Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

The thesis looks at the patterns, tendencies, and tensions that characterise the Anglo-American critical reception of the three peninsular woman authors Merce Rodoreda, Esther Tusquets, and Rosa Montero, generally assigned a representative role as feminist writers in the field of gender-centred Hispanism. The study begins with the recognition that there has been an increase in the level of awareness as to certain recurrent mechanisms of academic Hispanism in America, as is proved by the recent burgeoning of studies with an avowed metacritical slant. My analysis partakes in this trend but integrates also translational analysis, with a view to showing the validity of translated texts as critical artefacts, informed by similar operations and leanings. Ultimately, my aim is to shed light on the often downplayed complexities characterising ideologically inflected instances of cultural reception and diffusion, of which the Anglo-American critical response to women-authored, contemporary narrative in Spain is a case in point.

There is ample evidence of the crucial role of PI3K/Akt dependent signalling in cardiac function, cellular growth and cell apoptosis. The PI3K/Akt pathway mediates cardioprotective effects in experimental models of cardiovascular disease. For example, activation of this pathway ameliorates the sepsis-induced cardiac dysfunction, whereas its activation in myocardial ischaemia/reperfusion (I/R) limits cardiac injury. This thesis investigates the role of two drugs, which activate the PI3K/Aktpathway, namely the haematopoietic cytokine erythropoietin and the anti-malarial drug artesunate, in a mouse animal model of experimental sepsis-induced cardiac dysfunction and in a rat model of regional myocardial I/R injury, respectively. Using a clinically relevant model of caecal ligation and puncture in mice, I demonstrated that aged (8 months) C57BL/6 mice (receiving fluid resuscitation and antibiotic therapy) developed significant cardiac dysfunction (within 24 h), while younger mice (2 months) did not. Erythropoietin attenuated the impaired systolic contractility (in vivo and ex vivo) caused by endotoxaemia (lipopolysacchride 9 mg kg-1; young mice) and sepsis (aged mice). These beneficial effects were associated with activation of Akt and endothelial nitric oxide synthase survival pathways and inhibition of the glycogen synthase kinase 3β, nuclear factor-κB and interleukin 1β pro-inflammatory pathways, secondary to activation of the β-common receptor. A single bolus administration of artesunate at the start of reperfusion in a rat model of myocardial I/R significantly attenuated the infarct size. This effect was mediated via activation of pro-survival pathways (PI3K/Akt and ERK 1/2 and STAT-3) and inhibition of the glycogen synthase kinase 3β and nuclear factor-κB pro-inflammatory pathways. Thus, in this thesis I have demonstrated that pharmacological activation of the PI3K/Akt pathway by erythropoietin and artesunate in sepsis and myocardial I/R, respectively, plays a vital role in the amelioration of cardiac dysfunction and injury.

Translational regulation is an important mechanism for regulating gene expression. Regulation of specific transcripts is often mediated by elements present in the 3′ untranslated region (UTR) of the mRNA. In the Drosophila embryo, translational repression of nanos mRNA is mediated by Smaug protein bound to specific sequences present in the 3′ UTR of the mRNA. Here I show that Smaug recruits Cup protein to the mRNA and that Cup in turn binds to the translation initiation factor eIF4E that is present at the 5′. The interaction between Cup and eIF4E prevents formation of a productive translation initiation complex on the mRNA. Therefore, Smaug-dependent translational repression functions at the initiation step via the indirect interaction of Smaug with eIF4E, which is mediated by Cup. In the second example of translational regulation presented here, I show that an element present in the 3′ untranslated region of the Hsp83 mRNA mediates translational enhancement in the Drosophila embryo. I have identified three proteins, Hrp48, DDP1 and PABP that interact with this element and have demonstrated that Hrp48 and DDP1 function in translational enhancement.

Translational regulation is an important mechanism for regulating gene expression. Regulation of specific transcripts is often mediated by elements present in the 3′ untranslated region (UTR) of the mRNA. In the Drosophila embryo, translational repression of nanos mRNA is mediated by Smaug protein bound to specific sequences present in the 3′ UTR of the mRNA. Here I show that Smaug recruits Cup protein to the mRNA and that Cup in turn binds to the translation initiation factor eIF4E that is present at the 5′. The interaction between Cup and eIF4E prevents formation of a productive translation initiation complex on the mRNA. Therefore, Smaug-dependent translational repression functions at the initiation step via the indirect interaction of Smaug with eIF4E, which is mediated by Cup. In the second example of translational regulation presented here, I show that an element present in the 3′ untranslated region of the Hsp83 mRNA mediates translational enhancement in the Drosophila embryo. I have identified three proteins, Hrp48, DDP1 and PABP that interact with this element and have demonstrated that Hrp48 and DDP1 function in translational enhancement.

Background and objectives: Endothelial dysfunction and cancer are two of the important aspects of obesity-related medical complications, the prevalence of which is reaching an epidemic level worldwide. The protein kinase LKB1 has been shown to play opposite roles in these two metabolic diseases by promoting cellular senescence and inhibiting cell proliferation through regulating a series of its downstream targets. However, the molecular mechanisms wherebyLKB1 itself is regulated by its upstream molecules remains poorly understood. The major objectives of this study are to identify novel upstream regulators of LKB1 and to investigate how these upstream regulators modulate the subcellular localization and physiological functions of LKB1 by post-translational modifications. Key findings: 1. Our proteomic analysis demonstrated that LKB1 was modified by both acetylation and phosphorylation. The acetylation sites of mouseLKB1 include Lys48, Lys64and Lys312. The phosphorylation sites of mouseLKB1 include: Ser31, Thr32,Tyr36, Ser69, Thr71, Ser334and Thr336. 2. In both human embryonic kidney 293 (HEK293)cells and primary porcine aortic endothelial cells (PAECs), the nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase SirT1 attenuated the acetylation levels of LKB1,which consequently resulted in enhancedLKB1ubiquitination, thereby leading to the proteasome-mediated degradation of LKB1. 3. In primary PAECs, overexpression of SirT1 protected cells from cell cycle arrest and cellular senescence, whereas overexpression of LKB1 exhibited the opposite effects.SirT1 antagonizedLKB1-induced G1 phase arrest and cellular senescence by promoting the deacetylation and protein degradation of LKB1. 4. The in vitro phosphorylation assay and mass spectrometry analysis demonstrated that LKB1 could be phosphorylated by the Akt kinase at Ser334which was critical for the interaction between LKB1 and 14-3-3. The enhanced association between LKB1 and 14-3-3 subsequently attenuated the interaction between LKB1 and Ste20 related adaptor α(STRADα), which further promoted the nuclear accumulation of LKB1. 5. The cell proliferation and cell cycle distribution analysis of the stably-transfected MDA-MB-231 breast cancer cells demonstrated that overexpression of the LKB1 mutant S334D, which mimicked Ser334 phosphorylation and localized exclusively in the nucleus, completely lost its anti-tumor activities. On the other hand, the S334A mutation enhanced the tumor suppressive functions of LKB1. 6. Nude mice inoculated with the LKB1 S334A stably-transfected MDA-MB-231 cells exhibited delayed tumor onset, decreased tumor growth rate and tumor weight. By contrast, inoculation of nude mice with the MDA-MB-231 cells overexpressing LKB1 S334D mutation showed the opposite effects on these parameters. Conclusions: These results collectively suggest that the deacetylase SirT1 and the protein kinase Aktare the two important upstream regulators of LKB1. SirT1 prevents LKB1-induced cellular senescence and protect endothelial ageing by promoting proteasome-mediated degradation of LKB1. Akt inhibits the tumor-suppressive activity of LKB1 by enhancing the phosphorylation-dependent nuclear translocation. Further investigations on the precise mechanisms whereby SirT1 and Akt regulate LKB1 functions may help to design novel therapeutic strategies for treating obesity-related diseases, such as diabetes, cardiovascular disease and cancer. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis.

Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (sites viewed on August 10, 2006). PDF text of dissertation: 98 p. : ill. ; 1.16Mb. UMI publication number: AAT 3208087. Includes bibliographical references. Also available in microfilm, microfiche and paper format.