Dissertations / Theses on the topic 'Proteins – Metabolism'
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Ainsworth, Julia. "Comparison of p53 and MAGI-3 regulation mediated by the E6 protein from high-risk human papillomavirus types 18 and 33." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112368.
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The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation.In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein.
Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.
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Ring, Giselle Natasha. "Identification and characterization of TMEM 85, a novel suppressor of bax-mediated cell death in yeast." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112352.
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The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.
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Smits, Callum, and n/a. "Structures of the pro-survival protein A1 in complex with BH3-domain peptides." University of Otago. Department of Biochemistry, 2007. http://adt.otago.ac.nz./public/adt-NZDU20071218.131743.
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Protein:protein interactions are central to the regulation of the intrinsic programmed cell death (apoptosis) pathway. Opposing members of the Bcl-2 family of proteins, which have distinct sequence features, interact with each other on the outer mitochondrial membrane to regulate apoptosis. Pro-survival proteins such as Bcl-2, Bcl-x[L], Bcl-w, Mcl-1 and A1 protect cells from apoptosis and contain up to four regions of homology to Bcl-2 (Bcl-2 homology domains 1 - 4, BH1-4). Pro-apoptotic BH3-only proteins such as Bim, Puma, Noxa, Bad, Bmf, and Bid promote apoptosis by interacting with and inactivating pro-survival proteins, and contain just the BH3-domain. The pro-apoptotic proteins Bax and Bak are essential for apoptosis and contain three regions of homology to Bcl-2 (the BH1-, BH2- and BH3-domains).
In this study, two different sets of interactions involving pro-survival proteins were investigated. Initially, the pro-apoptotic protein Bnip3 was examined to determine if it was a mitochondrial anchor for the pro-survival protein Bcl-w. Secondly, to characterise the interactions between a pro-survival protein and different BH3-domains, structures were solved of the pro-survival protein A1 in complex with four different BH3-domains.
In the structure of Bcl-w, the hydrophobic C-terminus is bound to its own BH3-domain binding groove. This location of the C-terminus is consistent with the observation that Bcl-w is only loosely associated with the outer mitochondrial membrane in healthy cells. Upon interaction of Bcl-w with a BH3-domain, Bcl-w becomes tightly associated with the mitochondrial membrane, presumably due to displacement of the C-terminal residues by the BH3-only protein. In healthy cells it has been suggested that Bcl-w is associated with the membrane due to an interaction with an unidentified membrane protein, which preliminary experiments suggested may be Bnip3. Protein interaction experiments performed in vitro and in vivo did not reveal an interaction between Bnip3 and Bcl-w.
It was originally thought that each pro-apoptotic BH3-only protein could interact with all pro-survival proteins. However, it has recently become clear that there is selectivity within the pathway suggesting functional groupings. Bim and Puma behave as originally predicted and can interact with all pro-survival proteins and are potent killers. In contrast, Noxa and Bad interact with distinct subsets of pro-survival proteins. Noxa only binds Mcl-1 and A1, while Bad binds Bcl-2, Bcl-x[L] and Bcl-w. As a result, either Noxa or Bad acting alone is a weak killer, but together they are potent. Other BH3-only proteins bind tightly to some pro-survival proteins and weakly to others.
The diversity that exists between BH3-domain sequences precludes sequence-based identification of the determinants of specificity. In this study, crystal structures of A1:Puma BH3-domain, A1:Bmf BH3-domain, A1:Bak BH3-domain and A1:Bid BH3-domain complexes have been solved. Differences identified between these structures explain some of the variation in affinities observed in pro-survival protein:BH3-domain complexes. These observations, in combination with published data, suggest that BH3-domains bind weakly when the optimal interactions with conserved residues cannot be formed. Additionally, differences were observed in the A1:Bak BH3-domain structure that may be functionally important for the regulation of Bak.
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Spathaky, Jane Mary. "A novel method for the isolation of genes encoding peroxisomal matrix proteins." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361693.
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Charest-Marcotte, Alexis 1984. "Functional interaction between PROX1, ERR[alpha] and PGC-1[alpha] in the control of energy metabolism." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111571.
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Nuclear receptors play crucial roles in the transcriptional regulation of many biological processes such as development and cellular differentiation. ERRalpha is known, along with coactivator PGC-1alpha, to playa central role in the control of energy metabolism in cardiac and skeletal muscle. They activate the expression of many genes involved in mitochondrial oxidative metabolism. Here we identified PROX1, a factor that was previously shown to broadly influence metabolism, as a regulator of this pathway. Indeed, PROX1 interacts in vitro and in vivo with both ERRalpha and PGC-1alpha. To provide more insight on the hepatic functions of ERRalpha and PROX1, we performed ChIP-on-chip using mouse liver, identifying a large number of ERRalpha and PROX1 genomic targets and reinforcing their role in energy metabolism. Over 40% of the target genes were found to be common to both factors and we observed that PROX1 could be recruited to ERRalpha binding sites and act as a negative regulator o fthe ERRalpha/POC-1alpha pathway.
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Rossi, Merja. "Investigating cell type specific metabolism using GFP as a reporter protein." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:0c418362-63e7-496d-9ff6-584a0c54c127.
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Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells. The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.
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Faubert, Amélie. "Towards the identification of cellular and molecular regulators of hematopoietic stem cell self-renewal." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103195.
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Self-renewal is central to the expansion of normal and cancerous stem cells. Its understanding is therefore critical for future advances in transplantation-based therapies and cancer treatment. Although the molecular machinery controlling stem cell self-renewal remains poorly defined, a number of genes important to this process have recently been identified. Two prominent genes in this group are Hoxb4 and Bmi1. Members of our group led the way to demonstrate important regulatory functions of these genes in hematopoietic stem cell (HSC) self-renewal and expansion.The major goal of my thesis project is to dissect mechanisms that regulate self-renewal of HSCs. Our starting hypothesis was that HSC activity is regulated by complementary and independent self-renewal mechanisms: self-renewal of expansion and self-renewal of maintenance (Chapters 1-2). In order to further verify this theory, we have analyzed the genetic interaction between Hoxb4 and Bmi1. While Hoxb4 overexpression triggers HSC expansion, Bmi1 proper expression is essential to sustain long-term stem cell activity. We have also demonstrated that Hoxb4 and Bmi1 regulate distinct gene targets, likely suggesting a complementary and independent function for these two regulators in HSC activity (Chapter 3).
The second part of this thesis highlights efforts that were made in order to get a better understanding of self-renewal mechanisms. We have identified potential new regulators of stem cell activity by characterizing a stem cell leukemia population (Chapter 4) and by assessing the expression of asymmetrical distributed factors (Chapter 5) and selected nuclear factors of the Hematopoietic Stem Cell Nuclear Factor Database (Chapter 6) in stem cell-enriched sub-fractions.
This project will lead to a better understanding of the cellular basis regulating self-renewal of both normal and cancer stem cells and potentially to the future identification of new self-renewal determinants.
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Dzikaitė, Vijolė. "Studies of proteins in heme and iron metabolism /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-762-2/.
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Cervantes-Laurean, Daniel. "Preparation and Characterization of Model Conjugates for the Study of Proteins Modified by ADP-ribose." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc935701/.
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Modification of proteins by ADP-ribose has been shown to be a versatile modification with respect to the amino acid side chain. The results described here will allow the study of the biological importance of ADP-ribose glycation and also allow differentiation on crude extracts between enzymatic modifications from protein ADP-ribose glycation that can occur due to the presence of NAD glycohydrolases.
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Buscarlet, Manuel. "The neural progenitor to neuron transition : role and regulation of GrouchoTLE proteins." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115670.
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Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells.By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function.
Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism.
Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins.
Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins.
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Laberge, Marie-Kristine. "Nck1 is required for ER stress-induced insulin resistance and regulation of IRS1-dependent insulin signalling." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111950.
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Activation of the Unfolded Protein Response (UPR) following stress in the Endoplasmic Reticulum (ER) is an important mechanism by which obesity results in insulin resistance and type II diabetes. We uncovered a role for the adaptor protein Nck in modulating the UPR. In this study, we report that obese Nck1-/- mice, which show lower levels of UPR in liver and adipose tissue, present improved insulin signalling in these tissues. We established that the effect of Nck1 is cell autonomous by showing that HepG2 cells treated with Nck1 siRNA have reduced ER stress-induced UPR and Insulin Receptor Substrate-1 (IRS-1) serine phosphorylation. In these cells, we observed that the IRS-1 levels and activation of signalling components downstream of the insulin receptor were increased. This correlates with enhanced cell survival to stress and insulin stimulated glycogen synthesis. Overall, we demonstrated that Nck1 participates in ER-stress-induced insulin resistance and regulation of IRS-1-dependent signalling.
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Treitinger, Aricio. "Alterações metabólicas e do sistema de defesa antioxidante no plasma e em células mononucleares decorrentes da infecção pelo vírus da imunodeficiência humana." Universidade de São Paulo, 1996. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-10032015-110940/.
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No presente trabalho analisou-se um total de 101 indivíduos, sendo 26 não infectados e 75 infectados pelo HIV e classificados de acordo com o Walter Reed Army Institute (28 pacientes WR 1, 31 pacientes WR 2 e 16 pacientes WR 3/4). 05 indivíduos infectados pelo HIV apresentaram, nos estágios iniciais, uma diminuição progressiva do peso corporal, dos níveis séricos de uréia, albumina, colesterol total, HOL colesterol e LOL colesterol. Já os níveis séricos de proteínas totais, globulinas, IgG, IgA, α1 glicoproteína ácida, haptoglobina e as atividades enzimáticas da AST e da LD apresentaram elevação nos indivíduos infectados e em conseqüência da evolução da infecção. Os triglicérides demonstraram apenas tendência para aumento dos níveis séricos nos indivíduos estadiados como WR3/4. Os níveis de ferro sérico encontraram-se diminuídos nos indivíduos estadiados como WR 3/4, enquanto que a concentração de transferrina apresentou-se diminuída apenas no Grupo WR 2. Houve uma tendência para a elevação progressiva dos níveis médios de ferritina com a evolução da doença. Nenhuma alteração foi verificada nos níveis de proteína \"C\" reativa. A EC-SOO apresentou diminuição dos níveis de atividade nos indivíduos infectados pelo HIV, enquanto que em células mononucleares a SOD apresentou atividade diminuída nos indivíduos estadiados como WR 3/4. A GSH-Px não apresentou alteração de sua atividade em decorrência da infecção pelo HIV. Os níveis plasmáticos do α-tocoferol e do ascorbato apresentaram tendência para diminuição, enquanto o β-caroteno não apresentou alteração nos grupos estudados. Estes resultados sugerem que a haptoglobina, as globulinas e a IgA podem ser utilizadas para a avaliação da evolução da infecção pelo HIV. Por outro lado, os níveis dos constituintes do sistema de defesa antioxidante analisados indicam que os indivíduos soropositivos encontram-se menos protegidos contra a ação de espécies reativas de oxigênio, o que favoreceria a presença de um estresse oxidativo e a replicação viral.A total number of 101 individuals, including 26 controls and 75 patients classified according to the Walter Reed Army Institute (28 WR 1, 31 WR 2 and 16 WR 3/4) was studied. HIV infected individuals presented, during the early stages, a progressive reduction of body weigth, as well as urea, albumin, total cholesterol, HDL cholesterol and LDL cholesterol in blood serum. However, increased serum levels of total protein, globulin, IgG, IgA, α1 acid glycoprotein, haptoglobin, AST and LD were observed in HIV infected individuals during the evolution of infection. Decreased serum iron and a trend for increasing triglyceride was shown only for those individuals classified as WR 3/4. Transferrin was diminished only in the WR 2 group. A trend for enhancing serum ferritin following the progession of HIV infection was also observed. No alteration was observed on the levels of reactive \"C\" protein. Decreased EC-SOD activities were observed in HIV infected individuals as compared to controls, whereas in mononuclear cells the SOD activity was diminished only in WR 3/4 patients. HIV infection did not alter GSH-Px activity. A trend for decreasing α-tocopherol and ascorbate plasma levels was shown during the evolution of HIV infected patients, while no difference was observed for β-carotene levels in the studied groups. The above results suggest that haptoglobin, globulins and IgA can be used to assess the evolution of the HIV infection. Moreover, the decreased levels of the antioxidant defense system components observed in HIV infected patients may indicate that they are under an oxidative stress that could favor HIV replication.
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Marini, Wanda. "Comparing mutant p53 and a wild-type p53 isoform, p47 : rationale for the selection of mutant p53 in tumours." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116033.
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One of the major unresolved questions in cancer biology is why the majority of tumour cells express mutant p53 proteins. p53 is considered the prototype tumour suppressor protein, whose inactivation is the most frequent single genetic event in human cancer (Bourdon et al., 2005). Genetically-engineered p53-null knockout mice acquire multiple tumours very early on in life and human Li-Fraumeni families who carry germline mutations in p53 are highly cancer-prone (reviewed in Vousden and Lane, 2007). p53 mutant proteins have been found to acquire novel functions that promote cancer cell proliferation and survival, yet exactly why mutant p53s acquire oncogenic activity is still poorly understood. Mutant p53 has also been found to complex with wildtype p53, thus acting in a dominant negative way. However, this inhibition is incomplete since many cancers with mutant p53 alleles also have a loss of the second wild-type p53 allele and thus only express the mutant p53 (Baker et al., 1989). An N-terminal truncated p53 isoform, p47, arising from alternative splicing of the p53 gene (Ghosh et al., 2004) or by alternative initiation sites for translation (Yin et al. , 2002), has been described. Alternative splicing was found to be universal in all human multi-exon genes (Wang et al., 2008) and therefore determining the role of the p47 isoform with respect to the p53 gene is essential. Evidence in this study suggests that mutant p53 (p53RI75H) has a similar structure and function as p47, including the ability to complex with and impair both p53 and p73. Therefore, in addition to expressing a tumour suppressor protein, the p53 gene can also express an onco-protein (p47). This study therefore argues that tumours select for mutant p53 because it has gained the ability to function like p47, a wild-type p53 isoform.
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Prichard, Lisa. "The role of the IQ motif, a protein kinase C and calmodulin regulatory domain, in neuroplasticity, RNA processing, and RNA metabolism /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/6302.
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Jani, Klodiana. "The role of integrin-dependent cell matrix adhesion in muscle development /." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115688.
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Cell adhesion is essential to cell motility and tissue integrity and is regulated by the Integrin family of transmembrane receptors. Integrin binds to ligand extracellularly and provide anchor to the intracellular cytoskeleton via adhesion scaffolding proteins. In order to link cell to the surrounding matrix Integrin needs to be activated. Intracellular activation signals induce perturbations in Integrin cytoplasmic domain that are translated into a conformational change in extracellular region for high affinity ligand binding. Integrin engagement by matrix, in turn, triggers the assembly of adhesion complexes. Such early adhesions promote cytoskeletal organization with subsequent contractile activity that exerts forces against initial Integrin-matrix adhesions. In response to force, Integrin strengthens the interaction with matrix through its clustering and successive recruitment of additional adhesion components. These bidirectional regulatory loops mediated by such interactions are largely dependent on the unique function of Integrin adhesion components.We demonstrate a novel role for the PDZ/LIM domain protein Zasp as a core component of Integrin adhesions. Specifically, Zasp colocalizes with Integrins at focal adhesion in cultured cells and myotendinous junctions in Drosophila embryos. In both cases elimination of Zasp modifies Integrin function causing consequently defects in cell spreading and muscle attachment. Zasp supports Integrin adhesion to the extracellular matrix that is required to withstand tensile forces exerted during cell spreading and muscle contraction. Furthermore, we found that the distribution of Zasp in muscle Z-lines is essential to orchestrate the cross-linking of alpha-Actinin and Actin filaments. Disruption of Zasp leads to loss of muscle cytoarchitecture, pointing to a larger role for Zasp in sarcomere assembly. Finally, we demonstrate that Zasp, in addition to alpha-Actinin, physically interacts with the Integrin- and Actin-bound cytoskeletal protein Talin.
Collectively, our results point to a dual role for Zasp as a structural scaffold. First it regulates Integrin adhesion to the extracellular matrix by interacting with the head domain of Talin at the myotendinous junctions. Second, Zasp controls sarcomere assembly by tethering the presarcomeric alpha-Actinin component to the tail domain of Talin. Zasp finding as a crucial adhesion component provides further insights on the mechanism underlying Integrin-mediated adhesion.
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Sonnberg, Stephanie, and n/a. "Chordopoxviruses encode a novel class of F-box proteins." University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090120.145226.
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Poxviruses are an extensive family of large DNA viruses, including the human pathogen variola virus, which causes smallpox. Vertebrate poxviruses encode numerous proteins of unknown function that contain an ankyrin repeat (ANK) domain. ANK domains have been shown to mediate protein-protein interactions and are present in a large number of cellular proteins involved in pathways such as transcription, cell cycle regulation and development. Recently, an F-box-like motif, which conserved key residues of the cellular F-box motif, was identified at the C-terminus of these proteins. Most cellular F-box proteins are specificity factors of SCF1 ubiquitin ligases, recruiting substrate proteins to the SCF1 complex where they are poly-ubiquitinated. F-box proteins use their N-terminal F-box to bind the SCF1 complex, and a second interaction motif, such as a leucine-rich-repeat domain, but never an ANK domain, to recruit the substrate. Poxviral ANK/F-box-like proteins exhibit a reversed domain order compared to cellular F-box proteins, and a novel domain combination of ANK domain and putative F-box. The aim of this study was to examine the functionality of the poxviral F-box-like motif and to assess whether poxviral ANK/F-box-like proteins could function as specificity factors of cellular ubiquitin ligases.
Using immunoprecipitation of transiently expressed recombinant proteins, several poxvirus F-box-like motifs that represented the sequence variation and differences in motif length observed among poxvirus ANK/F-box-like proteins, were tested and found to be functional F-box domains. The poxviral ANK/F-box proteins interacted with all three subunits of the SCF1 complex, Skp1 (directly), Cul1 and Rbx1 (indirectly) regardless of the manner of SCF1 expression (transient over-expression or endogenous). A representative poxviral F-box was found to be both necessary and sufficient for binding SCF1, while the ANK domain of the same protein was expendable. SCF1 ubiquitin ligases bound by a poxviral ANK/F-box protein remained competent in forming poly-ubiquitin chains, indicating that these poxviral proteins are not inhibitors of SCF1.
This evidence strongly suggests that poxvirus ANK/F-box proteins function as specificity factors of SCF1 ubiquitin ligases. Individual chordopoxviruses each encode several discrete ANK/F-box proteins. The five ANK/F-box proteins of one poxvirus, orf virus, were competent in binding SCF1, and when transiently expressed, were found to exhibit different sub-cellular localization patterns that were consistent in two cell lines and in the presence and absence of orf virus infection. Furthermore, the ANK domains of the five orf virus ANK/F-box proteins were distinct in primary amino acid sequence and in predicted tertiary structure. Together theses findings indicate that the five orf virus ANK/F-box proteins interact with different binding partners.
In summary, this study identifies a novel class of F-box proteins encoded by chordopoxviruses that exhibits a reversed domain order and a novel domain combination of ANK domain with a truncated, but functional, F-box motif. Bioinformatic analysis, structure modeling, and microscopy findings suggest that chordopoxvirus ANK/F-box proteins function as specificity factors of SCF1 ubiquitin ligases. Several viruses use specificity factors to target cellular anti-viral factors to the ubiquitination system, a virulence mechanism that now seems likely to also exist in poxviruses.
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Dai, Tong. "Differential Role of CEACAM Proteins in Regulating Insulin Metabolism." University of Toledo Health Science Campus / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=mco1139336269.
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Ricard, Michelle. "Iron acquisition from porcine proteins by Actinobacillus pleuropneumoniae biotype 1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0034/MQ64438.pdf.
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Morris, Patricia Ann. "EXAFS of non-heme iron containing proteins." Diss., Georgia Institute of Technology, 1986. http://hdl.handle.net/1853/27402.
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Bian, Zhao. "Nucleator-driven assembly of curli organelles and their pathophysiological role in E. coli septic shock /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3688-9/.
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Chénard, Carol Anne. "Ribonucleoprotein complexes and protein arginine methylation : a role in diseases of the central nervous sytem." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115894.
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For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways.QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA.
Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes.
Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.
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Danai, Laura V. "Role of Protein Kinase Map4k4 in Energy Metabolism: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/791.
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Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D).
To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.
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Danai, Laura V. "Role of Protein Kinase Map4k4 in Energy Metabolism: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/791.
Full textAbstract:
Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D).
To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.
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Hamadeh, Mazen Jamal. "Methods for detecting abnormal adaptation to protein restriction in humans with special reference to insulin-dependent diabetes mellitus." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36948.
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Postprandial urea production in subjects with insulin dependent diabetes mellitus (IDDM) on conventional insulin therapy is normal when the previous diet is high in protein, but there is an incomplete adaptive reduction in urea production following protein restriction. To evaluate the nutritional implications of restricted protein intake in human diabetes mellitus, it is first necessary to establish a reliable method to measure changes in urea production and amino acid catabolism in response to changes in dietary protein intake. We therefore tested (1) the accuracy of the urea production rate (Ra) to depict changes in urea production, (2) whether sulfate production can be accurately depicted using tracer or nontracer approaches, after establishing the use of electrospray tandem mass spectrometry to measure sulfate concentrations and 34SO4 enrichments following administration of the stable isotope tracer sodium [34S]sulfate, (3) the reproducibility of urea and sulfate measurements following a test meal low in protein (0.25 g/kg) in subjects previously adapted to high (1.5 g/kg.d) and low (0.3 g/kg.d) protein intakes, and compared the metabolic fate of [ 15N]alanine added to the test meal with that of [15N] Spirulina platensis, a 15N-labeled intact protein, and (4) whether we could identify the differences in postprandial urea and sulfate productions between normal subjects and persons with IDDM receiving conventional insulin therapy previously adapted to high protein intake, when the test meal was limiting in protein. Under basal conditions, steady state urea Ra is an accurate measure of urea production. Following changes in urea production, both the tracer and nontracer methods seriously underestimated total urea Ra. The tracer method overestimated sulfate production by 20%, but the nontracer method provided an accurate measure of sulfate production and, hence, sulfur amino acid catabolism. Postprandial changes in urea and sulfate productions following normal ada
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Chan, Siu Chiu. "Regulation of cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway and characterization of Cidea's interacting proteins /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202007%20CHANS.
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Rossmann, Maxim [Verfasser]. "Structural analysis of proteins of human sphingolipid metabolism / Maxim Rossmann." Berlin : Freie Universität Berlin, 2009. http://d-nb.info/1023263297/34.
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Whittingham, Jane. "Cellular Roles for Proteins Linked to Phosphatidylinositol (3,5)-bisphosphate Metabolism." Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.502364.
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The phosphoinositide lipids Ptdlns(3)P and Ptdlns(3,5)P2 play important roles on the endocytic pathway. Ptdlns(3)P localises to early endosomes and multivesicular bodies (MVBs) and is proposed to recruit proteins involved in fusion of early endosomes and internalisation of ubiquitinated receptors. Ptdlns(3,5)P2 is proposed to be involved in terminal maturation of Iysosomes and endosome to Golgi transport, but the precise role of this lipid in mammalian cells remains unclear. Studies in yeast have identified various proteins associated with Ptdlns(3,5)P2 metabolism, for which mammalian homologues have been found. These are the Ptdlns(3)P5- kinase Fab1/PIKfyve, Fig4 a 5-phosphatase that dephosphorylates Ptdlns(3,5)P2, Vac14 which has been shown to act as an upstream activator of PIKfyve, and WIPI-2/Svp1 a putative downstream effector of Ptdlns(3,5)P2. In this study I have further characterised three of these proteins and examined the cellular roles of all four with respect to a variety of trafficking pathways in which Ptdlns(3,5)P2 has been implicated. siRNA was used to examine the knockdown phenotypes of each of these proteins. Furthermore, I directly compared for the first time the effects of knockdown of PIKfyve, with acute pharmacological inhibition of its enzymatic activity. Loss of PIKfyve activity caused a failure in the retrieval of a variety of different cargos to the trans-Golgi network (TGN), including mannose-6-phosphate receptors, responsible for efficient delivery of lysosomal enzymes, and the TGN resident protein TGN46, leading to their accumulation in dispersed punctae. A failure in tyrosine kinase receptor downregulation was also observed following combined knockdown of PIKfyve with either Vac14 or Fig4 or following pharmacological inhibition of PIKfyve. PIKfyve knockdown alone had no effect, suggesting a low threshold of Ptdlns(3,5)P2 is necessary and sufficient for this pathway. Svp1 is the best characterised Ptdlns(3,5)P2 effector in yeast and is also an autophagy-related gene (Atg18), thereby implicating Ptdlns(3,5)P2 in this process. A family of putative mammalian Svp1 homologues have been identified, known as the WIPI family. I investigated the role of Ptdlns(3,5)P2 and WIPI-2 in mammalian autophagy. By monitoring formation of the autophagosome marker GFP-LC3 II, PIKfyve and WIPI-2 were found to have opposite effects. Furthermore, WIPI-2 redistributes to punctate structures upon induction of autophagy, which partially colocalise with autophagosome markers, in a manner dependent not on PIKfyve but on PI(3)-kinase activity. The evidence presented suggests that Ptdlns(3,5)P2 may playa role in mediating the maturation of a subset of MVBs, leading to swelling of endosomal compartments and rendering the MVBllate endosome or autophagosomes refractory to fusion with the lysosome in cells depleted of PIKfyve.
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Wright, Sarah M. "The effects of dietary proteins on cholesterol and lipoprotein metabolism." Thesis, University of Nottingham, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262479.
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Tilley, Gareth John. "Electrochemical investigations into iron-sulfur cluster containing proteins." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365300.
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Rison, Stuart Christopher Gorthorn. "Of proteins and pathways : investigating protein functional classifications and the small molecule metabolism of escherichia coli." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397378.
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Andersson, Evalena. "Structure-function studies of enzymes from ribose metabolism /." Uppsala : Acta Universitatis Upsaliensis : Univ.bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3999.
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Philips, Brian John. "Protein interactions with the catechol estrogens 4-hydroxyestrone and 4-hydroxyestradiol in mouse tissue lysate : binding and metabolism studies /." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3036851.
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McDonald, Helen L. "Studies on the processing of rubella virus structural proteins by analysis of the endoproteolytic cleavage sites." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/28739.
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Rubella virus is a small enveloped positive strand RNA virus. Two species of viral RNA are found in infected cells: a full-length genomic RNA and a subgenomic species corresponding to the 3' one third of the genomic RNA molecule. The 24S subgenomic RNA specifies a polyprotein which is cotranslationally processed by endoproteolytic cleavage by host signal peptidase to yield three structural proteins, El, E2 and capsid. El and E2 are membrane glycoproteins forming the virion spikes, and C protein binds to 40S genomic RNA to form a nucleocapsid. El and E2 proteins contain N-linked oligosaccharide as a consequence of their passage through the endoplasmic reticulum (ER) and Golgi apparatus. According to the signal hypothesis, translocation of secretory and membrane proteins into the ER is mediated by a hydrophobic signal peptide. The signal peptides for E2 and El have been localized by in vivo expression of El and E2 cDNAs.
Oligonucleotide-directed mutagenesis was used to define the cleavage sites between C, E2, and El, as well as the effect of the cleavages on the transport and processing of E2 and El. The expression of the cleavage site mutants was studied in vitro and in vivo. It was found that uncleaved precursor polypeptides were retained in the ER and very little E2 or El polypeptide was observed at either the Golgi apparatus or the plasma membrane. The E2 and El polypeptides can cross the ER membrane without the cleavage of the signal peptide while the transport of E2 and El beyond the ER requires the cleavage of E2 from C and El from E2.
The C-termini of the C and E2 proteins, which were not previously defined, have been partially characterized. Capsid protein does not appear to undergo further proteolytic processing after it is cleaved from E2 by signal peptidase, but E2 may be processed at a second cleavage site at its C-terminus by a trypsin-like enzyme.Medicine, Faculty of
Medical Genetics, Department of
Graduate
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Birks, Stephen J. "Ketone metabolism in the purple non-sulphur bacterium Rhodobacter capsulatus." Thesis, University of Sheffield, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387852.
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Yamani, Lama. "Studies on transcobalamin in cultured fibroblasts from patients with inborn errors of cobalamin metabolism." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112320.
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Cobalamin must be metabolized intracellularly in order to bind two enzymes: methionine synthase in cytoplasm and methylmalonyl-CoA mutase in mitochondria. Defects in this process cause different inborn errors of cobalamin metabolism (cblA-cblG and mut). A previous study described a cobalamin-binding protein, in addition to methylmalonyl-CoA mutase, in crude mitochondrial fractions. The amount of [57Co]cobalamin bound to this protein was increased in cblB, mut and cblD variant2 cell lines, compared to control cell lines. In the present study, this protein was identified as transcobalamin (TC). Mitochondrial fractions from a cblB cell line were incubated with anti-TC antibodies, which precipitated the cobalamin-bound protein. Analysis of mitochondrial and cytoplasmic fractions isolated from a chloroquine-incubated cblF cell line showed that isolated mitochondrial fractions contain lysosomal material, suggesting that the identified TC is lysosomal. Quantification of cobalamin-bound TC levels in whole cell extracts showed significant increases in cblB and mut groups compared to control cell lines.
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Gelling, Cristy Lee Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Tetrahydrofolate and iron-sulfur metabolism in Saccharomyces cerevisiae." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/43270.
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Tetrahydrofolate-mediated one-carbon metabolism is required for the biosynthesis of many central metabolites, including some amino acids, nucleobases, and nucleotides, and hence dysfunction of one-carbon metabolism is associated with many human diseases and disorders. The mitochondrial glycine decarboxylase complex (GDC) is an important component of one-carbon metabolism, generating 5,10-methylene-tetrahydrofolate (5,10-CH2-H??4folate) from glycine. Previous work has shown that the genes encoding the unique sub-units of the Saccharomyces cerevisiae GDC (GCV1, GCV2 and GCV3) are regulated in response to changes in the levels of cytosolic 5,10-CH2-H??4folate (Piper et al., 2000). Given the centrality of 5,10-CH2-H??4folate to many aspects of metabolism, it was hypothesised that other genes may be regulated by the same mechanism. Using microarray analysis of S. cerevisiae under a number of conditions that affect 5,10-CH2-H??4folate levels, the ??one-carbon regulon??, a group of genes that were co-regulated with the GCV genes was identified. The one-carbon regulon corresponds closely to genes whose promoters are bound by the purine biosynthesis regulator Bas1p, but not all one-carbon regulon members are significantly purine regulated. Genetic approaches demonstrated that the one-carbon unit response and the purine response are distinct, though both depend on the presence of Bas1p. This demonstrated that the close metabolic connections of one-carbon and purine metabolism are reflected in over-lapping, but separable regulatory mechanisms. The identity of the sensor of one-carbon unit depletion remains unknown, but in the course of investigation of the candidate regulator Caf17p, it was demonstrated that Caf17p is in fact involved in Fe/S cluster protein maturation. Examination of the effects of Caf17p depletion revealed that Caf17p is required for the function and maturation of the related mitochondrial Fe/S proteins aconitase and homoaconitase, as well as the function of, but not de novo iron incorporation into, the mitochondrial radical-SAM Fe/S protein biotin synthase. Because other Fe/S proteins were unaffected, Caf17p appears to be a specialised Fe/S maturation factor. The presence of a putative H4folate binding site indicates that Caf17p may constitute a metabolic link between one-carbon and iron metabolism.
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Whytock, K. "An exploration into the proteins that regulate skeletal muscle lipid metabolism." Thesis, Liverpool John Moores University, 2019. http://researchonline.ljmu.ac.uk/9962/.
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Dysregulation of intramuscular triglyceride (IMTG) turnover in human skeletal muscle in sedentary and obese states leads to accumulation of lipid metabolites that contribute to skeletal muscle insulin resistance and ultimately progression to type 2 diabetes (T2D). People with T2D display low levels of IMTG turnover in comparison to insulin sensitive and trained individuals. IMTG stores are used as an energy substrate during 1 h of moderate-intensity exercise in trained individuals only and can be increased by consumption of a high fat, high calorie (HFHC) diet in sedentary and trained states. This thesis explores the metabolic and molecular regulation of proteins that regulate IMTG turnover, specifically focusing on the effects of 1) a HFHC diet and 2) a moderate-intensity exercise bout and 3) IMTG stores in different diseases states (lean, obese and T2D). Chapter 2 determined there were no sex-specific differences or main effects in functional outcomes of cardiovascular (arterial stiffness) and metabolic health (glucose tolerance and metabolic flexibility) in response to 7 days HFHC diet. Chapter 3 provides novel evidence that 7 days HFHC diet induces fibre type specific increases in IMTG stores primarily underpinned by an increase in perilipin-3 (PLIN3) protein expression and a redistribution of perilipin-2 (PLIN2) to lipid droplets (LD) storing IMTG. This occurred with no impairments in skeletal muscle insulin signalling and it is therefore proposed that increases in IMTG content assisted by PLIN2 and PLIN3 minimise the accumulation of lipid metabolites known to disrupt the insulin signalling cascade. Chapter 4 revealed that hormone sensitive lipase (HSL) preferentially redistributes to LD associated with perilipin-5 (PLIN5) following 1 h of moderate-intensity exercise. Chapter 5 developed a PLIN5 immunoprecipitation mass spectrometry protocol which identified phospholipase A2- group II, subgroup A (PA2GA) as a novel protein associated to PLIN5 in muscle from lean sedentary humans. In conclusion, this thesis presents novel data on key proteins that regulate IMTG turnover in human skeletal muscle.
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He, Didi. "Structural basis for iron (II) metabolism in encapsulated ferritin-like proteins." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23466.
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Ferritins are ubiquitous proteins that serve the dual-function of iron reservoir and sequestering the Fe(II) toxicity. The function of ferritins totally depends on the characteristic spherical structure with a di-iron centre performing the iron oxidation and a hallow cavity enclosing the iron minerals in a bioavailable form. I have characterised the structure, assembly and function of a new member of ferritin superfamily that is natively enclosed within an encapsulin shell. Encapsulin proteins are structurally-related to a virus capsid and form 60-meric or 180-meric icosahedrons. I show that this encapsulin associated ferritin-like protein (EncFtn) possesses two main alpha helices, which assemble in a metal-dependent manner to form a ferroxidase centre at a dimer interface. EncFtn adopts an annular decamer structure in contrast to the 24-meric classical ferritins or 12-meric mini-ferritin (DPS). The resemblance of the dimeric EncFtn and monomeric classical ferritins suggests that it is likely that classical ferritin evolves from EncFtn because of the gene duplication. EncFtn is a catalytically active ferroxidase but with only a limited iron binding ability due to its open structure. The encapsulin itself is not able to oxidise Fe(II), but is able to store about 2200 iron ions. I have demonstrated that the EncFtn must be housed in the encapsulin to achieve a maximum loading of approximately 4200 iron ions. The encapsulin nanocompartments are widely-distributed in both eubacteria and archaeon with distinct life styles and represent a distinct class of iron storage system, where iron oxidation and mineralisation are distributed between two proteins.
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Pedro, Roig Laia. "GlnK regulatory proteins and their role in Haloferax mediterranei nitrogen metabolism." Doctoral thesis, Universidad de Alicante, 2012. http://hdl.handle.net/10045/27319.
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Parolari, A. "LEGUME PROTEINS FOR THE MANAGEMENT OF CHRONIC DISEASES:HYPERLIPIDEMIA AND DIABETES." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/245903.
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Food proteins can be considered as source of bioactive peptides that can exert physiological functions to promote health and prevent chronic diseases, such as lipid disorders diabetes, hypertension cancer and obesity which are typical of industrialized societies. Soybean (Glycine max) and white lupin (Lupinus angustifolium) comprise the most widely grown legume crops in the world. In addition to being an invaluable source of oil and proteins for food and feed, many papers from our group pointed out the positive effect of soybean and white lupin proteins on lipid and glucose metabolism. The aim of the present work has been to evaluate in “in vitro” and “in vivo” experiments the ability of soybean and white lupin polypeptides to interact with the molecular mechanisms involved in the regulation of plasma and tissue lipids as well as on the glucose homeostasis. Soybean proteins: the α’ subunit of the soybean 7S globulin, the so called β-conglycinin, was shown to play a key role in the up-regulation of liver high affinity-LDL receptors, in “in vivo” and “in vitro” systems suggesting that biologically active peptides, capable of modulating lipid homeostasis, are likely to be produced by cell and gastrointestinal enzymes. Our research group has been following two different approaches to identify the active peptide/s involved in the lipid regulation. The first one has been to reduce the length of the polypeptide chain of α’ by a biotechnological process obtaining an extension form of α’ chain, roughly covering one third of the full-length polypeptide from N-terminus, which has proved active in the LDL-R up-regulation of Hep G2 cells. The second approach has been to make a screening of peptides with amino acid sequences occurring in α’, α and β subunits of soy β-conglycinin, and test their biological effect “in vitro”. These peptides have been evaluated for their effect on the expression of LDL-receptor, SREBP-2 and HMGCoA red mRNAs in HepG2 cells. Moreover, the peptide that proved more promising among the different compounds under study, has been tested in a rat model of human hypercholesterolemia in order to evaluate its potential on lipid homeostasis. gamma-Conglutin, a lupin seed glycoprotein, plays a key role on the glucose metabolism. Since the “in vitro” interaction of gamma -conglutin with mammalian insulin has been described, in the present study the effect of an oral dose of this protein was studied in an animal model of diabetes in order to evaluate its potential in the fine regulation of glucose homeostasis. Moreover in “in vitro” experiments the ability of gamma-conglutin to interact with cell compartment and to interfere in the insulin pathway has been followed in order to ascertain whether the protein was characterized by insulin-like property. Although the data obtained in this study should be confirmed by human studies, the potential of peptides from α’ subunit of soybean as well as that of lupin seed g-conglutin to control plasma lipids and glycaemia could be considered before developing new therapeutic strategies for the prevention or treatment of lipid/ glucose metabolism disorders.
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Högberg, Pi. "Disruption of vitamin A metabolism by dioxin /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-608-1/.
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Fang, Che. "Cytokines, alcohol metabolizing enzymes and stress-inducible ER proteins in alcoholic liver disease /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4160-2/.
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Stromme, Adrianna. "The characterization of the cytoskeleton and associated proteins in the formation of wound-induced contractile arrays /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116078.
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The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development.Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42.
The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure.
Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.
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Beauchamp, Pascal. "The functional role of the RNA-binding protein HuR in the regulation of muscle cell differentiation /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111586.
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Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.
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Bae, Dong-Hun. "The Effects of Iron Levels on the Interaction between Polyamine Metabolism and Iron Metabolism in Neoplastic Cells." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/18081.
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Iron is a crucial element that is associated with many metabolic pathways important for life sustaining processes. Polyamines are small positively charged polycations involved in various physiological functions. Both iron and polyamines levels are known to be high in cancer cells which suggests a possible unexplored link between the two metabolic pathways. For the first time, we demonstrate that iron-depletion robustly regulates the expression of 13 polyamine pathway proteins. Iron-depletion also decreased polyamine and S-adenosylmethionine levels (required for spermidine/spermine biosynthesis) and decreased 3H-spermidine uptake in accordance with expression of the polyamine importer, SLC22A16. The “reprogramming” of polyamine metabolism by iron-depletion showed dependence on the proto-oncogene, c-Myc, and tumour suppressor, p53 expression. Furthermore, the ability of iron chelators to inhibit proliferation can be rescued by polyamine supplementation. Collectively, these data demonstrate that iron and polyamine metabolism are closely linked at multiple levels. Moreover, we have identified that the mRNA and protein expression of the iron-containing enzyme, aci-reductone dioxygenase 1 (ADI1), was regulated by iron levels. Cellular iron depletion or deficient ADI1 metalation by the iron chaperone, PCBP1, promotes the proteasomal degradation of ADI1. Collectively, this demonstrates that cellular iron regulates ADI1 stability, a key enzyme involved in methionine salvage, polyamine biosynthesis and proliferation. In addition to regulating ADI1, poly(rC)-binding proteins (PCBPs) have been reported to function as iron-binding chaperones that deliver iron to ferritin. We observed that PCBP2 enhances, while PCBP1 inhibits ferritin 59Fe-loading and ferritin protein expression. Our results suggest that the regulation of ferritin iron-loading by PCBPs may involve a combination of translational regulation and/or iron chaperone activity. Overall this study has demonstrated for the first time the direct interaction between iron metabolism and polyamine metabolism, which is important in understanding how cancer cells can survive adverse environments.
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Sundaram, Priyanka. "The deubiquitinating enzyme USP19 negatively regulates the expression of muscle-specific genes in L6 muscle cells /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111547.
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Muscle wasting is a significant complication of many diseases including diabetes mellitus, renal and liver failure, HIV/AIDS, and cancer. Sustained loss of skeletal muscle can severely impair a patient's quality of life and often results in poor tolerance and responsiveness to disease treatments. The increased protein breakdown observed during muscle atrophy has been attributed to accelerated activity of the ubiquitin-proteasome pathway, but the precise mechanisms by which this activation stimulates muscle protein loss are poorly understood. Previous work showed that the deubiquitinating enzyme USP19 is upregulated in rat skeletal muscle in various forms of muscle wasting, including streptozotocin induced diabetes, cancer, and dexamethasone treatment. 1 To further explore the role of USP19 in muscle wasting, siRNA-mediated depletion of the enzyme was carried out in L6 myotubes. Knockdown of USP19 resulted in more rapid differentiation of myoblasts into myotubes, with a greater extent of myoblast fusion. It also produced tubes that were visibly larger than those formed by myoblasts transfected with a control siRNA. At the molecular level, silencing of USP19 increased the amount of myosin heavy chain (MHC) and tropomyosin proteins. It also increased levels of MHC transcript, suggesting that USP19 acts at the level of gene transcription or mRNA stability rather than protein degradation. USP19 may mediate its effects on muscle-specific gene expression through the myogenic transcription factor myogenin, since depletion of USP19 increased protein and mRNA levels myogenin but did not affect protein levels of the related transcription factor Myf5. Moreover, the increased tropomyosin and MHC observed upon USP19 knockdown could be abolished when myogenin was simultaneously depleted using siRNA. Collectively, these results suggest that USP19 functions to inhibit the synthesis of key muscle proteins and may therefore be a promising target for the treatment of muscle atrophy.
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Pertile, Tatiane 1982. "Estudo do crescimento do carcinossarcoma de Walter 256 em ratos jovens e adultos, suplementados com ácido eicosapentaenóico (EPA)." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314500.
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Orientador: Maria Cristina Cintra Gomes MarcondesDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O câncer pode promover a morte do hospedeiro, pois durante sua evolução há modificações da homeostasia dos processos metabólicos, promovendo profundas alterações caracterizadas como caquexia, que por sua vez relaciona-se à diminuição da qualidade e do tempo de vida do hospedeiro. Assim, no presente estudo, analisamos os efeitos da evolução do crescimento de neoplasia - carcinossarcoma de Walker 256 - em ratos jovens e adultos e os efeitos modulatórios do tratamento desses animais com EPA (ácido eicosapentaenóico) sobre o processo de caquexia e a concentração de citocinas anti e proinflamatórias no músculo gastrocnêmio, pois o tecido muscular é o tecido mais afetado no processo de caquexia. Foram utilizados 108 ratos Wistar machos, com idade de 30 dias (jovens) e 100 dias (adultos), os quais foram distribuídos de acordo com o local de implante tumoral, intraperitônio e subcutâneo, e tratamento ou não com ácido eicosapentaenóico, 100?g/Kg de peso corpóreo. Os animais receberam gavagem diária do EPA (animais tratados) ou de óleo mineral (grupos sem tratamento) até a fase pré-agônica. A partir dos órgãos coletados, foram calculados o ganho de peso corpóreo, os pesos relativos de cada órgão, do tumor e da carcaça. Com o objetivo de identificarmos a via de degradação protéica predominante nos grupos experimentais, foram avaliados, no tecido muscular, o teor de proteína muscular total e as atividades das seguintes enzimas: chymotrypsin-like, catepsinas B e H, calpaína e fosfatase alcalina,. A partir do sangue desses animais foram feitas análises fluorimétricas do fator de crescimento semelhante à insulina (IGF) e das citocinas - interleucinas 4 (IL-4), 6 (IL-6) e 10 (IL-10), interferon gama (INF-?) e leptina, utilizando-se kits específicos para citômetro de fluxo de fluorescência (Luminex). Também foi analisada a expressão gênica, no tecido muscular, por reação em cadeia da polimerase em tempo real (PCR-RT), para a via proteossômica e também para os fatores eucarióticos de inicialização. Os dados indicam efeitos modulatórios do EPA sobre o tecido muscular (manutenção da proteína e do peso), principalmente para os grupos jovens, e também no processo inflamatório crônico (aumento de citocinas pró e antiinflamatórias). Entretanto, efeitos mais expressivos do EPA não foram verificados na prevenção da espoliação de gordura (gordura perirrenal e leptina), no processo de síntese protéica (manutenção da expressão gênica de fatores eucarióticos de inicialização) ou também sobre a via proteossômica
Abstract: Cancer can promotes the host death, because during its evolution there are modifications in metabolic processes of homeostasis, promoting deep changes characterized as cachexia, which in turn relates with reduction in quality and lifetime of the host. Thus, in this study, we analyze the effects of development in Walker 256 carcinoma evolution - in young and adults rats and the modulatory effect of treatment with EPA (eicosapentaenoic acid) in gastrocnemius muscle as this tissue is the most affected in the cachexia process. Wistar males rats were used (n=108 animals), 30 days-old (young) and 100 days-old (adults), which were distributed according to the tumour implant, intraperitoneally and subcutaneously and treatment or not with eicosapentaenoic acid, 100 ?g/Kg of body weight. The animals receive daily EPA by gavage (treated animals) or nujol (sham groups) and were cared up to pre-agonic state. The bodies weight were measured and the body weight gain was calculated, as well the relative weights of tissues, tumour, and carcass. In order to identify the predominant pathway of protein degradation the total muscle protein content and proteolytic enzymes activities (chymotrypsin-like, cathepsin B and H, calpain and alkaline phosphatase) were measured in gastrocnemius muscle. The blood samples were assessed to measure the insulin-like growth factor 1 (IGF-1), leptin and the cytokines - interleukins (IL-4), 6 (IL-6) and 10 (IL-10), gamma interferon (INF-?), using specific kits for cytometer fluorescence (Luminex). It was also examined gene expression, in the muscle tissue, by real-time polymerase chain reaction (RT-PCR), assessing keys of ubiquitin-proteasome pathway and also on the eukaryotic initiation factors. The data indicated some modulatory effects of EPA on the muscle tissue (maintenance of protein and weight), mainly for the young rats, and also the chronic inflammatory process. However, more expressive effects of EPA have not been verified as preventing fat wasting (perirenal fat and leptin), nor in the process of protein synthesis (maintenance of eukaryotic initiating factors gene expression) or also in the ubiquitin-proteasome via
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular
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Farjo, Krysten Michelle. "The role of retinol dehydrogenase 10 in vitamin A metabolism." Oklahoma City : [s.n.], 2009.
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Behmoaram, Emy. "Biological studies of fascin function in cancer cell invasion and cancer progression." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111596.
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The process of metastasis is initiated through the acquisition of inherent and autonomous motile and invasive properties by tumor cells. These phenomena are initiated through a balance between forward cancer cell membrane protrusion and tail retraction, and occur via cell cytoskeleton remodeling, actin reorganization, and coordinated focal adhesion assembly and disassembly events. Among the vast network of cytoskeletal proteins, the actin-bundling protein fascin plays a major function in cell cytoskeleton remodeling. It is a 55-kDa protein involved in the formation of filopodia and cell migration, and found to be upregulated in many cancers. We report herein key functions for fascin in the regulation of prostate and breast cancer progression. Fascin expression is upregulated in localized and hormone refractory prostate cancer, responsible for a more aggressive clinical course. In addition, functional dissection of fascin reveals a novel function in the regulation of focal adhesion turnover dynamics, by modulating the phosphorylation state of central focal adhesion proteins through a potential collaboration with the protein tyrosine phosphatase, PEST. Together, our data support the importance of fascin in cancer cell invasion and as a significant prognostic marker and a potential therapeutic target for aggressive cancers.
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Yau, Tsz Wai. "Erythrocyte membrane dynamics and engineering : studies of the cell's morphology, metabolism, membrane flickering and transport." Thesis, The University of Sydney, 2011. https://hdl.handle.net/2123/28842.
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A cell is the fundamental unit of living things. All cells are encapsulated by a phospholipid bilayer called the cell membrane, which is important for enclosing cellular contents. The cell membrane contains cholesterol, proteins (both peripheral and integral), and carbohydrate chains that attach to either membrane proteins or phospholipids (glycoproteins, or glycolipid, respectively). The cell membrane is generally semipermeable to small solutes (e.g. water, 02, and CO2), and the transmembrane exchange is regulated extensively by various channels and transporters.
The central theme of this thesis is to understand the biophysics and biochemical properties of the cell membrane in relation to the cell's ability to deform and adapt to physical and chemical stress. I focus on the human red blood cell (RBC), using various cytotoxins as agents that alter membrane properties. The human RBC is a non-nucleated cell filled with haemoglobin,~ 150 g (L of whole blood)"1, which circulates around the body via the cardiovascular system, exchanging gases (oxygen and carbon dioxide) for various tissues in the human body. During circulation through narrow blood capillaries (sometimes ~2-3 µm in diameter) an RBC is able to withstand large shear stresses, which in addition to its simple cell structure, having no organelles, makes it an ideal model to study cell membrane biology.
The dynamics of RBC membrane fluctuation ("flickering") are believed to be an important contribution to the cell's ability to "wiggle" through narrow capillaries. There exists a controversy as to whether flickering is thermodynamically or metabolically driven. Differential interference contrast (DIC) microscopy was used to investigate RBC flickering under various biochemical stresses. A computer model of the RBC was used to simulate flickering data for comparison with the experimental DIC results.