are believed to improve antioxidant defence systems in living organizms. The aim of this work was to analize the effects of flavonoids (quercetin, myricetin and morin) in Hep 22a cells. Materials and methods: 1. Cell culture cytotoxicity studies; Flavonoids and the other components were obtained from Sigma, and used as received. 2. Study with fluorescence microscope 3. Statistical analysis Results and discusion: Hep 22a cell line is a mouse hepatoma cell line, which posseses the unlimited proliferation and migration features. Quercetin concentration for 50 % death of Hep 22a cells (cL50) was 160 µM, myricetin concentration was 60 µM, and morin concentration was 190 µM,. The citotoxity of flavonoids in Hep 22a cells was partly inhibited by catalase, by the antioxidant N,N‘-diphenyl-p-phenylene diamine DPPD, desferrioxamine and by dicumarol and an inhibitor of DT-diaphorase thus showing its prooxidant character. Inhibitors of cytochromes P-450, &#945... [to full text]