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1
Maxwell, Megan Amanda, and n/a. "PEX1 Mutations in Australasian Patients with Disorders of Peroxisome Biogenesis." Griffith University. School of Biomolecular and Biomedical Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040219.100649.
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The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed 137 T>C and 53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.
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Maxwell, Megan Amanda. "PEX1 Mutations in Australasian Patients with Disorders of Peroxisome Biogenesis." Thesis, Griffith University, 2004. http://hdl.handle.net/10072/366184.
Full textAbstract:
The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Stroobants, An Karin. "Studies on peroxisome biogenesis." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2001. http://dare.uva.nl/document/60799.
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Li, Xiaoling. "Peroxisome proliferation and division." Available to US Hopkins community, 2002. http://wwwlib.umi.com/dissertations/dlnow/3080712.
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Liu, Xiaoxi. "BEYOND PEROXISOME: ABCD2 MODIFIES PPARα SIGNALING AND IDENTIFIES A SUBCLASS OF PEROXISOMES IN MOUSE ADIPOSE TISSUE." UKnowledge, 2014. http://uknowledge.uky.edu/pharmacy_etds/41.
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ABCD2 (D2) has been proposed as a peroxisomal long-chain acyl-CoA transporter that is essential for very long chain fatty acid metabolism. In the livers of mice, D2 is highly induced by fenofibrate, a PPARα ligand that has been widely used as a lipid lowering agent in the treatment of hypertriglyceridemia. To determine if D2 is a modifier of fibrate responses, wild-type and D2 deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knock-down of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of dietinduced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes.
Although proposed as a peroxisomal transporter, the intracellular localization of D2, especially in adipose tissue, has not been validated with direct experimental evidence. Sequential centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but lacked well-known peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with evidence of a dense matrix and a diameter of ~200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment from adipose tissue confirmed the scarcity of PEX19. Proteomic profiling of the D2 compartment revealed the presence of proteins associated peroxisome, endoplasmic reticulum (ER), and mitochondria. We conclude that D2 is localized to a distinct subclass of peroxisomes that lack many peroxisome proteins and may physically associate with mitochondria and the ER.
6
Sadeghi, Azadi Afsoon. "Identification and characterization of novel signalling pathways involved in peroxisome proliferation in humans." Thesis, University of Exeter, 2018. http://hdl.handle.net/10871/33738.
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Peroxisomes represent crucial subcellular compartments for human life and health. They are remarkably dynamic organelles which respond to stimulation by adapting their structure, abundance, and metabolic functions according to cellular needs. Peroxisomes can form from pre-existing organelles by membrane growth and division, which results in peroxisome multiplication/proliferation. Growth and division in mammalian cells follows a well-defined multi-step process of morphological alterations including elongation/remodeling of the peroxisomal membrane (by PEX11β), constriction and recruitment of division factors (e.g. Fis1, MFF), and final membrane scission (by the dynamin-related GTPase Drp1) (Chapter 1). Although our understanding of the mechanisms by which peroxisomes proliferate is increasing, our knowledge on how the division/multiplication process is linked to extracellular signals is limited, in particular in humans. The classical pathway involved in peroxisome proliferation is mediated by a family of ligand-activated transcription factors known as peroxisome proliferator activated receptors (PPARs) (Chapter 1). This project focused on identifying novel signaling pathways and associated factors involved in peroxisome proliferation in humans. In this study, a cell-based peroxisome proliferation assay using the HepG2 cell model with spherical peroxisomal forms has been developed to investigate different stimuli and their ability to induce peroxisome proliferation (Chapters 2 and 3). In this system, peroxisome elongation has been used as the read-out for peroxisome 4 proliferation. We also showed that the number of peroxisomes increased after division of elongated peroxisomes indicating peroxisome proliferation. Different stimuli, such as fatty acids, PPAR agonists and antagonists, have been used in this study. PPAR agonists and antagonists had no stimulatory or inhibitory effect on peroxisome elongation in our assay, suggesting PPAR-independent regulatory processes. However, arachidonic acid and linoleic acid were able to induce peroxisome elongation, whereas palmitic acid and oleic acid were not effective. These findings indicate that general stimulation of fatty acid β-oxidation is not sufficient to induce peroxisome elongation/proliferation in HepG2 cells. Moreover, mRNA expression levels of peroxismal genes have been monitored during a time course in the HepG2 cell-based assay by qPCR. This analysis shows a regulation of expression of peroxins during peroxisome proliferation in human cells and suggests differences in the regulation pattern of PEX11α and PEX11β. In Chapter 4, motif binding sites for transcription factors in peroxisomal genes were analyzed. An initial map of candidate regulatory motif sites across the human peroxisomal genes has been developed (Secondment at the University of Sevilla, Spain with Prof. D. Devos). This analysis also revealed the presence of different transcription factor binding sites in the promoter regions of PEX11α and PEX11β, supporting different regulatory mechanisms. Based on the computational analysis, PEX11β contained a putative SMAD2/3 binding site suggesting a novel link between the canonical TGFβ signaling pathway and expression of PEX11β, a key regulator of peroxisome dynamics and proliferation. 5 Addition of TGFβ to HepG2 cells cultured under serum-free conditions induced elongation/growth of peroxisomes as well as peroxisome proliferation supporting a role for TGFβ signalling in peroxisomal growth and division (Chapter 5). Furthermore, to demonstrate that this induction is through a direct effect of TFGβ on the SMAD binding site found in PEX11β, we performed functional studies using a dual luciferase reporter assay with PEX11β wild type and mutated promoter regions (Secondment at Amsterdam Medical Center, Netherlands with Prof. H. Waterham). Whereas luciferase activity was induced by TGFβ stimulation with the PEX11β wild type promoter, mutation of the SMAD binding site abolished activation. In summary, this study revealed a new signaling pathway involved in peroxisome proliferation in humans and provided a tool to monitor peroxisome morphology and gene expression upon treatment with defined stimuli. Furthermore, I contributed to a study revealing that ER-peroxisome contacts are important for peroxisome elongation (Chapter 6). Our group identified peroxisomal acyl-CoA binding domain protein 5 (ACBD5), ACBD4 and VABP as a molecular linker between peroxisomes and the ER (Costello et al., 2017). Motif analysis of ACBD4 and ACBD5 promoter regions revealed that unlike PEX11β, these genes do not contain a binding site for SMAD, suggesting they are not co-regulated. Also, ACBD4 and ACBD5 do not share any common transcription factor binding sites suggesting different regulation. An interesting binding motif within the ACBD4 promoter is a glucocorticoid receptor binding site. In our study, we found potential glucocorticoid response elements (GRE) in other peroxisomal genes encoding β-oxidation enzymes. This may suggest an important role for glucocorticoid receptors in activating expression of peroxisomal genes resulting in the stimulation of fatty acid breakdown and energy production.
7
Bell, Alexander. "The molecular basis of peroxisome proliferation." Thesis, University of Nottingham, 1998. http://eprints.nottingham.ac.uk/10395/.
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Characterisation of expression of functional Peroxisome Proliferator Activated Receptora (PPARalpha)receptor in rodent species responsive and non-responsive to peroxisome proliferators is important for our understanding of the molecular mechanism of peroxisome proliferation and peroxisome proliferator induced hepatocarcinogenesis. In vitro electromobility shift assays, demonstrated that rodent liver nuclear proteins (LNP) bound to a Peroxisome Proliferator Response Element (PPRE) in a sequence specific manner and that LNP from methylclofenapate (MCP) treated mice do not have enhanced binding to a PPRE. These results demonstrate that in MCP treated mice, PPAR alpha levels with functional DNA binding do not increase. The diurnal expression of mouse PPAR alpha (mPPARalpha) protein in liver was examined by western blotting. There was no observable difference in the expression of mPPARalpha across a 24 hour period. In C57 BL/6 mice, PPARalpha protein levels are not regulated in a diurnal manner. A comparison of mouse and guinea pig LNP revealed a PPARalpha-immunoreactive protein in guinea pig. Guinea Pig PPARalpha (gPPAR a) was cloned and found to encode a 467 amino acid protein. Phylogenetic analysis of gPPARalpha showed a high substition rate: maximum likelihood analysis was consistent with rodent monophyly, but could not exclude rodent polyphyly (p~0.07). The gPPAR alpha cDNA was expressed in 293 cells, and mediated the induction of the luciferase reporter gene by the peroxisome proliferator Wy-14,643, dependent upon the presence of a PPRE. The gPPAR alpha mRNA and protein was expressed in guinea pig liver, although at lower levels compared to PPAR alpha expression in mice. The evidence presented here supports the idea that guinea pigs serve as a useful model for human responses to peroxisome proliferators. mPPAR alpha DNA binding domain (mPPARalpha-DBD) was cloned and expressed as a fusion protein. Both His*6-mPPARalpha-DBD and thioredoxin-mPPARalpha-DBD were produced as insoluble proteins when over expressed in E.coli. In vitro translated mPPAR alpha-DBD did not bind to a PPRE in an electromobility shift assay.
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Yaacob, Nik Soriani. "Molecular cell biology of peroxisome proliferators." Thesis, University of Surrey, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244831.
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Castro, Ines Gomes Oliveira. "Tail-anchored proteins at peroxisomes : identification of MIRO1 as a novel peroxisomal motility factor." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/24657.
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Peroxisomes are dynamic and multifunctional organelles, which are essential for human health and development. They are remarkably diverse, with functions that vary significantly between cells and organisms, and can dramatically change their size, shape and dynamics in response to cellular cues. In the past few years, several studies have significantly increased our understanding of the basic principles that enable peroxisome biogenesis and degradation, as well as their pivotal role in cellular signalling and homeostasis. However, several of these processes are still poorly understood. In this thesis we initially studied the peroxisome targeting mechanism of a group of C-terminally anchored membrane proteins, known as tail-anchored (TA) proteins. In order to investigate the molecular signals that enable TA protein targeting to cellular organelles, we analysed the physicochemical properties of a cohort of TA proteins both in silico and in vivo, and show that a combination of transmembrane domain (TMD) hydrophobicity and C-terminal tail charge determines organelle-specific targeting. Focusing on peroxisomes, we demonstrate that a balance between TMD hydrophobicity and high positive tail charge directs TA proteins to this organelle, and enables binding to the peroxisomal chaperone PEX19. These results allowed us to create a bioinformatical tool to predict the targeting of uncharacterised TA proteins and further develop our understanding of the molecular mechanisms involved in the targeting of this protein group. From our initial TA protein screen, we identified the TA protein MIRO1 at peroxisomes and looked at its role in the regulation of peroxisome motility. We show that endogenous MIRO1 localises to mitochondria and peroxisomes, and that dual targeting depends on the C-terminal tail. MIRO1 expression significantly increased peroxisome motility in several cell lines, and revealed a role for motility in peroxisome dynamics, by inducing organelle proliferation and elongation. These results reveal a new molecular complex at peroxisomes and provide us with a tool to further dissect the role of motility on peroxisome function.
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Anderson, Steven P. "Gene modulation during peroxisome proliferator-induced hepatocarcinogenesis." NCSU, 2001. http://www.lib.ncsu.edu/theses/available/etd-20011101-131940.
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ANDERSON, STEVEN PAUL. Gene modulation in peroxisome proliferator-induced hepatocarcinogenesis. (Under the direction of Russell C. Cattley and John M. Cullen). Recognition that peroxisome proliferator chemicals are potent hepatic mitogens and carcinogens in rats and mice has generated concern about possible human health risks associated with exposure to several of these chemicals, many of which have medical or commercial utility. Our broad objective was to improve the estimation of human health risk following peroxisome proliferator exposure by defining a subset of the molecular events associated with the rodent tumors. Our working hypothesis was that peroxisome proliferator-induced tumors in rodents result from specific, peroxisome proliferator-activated receptor-a(Ppara)-modulated changes in gene expression. The research was directed toward three specific aims. First, we sought to identify genes associated with hepatocarcinogenesis induced by the peroxisome proliferator, Wy-14, 643, in the rat. The principle conclusion of these studies - that peroxisome proliferators dysregulate expression of hepatic acute-phase protein genes - suggested possible perturbations in cytokine signaling networks that also regulate cell growth. Second, although Ppara is necessary for the rodent hepatocarcinogenesis induced by peroxisome proliferators, we were interested in identifying more proximate mediators of the increased cell proliferation. Thus, we examined cytokine signaling in mice treated with peroxisome proliferators. We found that peroxisome proliferator-induced increases in cell proliferation is not mediated via Tnfasignaling, but instead may be mediated through interleukin-1b or interleukin-6. Third, because Ppara is necessary for the cell proliferation that follows peroxisome proliferator exposure, we hypothesized that the receptor may play a role in hepatocellular proliferation induced by other stimuli. Following partial hepatectomy, liver regeneration in Ppara-null mice is transiently impaired, and may result from altered expression of genes regulating the G1/S cell cycle checkpoint in hepatocytes from these mice. Overall, our studies suggest that hepatic Ppara activation (1) alters inflammatory mediators, (2) modulates several potentially mitogenic cytokines, and (3) is necessary for normal liver regeneration after partial hepatectomy. Our data, compared with data from similar experiments on human hepatocytes, may provide further clues about the differences and similarities between peroxisome proliferator exposure in humans and laboratory animals.
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Nwosu, V. U. "Peroxisome enzymes in animal models of obesity." Thesis, University of Wolverhampton, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380662.
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Mitchell, Angela M. "Hepatic peroxisome proliferation : mechanisms and species differences." Thesis, University of Surrey, 1985. http://epubs.surrey.ac.uk/847808/.
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The present work has investigated the species differences and mechanisms involved in hepatic peroxisome proliferation. The suitability of primary hepatocyte cultures as an in vitro model for this phenomenon was determined. The effects of a range of peroxisome proliferators on cultured rat hepatocytes were both qualitatively and quantitatively similar to the effects observed in the liver of the rat in vivo. A species difference in the hepatic response to orally administered di-(2-ethyl hexyl)phthalate (DEHP) was demonstrated in the rat and the marmoset. This species difference phenomenon was further investigated using primary guinea pig and marmoset hepatocyte cultures. Peroxisome proliferation as seen in rat hepatocytes was not observed in guinea pig or marmoset hepatocytes with any agent tested. Similarly, peroxisome proliferation was not observed in cultured human hepatocytes. These observations are suggested to infer an inherent difference in sensitivity of the guinea pig, marmoset and human hepatocyte to peroxisome proliferation. The mechanism by which DEHP elicits peroxisome proliferation in the rat liver has been investigated. The metabolites of DEHP produced by o-1-oxidation of the monoester (metabolite IX [mono-(2-ethyl-5-hydroxy hexyl phthalate] and metabolite VI [mono-(2-ethyl-5 oxo hexyl phthalate]) were shown to be potent peroxisome proliferators in cultured rat hepatocytes. These metabolites produced a transient increase in intracellular neutral lipid in cultured rat hepatocytes. Metabolite Vis was further shown to interfere with hepatic lipid metabolism, causing an inhibition of mitochondrial medium chain acyl carnitine oxidation specifically. Hence it is proposed that the species difference in peroxisome proliferation is due to differences in response of guinea pig, marmoset and human hepatocytes to the accumulation of intracellular lipid.
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Nickkho-Amiry, Mahshid. "Peroxisome proliferator-activated receptors in endometrial cancer." Thesis, University of Manchester, 2011. https://www.research.manchester.ac.uk/portal/en/theses/peroxisome-proliferatoractivated-receptors-in-endometrial-cancer(2388ac43-ecd4-402f-a9c7-853be4902ec8).html.
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Endometrial cancer is a common gynaecological cancer. Improving outcomes for women with advanced disease remains a challenge and there is also a need to develop preventative strategies in those women at highest risk of developing disease. Peroxisome proliferator-activated receptors (PPARs) comprise of a group of transcription factors belonging to the nuclear hormone receptor subfamily. PPAR sub-types are involved in metabolic homeostasis and have been implicated in malignancy, particularly breast and colo-rectal malignancies both of which are associated with obesity. Endometrial cancer is also closely associated with both obesity and insulin resistance. The work described in this thesis examined the expression of PPARs in endometrioid endometrial cancer and investigated their effects on key pathways implicated in this disease. Immunoblotting revealed over expression of PPARα and loss of PPARγ in human endometrioid endometrial cancer tissues. Pull-down assays also demonstrated differential selectivity of different PPARs for heterodimerisation with different isoforms of the RXR family of transcription factors. PPARα was localized to tumour cells and vascular endothelium and ELISA demonstrated an increase in VEGF-A in PPARα silenced cells suggesting that PPARα may promote tumour angiogenesis. PPARγ was largely seen in epithelial cells and also macrophages within benign endometrium. Reduction of PPARγ expression in cultured endometrial cells led to increased proliferation and decreased apoptosis. Loss of PPARγ was correlated with a loss of the tumour suppressor PTEN in endometrial tissues. Furthermore, PPARγ silencing led to diminished expression of PTEN and a concomitant increase in phosphorylated AKT suggesting that PPARγ is protective against deregulated growth within the endometrium. Synthetic PPAR-specific ligands reduced proliferation and increased apoptosis in endometrial cell lines. These effects were present in PPAR-silenced cells too although reduced in magnitude, indicating that the actions of specific PPAR ligands are mediated via both receptor dependent and receptor independent pathways.In conclusion, this work has demonstrated the differential expression of PPARs and RXRs in endometrial cancers and identified possible mechanisms, both direct and indirect, by which these may modulate endometrial cancer growth. Different PPAR family members may provide targets for therapeutic intervention in endometrial cancer care and require further study in this regard.
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Harper, Courtney Christine. "Complex problems in peroxisome matrix protein import." Available to US Hopkins community, 2003. http://wwwlib.umi.com/dissertations/dlnow/3080674.
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Tinfo, Nicole Shivonn. "The peroxisome proliferator-activated receptor in ovarian biology." [Ames, Iowa : Iowa State University], 2007.
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Al, Kholaifi Abdullah. "The induction of liver growth by peroxisome proliferators." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/10443/.
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Peroxisome Proliferators (PPs) are a class of chemicals that cause a programme of augmentative liver growth, however, the mechanism which regulates the induction of hepatic DNA synthesis as a result of exposure to peroxisome proliferators is currently uncharacterized. This study sets out to characterise the induction of DNA synthesis in mouse by peroxisome proliferators, as a prerequisite for investigating and identifying the genes that are responsible for induction of DNA synthesis to control liver growth. Administration of BrdU in drinking water can reduce mouse body weight; an optimized protocol was devised, which does not lead to body weight loss, and which enables reliable measurement of DNA synthesis. Male 129S4/SvJae mice were treated with a single dose of ciprofibrate (100-400 mg kg-1) or methylclofenapate (25 mg kg-1) for two days. Although liver to body weight ratios increased significantly at all doses, no induction in DNA synthesis was observed within 2 days. Subsequent time course studies with ciprofibrate (100 mg kg-1day-1) or methylclofenapate (25 mg kg-1day-1) showed that liver-to-body weight ratio was significantly increased in treated groups by day 2, but that the induction of DNA synthesis was increased significantly only after three days of treatment, for both compounds. No induction of hepatic DNA synthesis was observed in PPARa null mice after treatment with ciprofibrate (100mg kg-1day-1) for 2 or 6 days, showing that the effect required the PPARa. A dose-response study with 0,1,3,10,30,100 or 200 mg kg-1 day-1 ciprofibrate for 3 days, or with 0,10,30,100 mg kg-1 day-1 ciprofibrate for 4 days revealed that liver to body weight ratios were significantly increased in 129S4/SvJae mice treated with 10mg kg-1day-1 and greater ciprofibrate at 3 and 4 days, whereas hepatic labelling index was significantly increased at 100 mg kg-1 day-1 ciprofibrate at 3 days after dosing, with progressive increases at doses of 30 and 100 mg kg-1 day-1 ciprofibrate at 4 days after dosing. In order to explain the early time course of induction of DNA synthesis reported by Styles [113] [164] in Alderley Park mice, a time course study was performed between 1-4 days in Alderley park mice using methylclofenapate (25mg kg-1day-1). The study showed that liver growth was induced by day 2, but DNA synthesis was significantly induced only after 3 days of dosing. To evaluate species differences, the time-course of induction of DNA synthesis was examined in F-344 rats treated with ciprofibrate (50mg kg-1day-1) for 1-4 days. The liver-to-body weight ratio was significantly increased in all time points, but DNA synthesis was significantly increased after 2 days of dosing. These findings demonstrate that there was a delay in induction of DNA synthesis by peroxisome proliferators in mouse by at least 48 hours. This delay in response is not due to strain differences. Moreover, induction of DNA synthesis in rat was earlier than those in mouse, which makes rats a feasible experimental model to study the immediate early genes/ proteins induced by peroxisome proliferators to induce liver growth.
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Browne, P. O. "Analyses of the peroxisome proliferator-activated receptor gamma." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597020.
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The peroxisome proliferator-activated receptor γ(PPARγ) plays a key role in the development of adipose tissue, and over the last few years has attracted increasing attention as the most likely target for a new and potent class for anti-diabetic drugs known as the thiazolidinediones (TZDs). The first part of this work investigated whether mutations in the gene encoding PPARγ are responsible for syndromes of aberrant adipose tissue development. The entire coding sequence of the PPARγ gene was screened in 9 individuals with inherited forms of lipodystrophy, and 98 children affected by severe early onset obesity were screened for activating mutations within the second exon of the PPARγ gene. Neither study identified any mutations that might contribute to these syndromes. The second part of this work investigated whether a common mutation in the amino-terminus of PPAR-γ (PPARγ Pro12A1a) was associated with body mass index (BMI) and/or insulin sensitivity in a UK Caucasian population. The results of this study suggest that there is a diet-dependent association of the PPARγ2 Prol2A1a mutation with BMI and as a consequence of this with insulin sensitivity. The final part of this work concerned the evaluation of an artificial dominant negative PPARγ mutant (PPARγm), and the generation of transgenic mouse lines in which PPARγ activity is blocked in skeletal muscle and adipose tissue by tissue specific expression of PPARγm. I hope that these transgenic will enable us to prove conclusively whether or not PPARγ is the mediator of insulin sensitisation by the TZDs.
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Dionisi, Mauro. "Endocannabinoid metabolism and peroxisome proliferator-activated receptor signalling." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11384/.
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The fatty acid amides (FAAs) family includes endocannabinoids, such as anandamide, as well as endocannabinoid-like molecules, such as N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA). Members of the FAA family show agonist activity at transmitter-gated channels (TRPV1), as well as peroxisome poliferator-activated receptors (PPARs). Given that FAAs appear to be hydrolysed principally through the action of the enzyme fatty acid amide hydrolase, inhibition of FAAH should lead to accumulation of a variety of FAAs. Therefore, in this study it was investigated whether pharmacological inhibition of FAAH could influence PPAR activity in SH-SY5Y human neuroblastoma cells or HeLa human cervical carcinoma cells. FAAH activity was assessed by monitoring liberation of [3H]-ethanolamine from labelled anandamide. FAAH protein and RNA expression were measured by immunoblotting and qRT-PCR respectively. Endocannabinoid levels were measured by LC-MS/MS. In order to evaluate PPAR activation, a PPRE-linked luciferase construct was co-transfected with expression plasmids for either PPAR α, β or γ. Binding to PPAR receptors was assessed with a competitor displacement assay (Invitrogen). In intact SH-SY5Y cells, sustained FAAH inhibition by URB597 (~75 %) led to accumulation of AEA, 2AG and PEA, but not OEA. Treatment with URB597, OL135 or PF750, three structurally and functionally distinct FAAH inhibitors, induced activation of endogenously expressed PPARs, while no activation was observed in FAAH-1 negative HeLa cells. Furthermore, exposure to URB597, OL135 or PF750 led to activation of over-expressed PPARs in SH-SY5Y cells. To rule out direct activation of PPARs by the FAAH inhibitors, cell-free binding assays showed that URB597, OL135 and PF750 could not bind to PPARα, PPARβ or PPARγ. Surprisingly, treatment with URB597 in HeLa cells led to intracellular accumulation of PEA but not AEA, OEA or 2AG. This might be due to inhibition of either FAAH-2 or NAAA, both of which are expressed in HeLa cells. Moreover, the presence of either URB597 or OL135 led to activation of PPARγ receptors over-expressed in HeLa cells. In conclusion, data in this study showed activation of PPAR nuclear receptors in vitro by inhibition of FAAH activity and subsequent augmentation of endocannabinoid tone. These data suggest that, at least in a model setup, it is possible to modulate the endocannabinoid tone without any previous external stimulus of their synthesis and trigger a functional effect.
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Ferreira, Joana Filipa Dias. "The role of STING on peroxisome-dependent signalling." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/21999.
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Mestrado em Biomedicina MolecularViruses are recognized by several cellular sensors from the innate immune system, activating signalling cascades which result in the production of interferons and other cytokines that affect the virus life cycle and hinder spreading to other cells. Although the RIG-I/MAVS and the STING pathways are assumed to signal, respectively, for RNA and DNA viruses, there is still some controversy on how these pathways interact with and influence each other. The interaction between STING and MAVS, previously reported to take place at mitochondria, supports a crosslink between these pathways. Our group has recently demonstrated that STING is also able to interact with the peroxisomal MAVS. With this work we aimed at studying in more detail the interplay between the STING pathway and the peroxisomal RIG-I/MAVS pathway. One of our approaches involved the knock-down of STING and stimulation of the RIGI/ MAVS pathway in cells that contained MAVS solely at peroxisomes, in order to study the importance of STING for the establishment of an effective peroxisome-dependent antiviral response. In parallel, we activated STING by transfecting 2’3’-cGAMP with the objective of performing RT-qPCR analysis of the peroxisome-dependent production of cytokines. The studies initiated with this thesis will contribute to the unravelling of the interplay between the STING pathways and the peroxisomal-dependent RIG-I/MAVS signalling.
Os vírus são reconhecidos por vários sensores do sistema imunitário inato, responsáveis pela ativação de cascatas de sinalização que levam à produção de interferões e citoquinas, impedindo o ciclo viral e a propagação da infeção às células vizinhas. Apesar de a via da RIG-I/MAVS e da STING serem respetivamente responsáveis pelo reconhecimento de vírus de ARN e ADN, existe ainda alguma controvérsia sobre como estas duas vias interagem. A interação entre a STING e a MAVS, anteriormente reportada nas mitocôndrias, sugere uma interligação entre as duas vias. O nosso grupo demonstrou recentemente que existe também uma interação entre a STING e a MAVS peroxisomal. Neste trabalho, o nosso objetivo consistiu em estudar a interligação entre a via da STING e a via RIG-I/MAVS peroxisomal. Começamos por silenciar a STING e a estimular a via RIG-I/MAVS numa linha celular que contem MAVS apenas nos peroxissomas, para estudar a influência da STING na resposta antiviral dependente dos peroxissomas. Por outro lado, tentamos ativar a STING através da transfeção da molécula 2’3’-cGAMP com o objetivo de analisar a produção de citoquinas e interferões dependentes da via peroxissomal por RT-qPCR. As experiências apresentadas nesta tese irão certamente contribuir para desvendar a interligação entre a via da STING e a via RIG-I/MAVS dependente dos peroxissomas.
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Heß, Katharina Andrea. "Peroxisome proliferator-activated receptors (PPARs) und die Lymphozytenmigration." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-56139.
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Al, Saryi Nadal. "Molecular studies of peroxisome biogenesis in Saccharomyces cerevisiae." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/13433/.
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Peroxisomes are subcellular organelles found virtually in all eukaryotic cells. They perform various functions depending on organism, cell type or environmental conditions. The importance of peroxisomes for human development and health is shown by the occurrence of inherited peroxisomal disorders. Peroxisome biogenesis involves a number of processes including peroxisomal membrane protein and matrix protein import, growth of the lipid bilayer and proliferation. Peroxisomal matrix proteins are directed to peroxisomes by the conserved targeting signals PTS1 and PTS2. However, a subset of matrix proteins neither contains a PTS1 nor PTS2. In this, study I describe how one of these latter enzymes, the nicotinamidase Pnc1 enters peroxisomes. We found that Pnc1 is co-imported with the PTS2-containing enzyme Gpd1 by a piggy-back mechanism. This mechanism requires the PTS2 receptor Pex7 and its coreceptor Pex21. In addition, we found that Pnc1 piggy-back import relies on homodimerisation of Gpd1. In the second part of this thesis, I describe the analysis of the AAA+ATPase Msp1 and the attempts to uncover its role in peroxisome biogenesis. A genome wide screen revealed a genetic interaction with two proteins; Pex25 and the novel PMP Ygr168c indicating a role of Msp1 in the regulation of peroxisome abundance. A preliminary analysis of Ygr168c is also presented. Together these studies further our understanding of peroxisomal protein import and biogenesis and provide novel areas for future exploration.
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Marcus, Sandra L. "Transcriptional regulation of rat peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase by peroxisome proliferators." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34808.pdf.
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Anderson, I. W. "Permeability of leaf peroxisomes to photorespiratory metabolites." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379882.
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Makowska, Janet Mary. "Species differences in the hepatic and renal responses to ciprofibrate." Thesis, University of Surrey, 1988. http://epubs.surrey.ac.uk/847765/.
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The influence of pharmacokinetic parameters on the hepatic biochemical responses to the peroxisome proliferators, ciprofibrate, bezafibrate and clofibrate, was studied in the Fischer rat following a 26-week treatment period. With once daily dosing, the induction profiles of these compounds were dissimilar and the order of response was ciprofibrate > bezafibrate > clofibrate. By adjusting the frequency of dosing with respect to drug half life, i. e. clofibrate and bezafibrate twice daily and ciprofibrate once every 48 hours, the differences in response were ablated. The effect of short term ciprofibrate administration on hepatic enzyme parameters was studied in different rat strains and species. The rat (all strains), mouse, hamster and rabbit were termed responsive due to a coordinate induction of cytochrome P-452, carnitine acetyltransferase and peroxisomal beta-oxidation. No treatment related changes were observed in the guinea pig. In the marmoset a slight increase in peroxisomal beta-oxidation was demonstrated with no induction of cytochrome P-452 and carnitine acetyltransferase. In responsive species increased 12-hydroxylation of lauric acid correlated with an increase in mRNA hybridising to a cytochrome P-452 cDNA probe. The guinea pig and marmoset were designated non-responsive. In the marmoset and Fischer rat the hepatic enzyme responses to a 14-day and 26-week ciprofibrate administration were similar. A 4-week recovery group was included in the marmoset chronic study and the increase in peroxisomal beta-oxidation was found to be readily reversible whereas mitochondrial and microsomal changes were not. Electron microscopy revealed no peroxisome proliferation in the marmoset. Renal enzyme parameters were examined in ciprofibrate treated animals and considerable rat strain and species differences were observed. Renal enzyme changes were minimal. The response to ciprofibrate appeared to be largely specific for the liver in responsive species. From these results it is clear that the rat is not a suitable animal model to predict the hepatic response in the marmoset. If it is assumed that the marmoset resembles man more closely than the rat, extrapolation would indicate that peroxisome proliferators are not a toxicological hazard to man.
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Mehl, Isaac R. "Regulation of gene expression by peroxisome proliferator activated receptors." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3249923.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed April 4, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Gootjes, Jeannette. "Molecular, biochemical and clinical aspects of peroxisome biogenesis disorders." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2004. http://dare.uva.nl/document/74957.
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Montgomery, S. "The biochemistry and ultrastructure of glyoxysome and peroxisome development." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376933.
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Owens, Joanna. "Regulation of peroxisome proliferator-activated receptor-alpha (PPAR#alpha#)." Thesis, University of Surrey, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341337.
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Choudhury, Munim. "PPARalpha in peroxisome proliferation : molecular characterisation and species differences." Thesis, University of Nottingham, 2000. http://eprints.nottingham.ac.uk/10396/.
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Peroxisome proliferators (PPs) cause proliferation of peroxisomes and hepatocarcinogenesis in rodent liver, mediated by Peroxisome Proliferator-Activated Receptor-alpha (PPARalpha). There are marked species differences in peroxisome proliferator-induced responses, and the functionality of PPARalpha may be an important determinant factor in species sensitivity to PPs. Primary hepatocytes were investigated for a highly responsive marker induced by PPs to study the effects of transfected PPARalphaCYP4A1 was highly induced in rat hepatocytes that require hydrocortisone for maximal induction. Hepatocytes were cultured in hydrocortisone-deficient media to determine if reduced endogenous PPARalpha was associated with lowered induction of CYP4A1. However, there was residual induction of CYP4A1 by peroxisome proliferators. Primary hepatocytes from PPARalpha knock-out (-/-) mice were investigated as they lack endogenous PPARalphaIn vitro and in vivo studies demonstrated that Cyp4a10 and 14 were highly inducible by PPs in the hepatocytes of wild-type but not in -/- mice. However, addition of either mouse or guinea pig PPARalpha in -/- hepatocytes did not induce the expression of these marker genes, although both receptors showed trans-activation ability in a reporter assay. The failure of added PPARalpha to activate endogenous genes responsive to PPs, whilst at the same time activating episomal DNA containing response elements of PP-inducible gene, suggests that the endogenous genes require PPARalpha to remain in an accessible conformation. Although hamster is considered to be a partially-responsive species to PPs, their response to PPs is poorly characterized. Three CYP4A genes (CYP4A17, 18 and 19) were cloned from hamster liver cDNA, and hepatic CYP4A17 was found to be highly inducible by PPs. In addition, PPARalphawas cloned from hamster liver and shows higher identity to rat and mouse PPARalpha than to human and guinea pig. Hepatic expression of PPARalpha mRNA was compared between mouse, hamster and guinea pig. The level of PPARalpha transcript was found to correlate well with species response to PPs, i.e. mouse (highly responsive species) has the highest level and guinea pig (non-responsive) the lowest, while hamster (partially-responsive) has an intermediate level. This is consistent with a model where the level of expression of hepatic PPARalpha determines species response to PPs. Expression of PPARalpha and transcriptional coactivators, such as PBP, SRC-1 and CBP/p300, were confined to mouse liver at the RNA level, but in each case expression showed homogenous distribution within the liver acinus and was non-inducible by PPs. Mouse PPARalpha ligand binding domain (LBD) was bacterially expressed as a histidine-tagged protein and soluble proteins were purified using affinity and column chromatography. Functional LBD may serve as a useful bait in protein-protein interaction studies for the identification of any novel PPARalpha interacting coactivator protein.
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Savory, Richard. "PPAR#alpha# : inducibility and species differences in expression." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337248.
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Xie, Weiqiao Hope Lila W. "Isolation and characterization of a gene required for peroxisome biogenesis." Oregon Health & Science University, 1993. http://content.ohsu.edu/u?/etd,234.
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M.S.Molecular Biology
This thesis describes the cloning and analysis of PER6, a gene required for peroxisome biogenesis in Pichia pastoris. The gene was cloned by functional complementation of a per6 P. pastoris mutant strain that was one of a number of peroxisome-deficient mutants isolated in this laboratory. The complementing activity was localized to a small DNA fragment by subcloning and Northern filter hybridization analysis and the DNA sequence of the fragment was determined. The sequence revealed a 1296-bp open reading frame which potentially encodes a 432-amino acid protein of 49 kD. The gene was transcribed into a message of 1.4 kilobases that was constitutively expressed but induced several-fold in cells growing on methanol. A mutant strain with a deletion of a large portion of the open reading frame was constructed and used to genetically demonstrate that the cloned gene was identical to the defective gene in the originally isolated per6 mutant. The predicted amino acid sequence of the PER6 product revealed several interesting features, including a significant regional similarity to PAF-1, a gene known to be defective in some patients with Zellweger syndrome, a lethal human genetic disease caused by peroxisome deficiency. Finally, the PER6 product was produced in E. coli and purified to serve as antigen for antibody production.
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Xie, Weiqiao. "Isolation and characterization of a gene required for peroxisome biogenesis /." Full text open access at:, 1993. http://content.ohsu.edu/u?/etd,234.
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Brown, Trevor Wayne. "A study of peroxisome biogenesis in the yeast Yarrowia lipolytica." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0014/MQ60095.pdf.
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Amer, Abeer H. A. "Mechanism of action of liver growth induced by peroxisome proliferators." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/11947/.
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Humans are ubiquitously exposed to peroxisome proliferators including hypolipidemic agents, industrial solvents and atural products. Because of this and the fact that peroxisome proliferators cause non-genotoxic hepatocarcinogenesis in rodents, it is of importance to elucidate the mechanism of action of the peroxisome proliferators in order to provide an assessment of the hazard, if any, of these compounds to humans. It is also known that the peroxisome proliferators begin their actions by inducing hepatic DNA synthesis. Thus, the aim of this thesis was to find genes that could be responsible for triggering the induction of hepatic DNA synthesis caused by peroxisome proliferators, specifically ciprofibrate. First, it was important to indicate when the induction of hepatic DNA synthesis actually happens. This was done with BrdU immunohistochemical procedures. The induction of hepatic DNA synthesis with ciprofibrate in mice was observable only after 4 days making it difficult to specify when the induction actually happened. In rats the induction of hepatic DNA synthesis was found to peak at 24 hours and this system gave the better opportunity to find the genes responsible. The difference in the timing of induced hepatic DNA synthesis betweenmice and rats implied that there could be a species difference in the mechanism of each species’ response to PPAR. With immunohistochemistry it was noticed that there was a difference in the lobular localization of hepatic DNA synthesis in the liver tissues of rats and mice dosed with different inducers, with the rat livers exhibiting periportal distribution while hepatic DNA synthesis in the mice seemed to be distributed throughout the liver tissue. The effects of ciprofibrate or cyproterone acetate on liver gene expression in rats were studied, using cDNA microarrays, transcriptome sequencing and quantitative real- time PCR. A 1- 5 hour treatment period was chosen to detect the immediate early gene response, while a 24 hour time point was chosen to elucidate the confounding effects from the hepatic DNA synthesis seen during the 24 hour stimulation. The results showed that ciprofibrate altered the expression of numerous genes including previously known PPARa agonist-responsive genes involved in processes such as PPAR signalling pathways, fatty acid metabolic pathway, cell cycle, palmitoyl-CoA hydrolase activity, lipid metabolism, inflammatory responses, and stress responses, in addition to a large number of novel candidate genes. Three novel induced genes G0s2, Ccnd1 and Scd1, (and two marker genes CYP4A1 and CYP3A1) were confirmed with quantitative real- time PCR. The G0s2, Ccnd1 and Scd1 were found to be up-regulated at the hours 1 and 3 after dosing and not 24 hours, and the G0s2 and Scd1 were specific for the ciprofibrate suggesting they were involved in a distinct PPARa pathway responsible for the hepatic DNA synthesis. The complete database of the transcriptional response provided here opens doors of opportunity for further research to identify genes responsible for the liver growth induced by peroxisome proliferators.
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De, Mora Kim Stephen. "Foundational technologies in synthetic biology : promoter measurement and peroxisome engineering." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/7870.
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The confluence of next generation DNA sequencing and synthesis when combined with the application of concepts such as standardization and modular design has led to the genesis of a new discipline. The nascent field of Synthetic Biology concerns the rational design and construction of genetic circuits, pathways, machines and eventually whole organisms. The immaturity of this field dictates that early research efforts, including this Thesis, describe foundational work towards the creation of tools which make biology more amenable to being engineered. The first part of this Thesis describes an attempt to standardize the measurement of transcriptional promoter activity in E. coli. A method to measure in vivo promoter activity was developed for E. coli and tested in a multi-institution trial. Comparable results were achieved with less than a two-fold range for the measured promoters across eight laboratories. A standardized measurement kit was created and distributed for use by the teams participating in the 2008 international Genetically Engineered Machines competition. Techniques learned measuring the activity of E. coli promoters were applied to a collection of S. cerevisiae strains. Several promoters were measured in synthetic dextrose media and ADH1 was measured in multiple media conditions. The outcome of these experiments is to consider proposing ADH1as the reference promoter in S. cerevisiae. The second aspect of this Thesis describes the construction of artificial organelles in S. cerevisiae. Artificial organelles hold the prospect of being able to insulate synthetic genetic pathways from the cell. Two proteins are essential for the biogenesis of the peroxisome organelle in humans and yeast, Pex3p and Pex19p. Pex3p functions as a peroxisomal membrane receptor for Pex19p, while Pex19p shuttles other peroxisomal proteins to the membrane, including Pex3p, creating a feedback loop. Human Pex19p has previously been shown to dock to yeast Pex3p and a version of yeast Pex19p has been shown to work with human Pex3p as a high degree of evolutionary conservation exists between these proteins. Because of these inter-species protein docking characteristics, there exists the possibility of creating bimodality: the ambition of the work was therefore to create a cell strain which possessed both synthetic “humanized” and natural yeast peroxisomes. An S. cerevisiae BY4741a derivative strain was engineered with fluorophore tagged versions of human (CFP) and yeast (YFP) Pex3p and untagged yeast and human Pex19p proteins. The results indicated the creation of a single population of peroxisomes when a measure of fluorescently imaged CFP and YFP peroxisomes were plotted on a scatter plot. A log of the ratio of CFP to YFP peroxisomes was plotted on a histogram and a normal distribution was found to best fit the curve, indicating a lack of bimodality. Finally, microscopy images of this strain were reviewed and by visual inspection, showed no evidence of distinct human or yeast peroxisomes. This experiment therefore produced no evidence of bimodality when examining the interactions of human and yeast Pex3p and Pex19p proteins. However, the four proteins were shown to interact closely to produce a single population of chimeric human-yeast peroxisomes. The peroxisome-deficient mutant phonotype strain was rescued using human Pex3p and Pex19p.
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Kaplan, Claude Paul. "Isolation and characterisation of genes involved in plant peroxisome biogenesis." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627157.
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Nguyen, Tam Hong. "Pex13 Mutant Mice as Models for the Peroxisome Biogenesis Disorders." Thesis, Griffith University, 2008. http://hdl.handle.net/10072/366797.
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Zellweger syndrome (ZS) is the most severe form of a spectrum of disorders resulting from mutations in PEX genes, genes that encode proteins necessary for peroxisome biogenesis. Loss of functional peroxiosmes leads to disruption of multiple metabolic pathways involving the peroxisome, including very long chain fatty acid oxidation and plasmalogen and bile acid synthesis. ZS patients exhibit a range of clinical abnormalities, including facial dysmorphism, cataracts, hypotonia, seizures, psychomotor retardation, and hearing impairment. In terms of tissue pathology, there are also wide ranging effects, including neuronal migration defects, hepatomegaly, retinopathy, and renal cysts. Pex13 encodes a peroxisomal membrane protein that is essential for peroxisome biogenesis. Previous work in this laboratory resulted in the generation of a Pex13-null mouse model for the purpose of investigating the pathogenesis of Zellweger syndrome. The work in the first part of this thesis extends these studies and describes the generation and initial characterisation of tissue-specific Pex13 mouse models. These tissue-specific models are expected be useful tools for analysis of the impact of localised, brain- and liver-specific elimination of peroxisomes on the pathogenesis of ZS. In addition, in the second part of the thesis, a separate and novel hypothesis is addressed as an explanation for the molecular pathogenesis of ZS, through investigating the relationship between reduced peroxisome abundance and microtubule-mediated peroxisome trafficking.
Pex13 brain mutant mice were generated by mating the previously generated Pex13-floxed mice with mice expressing Cre recombinase under the control of the neuron-specific rat nestin promoter. Pex13 brain mutant mice displayed growth retardation beginning at day 5 postnatal, with gradual deterioration until death at approximately day 22 postnatal. Other clinical features included contracted postures, under-developed lower body mass, abnormal and unsteady gait, and abnormal motor coordination. In terms of brain metabolic function, these mice exhibited significant defects in plasmalogen synthesis, but, surprisingly, VLCFA levels were similar to those of littermate control mice. Significantly, peroxisome elimination in brain resulted in increased levels of plasmalogen levels in liver of Pex13 brain mutant mice. Consistent with the expected pathology resulting from deficiency of brain peroxisomes, brain mutants exhibited defective neuronal migration characterised by increased cellular density in the intermediate zone of the neocortex. Microarray analysis of total brain RNA from Pex13 brain mutants revealed several functionally-linked pathways associated with the differentially expressed genes, including cell-cell signalling, cell compromise/death, lipid metabolism, cell movement, and serotonin synthesis.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Smith, Steven Andrew. "The role of Peroxisome proliferator-activated receptors in the rat brain /." St. Lucia, Qld, 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17982.pdf.
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Roberts, Lee D. "Defining the metabolic effect of peroxisome proliferator-activated receptor δ activation." Thesis, University of Cambridge, 2010. https://www.repository.cam.ac.uk/handle/1810/226743.
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Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that function as ligand activated transcription factors. There are three identified isotypes: PPAR alpha, PPAR gamma and PPAR delta, together controlling the expression of genes involved in inflammation, cell differentiation, proliferation, lipid and carbohydrate metabolism and energy homeostasis. The PPARs are potential targets for the treatment of dyslipidaemia, type II diabetes mellitus and the metabolic syndrome. This thesis uses a multi-platform metabolomics approach, 13C-isotope substrate flux analysis, respirometry and transcriptomics to determine the role PPAR delta and PPAR gamma play in metabolic control both in adipose tissue and systemically. To achieve this, the metabolic phenotype of the 3T3-L1 adipocyte cell line was defined to generate a metabolically phenotyped in vitro model of adipose tissue. The importance of fatty acid alpha-oxidation in the differentiation of adipocytes was emphasised The effects of PPAR delta and PPAR gamma activation in white adipose tissue from the ob/ob mouse model of insulin resistance, and in the phenotyped 3T3-L1 adipocyte model, were investigated. PPAR delta activation was distinguished by oxidative catabolism of fatty acids and citric acid cycle intermediates. Conversely, PPAR gamma activation was identified by the sequestration of lipids into adipose tissue. Moreover, to address the systemic influence of PPAR activation, with a focus on the Cori cycle and the interactions of the liver and skeletal muscle, the metabolic changes that occur in these tissues following PPAR delta and PPAR gamma activation in the ob/ob mouse were examined. PPAR delta activation was characterised by the mobilisation and release of triacylglycerols (TAGs) into circulation as an energy source for peripheral tissues whereas PPAR gamma activation was defined by a reduction and sequestration of circulating TAGs. This thesis has better characterised the role of the PPARs as master regulators of metabolism and emphasised their potential as therapeutic targets for metabolic diseases of global importance.
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Eitzen, Gary A. "An analysis of peroxisome assembly mutants of the yeast Yarrowia lipolytica." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq22978.pdf.
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Lagali, Pamela Sarita. "Studies of peroxisome proliferator-activated receptors in transcriptional processes and disease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ34386.pdf.
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Cheng, Wai. "The relationship between peroxisome proliferator-activated receptors (PPARs) and cell proliferation /." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36433937.
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Luense, Lacey Jeanne. "The role of peroxisome proliferator activated-receptor gamma in ovarian function." [Ames, Iowa : Iowa State University], 2007.
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Hughes, Robert Ian. "The role of peroxisome proliferator activated receptor agonists in Cardiovascular disease." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504931.
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Cheng, Wai, and 鄭蔚. "The relationship between peroxisome proliferator-activated receptors (PPARs) and cell proliferation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B45010614.
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Shiau, Chung-Wai. "Thiazolidinediones: from peroxisome-proliferator-activated receptor γ(PPARγ) to anticancer agents." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1128111032.
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Wei, Shuo. "Peroxisome Proliferator-Activated Receptor γ (PPARγ)-Independent Antitumor Effect of Thiazolidinediones." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1259167390.
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Brown, Emily Lauren. "Regulation of Peroxisome Proliferator-Activated Receptor Alpha by Selected Beta-Apocarotenoids." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1275401911.
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Rangwala, Shamina M. "In vitro mechanistic studies of peroxisome proliferation by chiral clofibrate analogs /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487946776024538.
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Pinho, Sónia Andreia de Almeida. "Induction and determination of ROS and their effect on peroxisome dynamics." Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/3160.
Full textAbstract:
Mestrado em Métodos BiomolecularesPeroxissomas são organelos celulares de membrana simples, os quais têm importantes funções metabólicas, como por exemplo metabolismo de lípidos e ROS, sendo assim indispensáveis para a saúde e desenvolvimento humano. Os peroxissomas são organelos altamente flexíveis e dinâmicos que rapidamente se agregam, multiplicam e degradam em resposta a necessidades metabólicas. Em cultura celular, o stress oxidativo e outros estímulos externos (ex. factores de crescimento, ácidos gordos, despolimerização) têm mostrado induzir processos de crescimento (alongamento) e divisão de peroxissomas, os quais estão relacionados com a sua proliferação. Considerando que alguns dos componentes moleculares da maquinaria de crescimento e divisão têm sido identificados nos últimos anos, as vias de sinalização e regulação que medeiam a proliferação de peroxissomas são largamente desconhecidas. O objectivo desta dissertação de mestrado foi examinar o efeito de diferentes estímulos externos promotores de ROS na indução do crescimento/proliferação do compartimento peroxisomal. Foi seleccionado um sistema de cultura de células de mamífero que apresentam um compartimento peroxisomal dinâmico. Análises baseadas em fluorescência para a detecção da produção de ROS e alterações nos níveis de GSH intracelular foram estabelecidos. Estes procedimentos foram usados primeiro para esclarecer se o alongamento de peroxissomas observado após despolimerização de microtubulos (pelo nocodazole) é mediado por ROS. O alongamento de peroxissomas e a despolimerização dos microtubulos após tratamento com nocodazole foi monitorizado e quantificado por microscopia de imunofluorescência. O Nocodazole induziu um aumento dos níveis de ROS intracelular e apesar do pré tratamento com antioxidantes ter baixado os níveis de ROS, não preveniu o alongamento peroxisomal. Estes resultados demonstram que as alterações morfológicas do compartimento peroxisomal induzidas pelo nocodazole são independentes da produção de ROS. Para além disso, foi examinado o efeito de alterações nos níveis de glutationa (GSH) celular no compartimento peroxisomal. Interessantemente, a redução dos níveis de GSH intracelular pelo BSO, um inibidor da enzima γ-glutamylcystein synthetase (γ-GCS), resultou num aumento acentuado de crescimento/proliferação de peroxissomas. Os níveis de ROS e GSH foram determinados por análises baseadas em fluorescência. Pré tratamento com antioxidante preveniu o alongamento de peroxissomas indicando que a alteração no estado redox celular (citoplasmatico) levou à proliferação de peroxissomas a qual exerce, supostamente, uma função protectora para a célula. Finalmente, foi investigado se a inibição da cadeia respiratória mitocondrial e com isso, se ROS provenientes das mitocondrias foi capaz de induzir crescimento e divisão dos peroxissomas. Entre os inibidores analisados, apenas a rotenona, inibidor do complexo I, teve um efeito proeminente na elongação de peroxissomas. Todavia, foi demontrado que o seu efeito é devido à acção que a rotenona tem na despolimerização dos microtubulos. Assim, apesar da relação de proximidade entre mitocondria e peroxissomas, as ROS provenientes das mitocondrias não são prováveis de induzir alterações no compartimento peroxisomal. Os nossos resultados também indicam que estudos in vivo usando a rotenona têm de ser interpretados com muito cuidado. Além disso, os resultados mostram que as ROS podem alterar a dinâmica do compartimento peroxissomal, mas têm de vir de locais específicos dentro da célula (por exemplo, do citosol). ABSTRACT: Peroxisomes are single membrane bound subcellular organelles, which fulfill important metabolic functions, for example in lipid and ROS metabolism, and are thus indispensable for human health and development. Peroxisomes are highly flexible organelles that rapidly assemble, multiply and degrade in response to metabolic needs. In cultured cells, oxidative stress and other external stimuli (e. g. growth factors, fatty acids, microtubule depolymerization) have been shown to induce processes of growth (elongation) and division of peroxisomes, which are related to peroxisome proliferation. Whereas some of the molecular components of the growth and division machinery have been identified in the last years, the regulatory and signaling pathways mediating peroxisome proliferation are largely unknown. The aim of this master thesis was to examine the effects of different ROS-producing external stimuli on the induction of growth/proliferation of the peroxisomal compartment. A suitable mammalian cell culture system with a dynamic peroxisomal compartment was selected and fluorescent-based assays for the detection of ROS production and changes in the intracellular GSH levels were established. The setup was first used to clarify if peroxisome elongation observed after depolymerization of microtubules (by nocodazole) is mediated by ROS. Peroxisome elongation and microtubule depolymerization after nocodazole treatment was monitored and quantified by immunofluorescence microscopy. Although nocodazole induced an increase in cellular ROS levels, a pre-treatment with antioxidants and lowering of intracellular ROS levels did not prevent peroxisome elongation. These findings demonstrate that the morphological changes of the peroxisomal compartment induced by nocodazole are independent of ROS production. Furthermore, I examined the effect of changes in the cellular redox state on the peroxisomal compartment. Interestingly, the reduction of intracellular GSH levels by BSO, an inhibitor of γ- glutamylcystein synthetase (γ-GCS), resulted in a prominent increase in peroxisomal growth/elongation. ROS and GSH levels were determined by fluorescent-based assays. Pre-treatment with antioxidants prevented peroxisome elongation indicating that changes in the cellular (cytoplasmic) redox state lead to peroxisome proliferation, which is supposed to have a protective function for the cell. Finally, I investigated whether inhibition of the mitochondrial respiratory chain and thus, mitochondriaderived ROS were capable of inducing peroxisomal growth and division. Among the inhibitors tested, only rotenone, a complex I inhibitor, had a prominent effect on peroxisome elongation. However, I demonstrated that this effect is due to a microtubule-depolymerizing activity of rotenone. Thus, despite the close peroxisomemitochondria relationship, mitochondria-derived ROS are unlikely to induce changes of the peroxisomal compartment. Our findings also indicate that in vivo studies using rotenone have to be interpreted with great care. In addition, the results show that ROS can alter the dynamics of the peroxisomal compartment, but have to come from specific locations (e.g. the cytosol) within the cell.