Dissertations / Theses on the topic 'Mammalian'
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Godliman, N. I. "Mammalian transsulphuration." Thesis, Bucks New University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378101.
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Meijaard, Erik, and emeijaard@tnc org. "Solving Mammalian Riddles." The Australian National University. Faculty of Arts, 2004. http://thesis.anu.edu.au./public/adt-ANU20050924.221423.
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Since the mid 19th century, the biogeography of island South-East Asia has been the subject of much study. Early researchers explained many of the species distribution patterns by the rise and fall of sea levels and land. This and the work of other researchers culminated in a theory that emphasized the role of Pleistocene sea level low stands in species evolution. With the advent of newly developed molecular techniques, however, it became clear that many species divergence events had taken place before the Pleistocene and a biogeographical theory focusing on Pleistocene sea level changes was inadequate. In this research, I have developed a new biogeographic model that explains present-day distribution patterns and evolutionary relationships between species. I use this new model to explain 10 ‘mammalian riddles’, i.e. evolutionary or distribution patterns in selected mammal species groups that could not be explained with the existing theories. I developed the new model by analyzing the geological literature for this region, and by mapping palaeogeographical and palaeoenvironmental changes for the last 20 million years. In addition I compiled information on the palaeontological record for the region and on divergence times between taxa using a molecular clock assumption. These phylogenetic data were compared with the palaeomaps to assess whether particular divergence events could be correlated with certain palaeogeographical or palaeoenvironmental changes. The combination of these two information sources has resulted in a much-improved understanding of mammalian evolution in island SE Asia. Using this model it is now possible to relate important palaeoenvironmental events, such as the Late Miocene cooling, an Early–Middle Pliocene highstand, or the emergence and submergence of a land bridge between the Malay Peninsula and Java to evolutionary changes in species. I test the accuracy of the new model by analysing the relationships within several mammal groups using craniometric and molecular analysis. The observed relationships and deduced timing of divergence between taxa could in many cases be explained by the model, which indicates that it is relatively accurate. In addition, with the new model I have been able to find solutions to most mammalian riddles, although these results require further testing. Overall, I therefore believe I have made a significant contribution to the biogeographical understanding of island SE Asia.
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Whitford, C. "Mammalian somatostatin receptors." Thesis, University of Newcastle Upon Tyne, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356818.
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Palczewski, Grzegorz. "Mammalian Carotenoid Metabolism." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1467993233.
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Yurttas, Piraye. "Peptidylarginine deiminase 6 and the cytoplasmic lattices : mammalian regulators of maternal factor storage and localization necessary for embryonic genome activation and development /." Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1528353791&sid=6&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Croft, S. M. "Mammalian-wide interspersed repeats (MIRs) and their role in mammalian gene function and evolution." Thesis, Nottingham Trent University, 2009. http://irep.ntu.ac.uk/id/eprint/104/.
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Transposable elements (TEs) are ubiquitous components of plant and animal genomes and constitute more than ~45% of the human genome. Though originally considered as 'parasitic' or 'junk' DNA, TEs are now thought to have played a role in shaping genomes during evolution, contributing to genome plasticity and diversity. All classes of retrotransposons accumulate in the genome via a process termed retrotransposition, wherein the elements are reverse transcribed into RNA and inserted into the genome as DNA. Exaptation of these elements can provide additional or novel function for endogenous genes. Mammalian-wide interspersed repeats (MIRs) are short interspersed nuclear elements (SINEs), belonging to the non-autonomous class of retroelements and are found in all mammals. The recruitment of an MIR element by a gene may provide insight into mammalian evolution and gene function. The human genome was screened for genes that have exaptated MIR elements and the compiled dataset was analysed to determine any commonality which may suggest conserved function(s). Subsequently 1359 genes were identified that have exaptated MIR elements, constituting 5% of the total genes in the human genome. MIR elements may be multifunctional, as 1% of the total human genes contain MIRs that are spliced and/or are contributing to protein coding sequences. Subsequently sequence motifs were identified in the MIR consensus sequences which resemble canonical mammalian splice sites; therefore MIR elements recruited in the 5'-UTR and coding sequence may be a result of the exonisation of intronic elements. The MIR-containing transcripts are frequently expressed in neurological tissue, suggesting a role in neuronal function. Moreover a number of MIR-containing mRNA transcripts are known to be localised to the dendritic compartment of the neurone, and ciliated region of photoreceptors. Some of the localised mRNAs contain putative microRNA binding sites within the MIR sequence, and possible dsRNA structures were noted between MIR elements. It is proposed that exaptated MIR elements may be a source of cis-acting regulatory elements, involved in post-transcriptional control of gene expression, including localisation of mRNA to distinct intracellular compartments.
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Marcet, Ortega Marina. "Surveillance mechanisms in mammalian meiosis." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/387429.
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Per tal de protegir les cèl·lules germinals de sofrir inestabilitat genòmica, diversos mecanismes de control s’encarreguen de que la progressió de la meiosis sigui correcte. En mamífers, els espermatòcits que presenten defectes de recombinació o de la formació de la vesícula sexual pateixen un bloqueig a l’estadi de paquitè. Estudis previs del nostre laboratori descriuen que la via complex MRE11-ATM-CHK2 activa l’arrest dependent de recombinació en presència de trencaments de doble cadena (DSBs) no reparats. L’objectiu d’aquest treball ha estat identificar si els membres de la família p53, els quals són possibles substrats de ATM i CHK2, participen en l’activació del arrest depenent de recombinació. En una aproximació genètica, hem obtingut ratolins doble mutants portadors d’una mutació de un membre de la família p53 (p53, Tap63 o p73) en un fons defectiu per Trip13. La mutació de Trip13 causa defectes de recombinació, el qual activa l’arrest depenent de recombinació en els espermatòcits a l’estadi de paquitè. Per tant, hem estudiat com l’absència d’algun membre de la família p53 afectava aquest fenotip d’arrest el espermatòcits Trip13mod/mod. Els nostres resultats demostren que tant la deficiència de p53 com Tap63, però no p73, permeten que els espermatòcits progressin més enllà i arribin a l’estadi de paquitè tardà tot i acumular nombrosos DSBs no reparats. Addicionalment, l’absència de p53 o Tap63 resulta en una disminució del nombre d’espermatòcits apoptòtics a l’estadi de paquitè primerenc. Així, els nostres resultats indiquen que p53 i TAp63 són responsables d’activar l’arrest dependent de recombinació en els espermatòcits de ratolí. Tot i així, els espermatòcits doble mutants encara presenten un bloqueig a l’estadi de paquitè. Per tal d’estudiar si els espermatòcits doble mutants arresten a causa de l’activació de l’arrest depenent de la correcta formació de la vesícula sexual, hem analitzat la funcionalitat del MSCI en els mutants Trip13. Per tant, el fet de saltar-se l’arrest dependent de recombinació ens ha permès elucidar el paper de TRIP13 en el silenciament meiòtic, de manera que al fallar la vesícula sexual es desencadena l’apoptosi i bloqueig dels mutants Trip13. Aquests resultats infereixen que el bloqueig depenent de recombinació i el depenent de la correcta formació de la vesícula sexual, són mecanismes que s’activen per mecanismes genèticament separats. A partir de l’observació que TRIP13 és necessari per implementar el silenciament del MSCI, he dut a terme un anàlisis exhaustiu de la transcripció en els mutants de Trip13. Els nostres resultats de marcatge de RNA amb EU i activació de la RNA polimerasa II fosforilada (S2) suggereixen que la expressió de RNA en els espermatòcits mutants per Trip13 es troba incrementada en els estadis inicials de la meiosis. Addicionalment, la seqüenciació del RNA ha permès observar que els gens dels cromosomes sexuals i gens pre-meiòtics es troben sobre expressats en els mutants de Trip13, suggerint que TRIP13 és necessari per mantenir l’expressió d’aquests gens a nivells baixos. En conjunt, els resultats presentats en aquest treball contribueixen a entendre com els mecanismes de control regulen diversos passes crucials de la progressió de la profase meiòtica en els espermatòcits de mamífer.In order to protect germinal cells from genomic instability, surveillance mechanisms ensure that meiosis occurs properly. In mammals, spermatocytes that display recombination or sex body defects experience an arrest at pachytene stage. Previous studies from our lab described that the MRE11 complex-ATM-CHK2 pathway activates the recombination-dependent arrest in the presence of unrepaired double strand breaks (DSBs). In this work we aimed to identify if p53 family members, which are putative targets of ATM and CHK2, participate in the activation of the recombination-dependent arrest. As a genetic approach, we bred double mutant mice carrying a mutation of a member of the p53 family (p53, TAp63, p73) in a Trip13 defective background. Trip13 mutation causes recombination defects, which activate the recombination-dependent arrest in pachytene-stage spermatocytes. Thus, we studied how the absence of p53 family members affected the arrest phenotype of Trip13mod/mod spermatocytes. Our data showed that p53 and TAp63 deficiency, but not p73, allowed spermatocytes to progress further into late pachynema, despite accumulating numerous unrepaired DBSs. In addition, lack of p53 or TAp63 resulted in a decrease of apoptotic spermatocytes at early pachytene stage. Therefore, our results indicate that p53 and TAp63 are responsible to activate the recombination-dependent arrest in mouse spermatocytes. Even though, double mutant spermatocytes still arrested at pachytene stage. To study if double mutant spermatocytes were arresting due to the activation of the sex body deficient arrest we analyzed MSCI functionality in Trip13 mutants. Thus, by bypassing the recombination-dependent arrest has allowed us to elucidate a role for TRIP13 protein in meiotic silencing, which consequently triggers apoptosis in double mutants at late pachytene stage due to sex body impairment. These results infer that the recombination-dependent and the sex-body deficient arrest are activated by two genetically separated mechanisms. From the observation that TRIP13 is required to implement MSCI silencing, we performed an exhaustive analysis of transcription in Trip13 mutants. Our results suggested that RNA expression in Trip13 mutants was increased in early meiotic stage spermatocytes, assessed by EU-labeling RNA and phosphorylated(S2)-RNA polymerase II. Moreover, RNA sequencing data highlighted the observation that sex chromosome genes and pre-meiotic genes are overexpressed in Trip13 mutants, suggesting that TRIP13 is required to maintain the expression of these genes at low levels. Overall, the data presented in this work contributes to the understanding on how surveillance mechanisms control several crucial steps of meiotic prophase progression in mammalian spermatocytes.
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Tsirigotis, Maria. "Mutational analysis of mammalian ubiquitin." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/29267.
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Much of what is known about the ubiquitin/proteasome pathway has been deduced from mutational analysis performed in the yeast model system. From the high level of conservation between yeast and mammalian ubiquitin it would be expected that expression of analogous ubiquitin isoforms in higher eukaryotes would result in similar phenotypes. A site directed mutagenesis approach was employed to investigate the phenotypes of expression of mutant ubiquitin in higher eukaryotes and in the in vivo setting of novel ubiquitin transgenic mice. It was found that Ub-EGFP fusion proteins are efficiently recognized and processed by ubiquitin specific proteases both in mammalian cells and in transgenic mice; the transgene-derived ubiquitin moiety was found to substitute for endogenous ubiquitin in poly-Ub chain assembly and ubiquitinated conjugates were recovered using standard purification methodologies. The expression of chain-terminating ubiquitin derivatives (K48R and K63R) predisposed cells to the toxic effects of misfolded proteins and sensitized cells to DNA damaging agents. In transgenic mice, the expression of K48R mutant ubiquitin was found to confer protective effects and delay the deterioration of Purkinje neurons in a mouse model of SCA-1. The neuroprotective effect of K48R mutant ubiquitin may be mediated though stabilization of key transcription factors whose loss figured in the normal course of the SCA1 disease.
The expression of C-terminal variants in yeast has been proposed to have profound effects on ubiquitin metabolism. A mechanistically related mechanism has been proposed to contribute to the pathogenesis of Alzheimer's disease wherein transcriptional frameshifting of the ubiquitin B mRNA generates an aberrant ubiquitin protein (termed UBB+1) with an altered C-terminus. To investigate the constraints with regard to processing/conjugation and recycling of ubiquitin in higher eukaryotes a plethora of C-terminal ubiquitin variants were generated and introduced in mammalian cells as linear fusions with EGFP. Mutations that inactivate yeast ubiquitin did not abolish the function of ubiquitin in higher eukaryotes; C-terminal ubiquitin variants were processed by deubiquitinating enzymes and in some cases were found to conjugate to cellular proteins. The tolerance of mammalian cells to mutant ubiquitin may be attributable to loosened constraints that exist at the C-terminus due to mechanisms that couple deubiquitination, targeting and destruction of Ub-EGFP fusion proteins. Preliminary data suggest that prolonged exposure of cells of neuronal lineage to C-terminal ubiquitin variant as assessed in transgenic mice may result in perturbed ubiquitin homeostasis, a feature observed in the pathogenesis of Alzheimer's disease.
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Lau, Stephen S. K. "Gene silencing in mammalian cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28435.pdf.
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Lee, Douglas P. "Glycerolipid metabolism in mammalian tissues." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2002. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ62649.pdf.
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Zhong, Liangwei. "Selenium in mammalian thioredoxin reductase /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4243-9/.
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Renglin, Lindh Anna. "Mitotic aberrations in mammalian cells /." Stockholm : Dept. of genetics, microbiology and toxicology, Stockholm university, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-522.
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Dou, Dengfeng. "Mammalian and viral protease inhibitors." Diss., Wichita State University, 2010. http://hdl.handle.net/10057/3281.
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Chronic Obstructive Pulmonary Disease (COPD) is currently the fourth leading cause of death in the US. COPD is a multi-factorial disorder characterized by an oxidant/antioxidant imbalance, inflammation, a protease/antiprotease imbalance and apoptosis. This dissertation describes a general strategy for the design, synthesis and biochemical evaluation of dual function inhibitors which could potentially interrupt the above disorder, thereby enhancing the treatment of COPD. An example of this type inhibitor based on the 1,2,5-thiadiazolidin-3-one scaffold has been proven effective against both human neutrophil elastase (HNE) and caspase-1, two key enzymes responsible for elastin degradation and inflammation, respectively. In addition, an X-ray crystal structure and a high resolution mass spectrum of inhibitor bonded HNE have proven the proposed mechanism of HNE inactivation. Furhtermore, simple reversible competitive inhibitors of COPD-related enzymes (HNE and proteinase 3) have also been designed, synthesized and evaluated biochemically.
West Nile virus and Dengue virus are recognized as a major health threat that affects millions of people worldwide. However, there is currently no treatment or vaccine available for the virus infection. This dissertation describes the design, synthesis and biochemical evaluation of reversible competitive inhibitors of both West Nile virus and Dengue virus NS2B-NS3 protease. Combinatorial chemistry and click chemistry methods have been used in the design of the protease inhibitor and the identified hit was optimized using computational programs (AutoDock4 and SYBYL). Several more hits were identified during the optimization and further development could potentially lead to very potent inhibitors of NS2B-NS3 protease with good pharmacokinetics and oral bioavailability.Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Xue, Yue 1978. "Iron metabolism in mammalian cells." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79216.
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Iron, known for its versatility, is an essential element in the metabolism of mammalian cells. One of the most common iron disorders is autosomal recessive disease---hereditary hemochromatosis, which leads to the iron overload in population of northern European descent. During years of my graduate research, I focused on the study of Hemochromatosis gene Hfe and a point mutation C282Y that leads to more than 80% of all hemochromatosis cases.Iron Regulatory Proteins (IRPs), which serve as main posttranscriptional regulators of cellular iron homeostasis, are the other interest of research. Iron regulatory proteins reversibly interact with iron regulatory elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. The binding ability of IRPs is under tight control so that they respond to the changes in the intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Besides intracellular iron levels, some other stimuli, such as oxidative stress, are capable of regulating this RNA-protein interactions.
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Theil, Ian. "Anchorage-dependent mammalian cell culture." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56768.
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Genetically engineered anchorage-dependent human embryonic kidney (293) cells were cultured at 37$ sp circ$C on 1 mm thick sheets of a fibrous polymeric matrix having an average fibre diameter of 10.2 $ mu$m and a void fraction of 0.81 using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2.5 mM glutamine. Immobilization efficiencies above 70% were observed when cells were added to 100 mL spinner flasks (operating at 60 rpm) containing 70 mL of medium and two 1 x 1 cm squares of matrix (total gross area of 2 cm$ sp2$) fastened to the base of the stirrer shaft. Loadings in excess of 2.4 $ times 10 sp7$ cells per cm$ sp2$ of matrix were measured after 2 h.The state of the cultures was followed by measuring the consumption of glucose and glutamine and the production of lactate and ammonium.
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Grünert, Stefan. "Translation initiation in mammalian systems." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390257.
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Vavrova, Jana. "Regulation of mammalian SINE transcription." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/433/.
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Despite the abundance of the templates, both human and rodent SINEs are normally expressed at a very low level. DNA methylation-mediated silencing has been proposed as a possible cause of their transcriptional repression. The effect of DNA methylation and the effect of DNA methylation-dependent methyl-CpG-binding domain proteins (MBD proteins) on SINE transcription were studied here. It was shown that both human and rodent SINEs are bound by MeCP2, MBD1 and MBD2. Both human and rodent SINEs were also shown to be occupied by HDAC1, HDAC2 and a component of SWI/SNF complex, Brahma. Human Alus were also found to be occupied by components of two corepressor complexes, SIN3 and NuRD. Whether MBD proteins repress SINE transcription in a DNA methylation-dependent manner was further investigated using systems with low or near absent DNA methylation and, in the case of MeCP2 protein, by its direct removal. MeCP2 was found to have no repressive effect on B1 and B2 expression. RT-PCR analysis showed no increase in B1 and B2 RNA levels in MeCP2 null mice kidneys. ChIP analysis of Dnmt1n/n p53-/- embryonic fibroblasts, which have less than 5% of the normal DNA methylation level, showed significant reduction in MeCP2 and MBD2 binding, confirming that their presence is DNA methylation-dependant. RT-PCR comparison of Dnmt1+/+ p53-/- and Dnmt1n/n p53-/- cells, however, detected no increase in B1 or B2 RNA levels. This was consistent with results obtained from MeCP2 null mice, where lack of MeCP2 did not result in increased B1 and B2 expression and with a previous study involving human Alus (Yu et al., 2001). MBD2 also does not seem to repress SINE activity, as its release following loss of DNA methylation did not result in increased SINE RNA levels. Strikingly, all human and rodent SINEs studied here were found to be bound by transcription factors TFIIIB and TFIIIC at comparable levels with actively transcribed genes. Some RNA polymerase III was also detected, but at levels significantly lower than on active genes, suggesting a defect in RNA polymerase III loading onto SINEs. This occupancy of the transcriptional complex was comparable in cells with normal levels of DNA methylation and in cells with significantly reduced levels of DNA methylation, suggesting that the occupancy is not affected by methylated DNA or DNA methylation-dependent components of chromatin. Indeed, removal of 50% of histone H1 did not result in increased B1 or B2 expression in this study. The fact that all tested SINEs are occupied by TFIIIB and TFIIIC also brings an unprecedented insight into the number of these transcription factors present in the cell.
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Hawley, Patricia. "Oligodeoxynucleotide interaction with mammalian cells." Thesis, University of East Anglia, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338058.
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Gohari, Nasrollah Saleh. "Homologous recombination in mammalian cells." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414660.
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Savitsky, M. J. "Studies on mammalian hyaluronidase enzymes." Thesis, Bucks New University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382493.
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Jenkin, L. "Oxytocin in the mammalian prostate." Thesis, University of Bristol, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246289.
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Liakath-Ali, Kifayathullah. "Phenogenomics studies in mammalian skin." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709260.
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Nathubhai, Amit. "Molecular probes for mammalian chitinases." Thesis, University of Bath, 2010. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518107.
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Chitin is a glycopolymer consisting of +-(1, 4)-linked N-acetyl-D-glucosamine residues that occurs widely in nature and is a constituent of many organisms that are pathogenic to humans, including insects, fungi, and parasitic nematodes. As these organisms depend on the ability to break down chitin at key points of their life-cycle, inhibitors of the enzymes termed chitinases that catalyse the hydrolysis of chitin, are of considerable interest as potential drugs and insecticides. Although chitin is absent from mammalian physiology, two human chitinases along with several chitin-binding proteins (chi-lectins) have been associated with the onset or transmission of several major human diseases such as asthma, Legionnaire’s disease and osteoarthritis. Therefore, selective inhibitors of chitinases are now of considerable interest as new drug leads and biochemical probes. Until recently, few broad spectrum chitinase inhibitors had been identified. The natural cyclopentapeptide, argifin, has been shown to be a potent inhibitor of several bacterial-type family 18 chitinases including Aspergillus fumigatus chitinase B1 (AfChiB1). With the aid of high resolution X-ray structures we have designed and prepared linear fragments of the natural product cyclopentapeptide argifin using a combination of SPPS and solution phase synthesis. This has allowed us to determine that the Nmethyl guanylurea motif serves as a starting point for the generation of novel, drug-like, peptidomimetic inhibitors family 18 chitinases. We have also demonstrated that the cis configuration about the Arg(MC)-N-MePhe peptide bond is essential to retain any significant biological activity. This dipeptide motif is also found in another naturally occurring cyclopentapeptide, banyasin A, extracted from the cyanobacteruim Nostoc sp. Banyasin A also contains a rare +-amino acid, 3-amino-2-methyl-5E-octenoic acid (Amoa), in which the stereochemistry at the C3 and C5 of Amoa has not been resolved. The diastereoselective synthesis of Amoa for the preparation of banyasin A has also been established using chiral oxazolidinone-based aldol chemistry, which has allowed us to successfully prepare a single diastereoisomer of the natural product cyclopentapeptide incorporating this +-amino acid. New methodology for the preparation of argifin has also been developed to reduce the propensity towards the formation of undesired side products and to prepare the natural product on a larger scale.
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Mikkelsen, Tarjei Sigurd 1978. "Mammalian comparative genomics and epigenomics." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/52808.
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Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
The human genome sequence can be thought of as an instruction manual for our species, written and rewritten over more than a billion of years of evolution. Taking a complete inventory of our genome, dissecting its genes and their functional components, and elucidating how these genes are selectively used to establish and maintain cell types with markedly different behaviors, are key challenges of modern biology. In this thesis we present contributions to our understanding of the structure, function and evolution of the human genome. We rely on two complementary approaches. First, we study signatures of evolutionary processes that have acted on the genome using comparative sequence analysis. We generate high quality draft genome sequences of the chimpanzee, the dog and the opossum. These species share a last common ancestor with humans approximately 6 million, 80 million and 140 million years ago, respectively, and therefore provide distinct perspectives on our evolutionary history. We apply computational methods to explore the functional organization of the genome and to identify genes that contribute to shared and species-specific traits. Second, we study how the genome is bound by proteins and packaged into chromatin in distinct cell types. We develop new methods to map protein-DNA interactions and DNA methylation using single-molecule based sequencing technology. We apply these methods to identify new functional sequence elements based on characteristic chromatin signatures, and to explore the relationship between DNA sequence, chromatin and cellular state.
by Tarjei Sigurd Mikkelsen.
Ph.D.
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Mao, Chih-Chieh. "Dissecting the mammalian mitochondrial nucleoid." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614071.
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Mahon, Annette. "Mammalian body size and phylogeny." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616106.
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Khojah, Sohair Mohammed. "Ageing in the mammalian brain." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6244/.
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With a globally ageing population diseases associated with this natural process are becoming major issues worldwide. Research into the process of ageing and its concomitant issues is rapidly expanding; the need for new tools and models to investigate this rapidly expanding arena of research is paramount. The discovery of a spontaneous mutation in the AS rat strain which introduces a premature stop mutation into the gene encoding protein kinase C γ (PKCγ) lead to the development of a model for one such age related disorder, Parkinson's disease. Consequently, this model has been selected to investigate age related changes in specific areas of the brain (the cerebellum, basal ganglia, cerebral cortex and brainstem). These regions were selected because they have previously been shown to demonstrate changes with age (cerebellum, cerebral cortex and basal Ganglia), they show differences between the AS and AS/AGU strains (basal ganglia) or they show differences in PKCγ knockout models (cerebellum). The Brainstem was selected as it shows little change due to age and shows no differences in PKCγ knockouts or AS/AGU rats. This study used established qPCR methods to measure a validated biomarker of ageing, CDKN2A (the transcript for p16INK4a) in the brains of these rats to determine whether this model is in fact a genuine model for accelerated ageing. This thesis demonstrates that CDKN2A expression, in combination with senescence-associated β-galactosidase staining, provides clear evidence of accelerated ageing in the brains of AS/AGU rats when compared with the parent AS strain. These investigations were furthered by an investigation of members of the Sirtuin family of proteins. The changes in expression of these Sirtuins indicates that there may be increased levels of cellular stress, disruption of metabolism and DNA damage in the AS/AGU rats, this would be congruent with the accelerated ageing phenotype present in this strain. Furthermore, the levels of these Sirtuins were in line with the predictions from the MTR trinity in regards to the accelerated ageing phenotype. Whilst some of the changes in senescence and metabolic disruption may be attributable to the PKCγ mutation in the AS/AGU rats, it would appear that there is some element of accelerated ageing that is independent of this mutation.
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Piergiovanni, G. M. M. "Functional characterisation of mammalian GEMC1." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1420499/.
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Chromosomal DNA must be faithfully duplicated only once per cell cycle in order to ensure genome stability. At the end of mitosis and during G1 phase of the cell cycle, eukaryotic cells assemble pre-replicative complexes (pre-RCs) on multiple replication origins distributed along their chromosomes in a process called origin licensing. During S phase, licensed origins are activated, DNA is unwound and the replicative machinery is recruited triggering origin firing. Cyclin-dependent kinases activity (CDKs), ubiquitin-dependent protein proteolysis and licensing factors inhibitors cooperate to promote origins firing and prevent replication origins from being reused within the same cell cycle. Mis-regulation of origin licensing contributes to genetic instability and is commonly observed in a variety of cancers. GEMC1 is a recently identified CDK target involved in the control of DNA replication in Xenopus laevis. GEMC1 is highly conserved in vertebrate organisms but nothing is currently known about its biological role in mammalian cells. In this study, the first characterisation of mammalian GEMC1 is presented. GEMC1 is a nuclear protein that is highly regulated during the cell cycle by a series of post- translational modifications, which include CDK-dependent phosphorylation and ubiquitination. Similar to its Xenopus homologue, GEMC1 interacts with TopBP1 and Cdc45 in S phase and its depletion strongly impairs origins firing, leading to the accumulation of DNA double-strand breaks (DSBs) in replicating mouse and human cells. These results confirm that mammalian GEMC1 has a positive role in chromosomal DNA replication. GEMC1 also forms a complex with the licensing inhibitor Geminin, both in vivo and in vitro, through its coiled-coil domain. Strikingly, GEMC1 overexpression in human U2OS cells induces DNA re-replication, chromosomal breakages that cause a G2/M arrest and centrosome amplification. All together, these results suggest that GEMC1 is a powerful DNA replication promoting factor that functions by stimulating origin firing and by antagonising Geminin dependent inhibition of origin licensing in vertebrate cells. These data also indicate that regulation of GEMC1 protein levels is critical for the maintenance of genome stability.
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May, Joel. "Gadd45g and mammalian testis determination." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:6ce4cf97-83da-4cc6-a433-25947f53a7f3.
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Mammalian sex is determined by the presence or absence of Sry. This gene, located on the Y chromosome, promotes the development of a testis from the biopotential gonad by initiating a transcriptional programme that results in Sertoli cell formation. SRY is required to reach a specific threshold level during a critical time-period in order to induce the testicular differentiation pathway. We have previously shown that XY mouse embryos lacking Growth arrest and DNA damage inducible 45 gamma (Gadd45g) exhibit gonadal sex reversal due to a delay in Sry expression. Efforts to study the role of GADD45γ in sex determination have been stymied by the lack of a working antibody. As an alternative, I have produced a BAC transgene produces a similar expression profile to endogenous Gadd45g. This BAC was also shown to rescue the sex reversal found in B6-YPOS gonads, though further research is required to understand the molecular mechanisms through which this occurs. As a result, this BAC was chosen for modification using BAC recombineering to contain the sequence for a poly-His/FLAG epitope tag. Though this edited BAC was generated successfully, no transgenic lines have yet been produced. Members of the Gadd45 family have previously been associated with active DNA demethylation. Assuch, I have conducted whole genome bisulphite sequencing on DNA from XY wild-type, XX wild-type and XY Gadd45g-/- embryos at the 18 tail somite stage, where Sry expression is at its peak. Gadd45g and Sry are only expressed in the somatic cells of the gonads, which have been purified through fluorescence-activated flow cytometry using the transgenic line Sf1-EGFP. Due to the low number of cells that are purified from the embryonic gonad at this stage, sequencing libraries were generated using post-bisulphite adaptor tagging (PBAT). Analysis of the acquired sequences revealed that there are no significant differences between the methylomes of XY wild-type, XX wild-type and XY Gadd45g-/- mutant gonadal somatic cells. It therefore appears that GADD45γ does not have a role in establishing the methylome at this particular stage of sex determination.
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Jiang, Lei. "Mitochondrial Distribution in Mammalian Cells." University of Dayton / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1259968456.
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Imai, Takeshi. "Studies on Mammalian Selenite Metabolism." Kyoto University, 2013. http://hdl.handle.net/2433/175070.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第17641号
農博第2003号
新制||農||1012(附属図書館)
学位論文||H25||N4762(農学部図書室)
30407
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 阪井 康能, 教授 平竹 潤
学位規則第4条第1項該当
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Heikinheimo, Liisa. "Phosphatidylserine translocation in mammalian cells." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/heikinheimo/.
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Zheng, Wenxuan. "Properties of mammalian P2X₇ receptors." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/properties-of-mammalian-p2x7-receptors(1ff23f4a-47eb-4d06-b54c-bd7488c9700c).html.
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To establish comprehensive pharmacology of P2X₇ receptors, membrane current recording, intracellular calcium transient recording and ethidium bromide uptake were carried out to examine several selective (A-740003, A- 438079) and non-selective (suramin) P2X₇ antagonists across mammalian P2X₇ receptors (human, mouse and rat). These P2X₇ receptors demonstrated species-dependent sensitivities to antagonists. In each species, A-740003 revealed variant IC50 values with different assays, indicating the assay- dependent pharmacology of P2X₇ receptors. Conventionally, pharmacology can be used to define a native current but not in the case of the human breast cancer cell line, Hs578T. It is found that P2X₇ was expressed at both mRNA and protein level. The ATP-evoked currents recorded from Hs578T cells were P2X₇-like with distinctive electrophysiological features. But the pharmacology profile of the currents did not fit with P2X₇ receptor. Further experiments are needed to either include or exclude the existence of functional P2X₇ receptors in Hs578T. Transmembrane domain 2 (TM2) is known as the pore-forming region for P2X receptors. TM2 of P2X₇ receptor was investigated with cysteine substitution scanning. The predicted α-helix structure of the TM2 segment was in good agreement with the results from the substituted cysteine accessibility method (SCAM). Thr336, Ser339, Tyr343, Phe344 and Thr348 were found important for both channel dilation and aqueous pore formation. Ser339 was further studied. Various substitutions at Ser339 were explored. The results suggest that the polarity of the side chain at Ser339 is essential for the channel dilation. Furthermore, disulfide bond formation was identified between S339C in the trimeric receptor, implying that the side chains of Ser339 might turn very close to each other during the channel opening and dilation.
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Sarwar, Muhammad. "Measurement of mammalian cell adhesion." Thesis, University of Bath, 1992. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314527.
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Krippaehne, Suzanne Louise. "Three Dimensional Mammalian Skull Morphology." PDXScholar, 1992. https://pdxscholar.library.pdx.edu/open_access_etds/4601.
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This thesis deals with archiving morphological data utilizing a three dimensional coordinate system. Morphological reference points are archived via rectangular position coordinates, rectangular position vectors, and spherical position vectors. The concepts of translation trajectories, translation vectors, and relative position vectors are developed. Analysis of three dimensional coordinate data utilizing translation trajectories and translation vectors is described. In order to test the methodology developed, the method is applied to an analysis of harbor porpoise, Phocoena phocoena L., skull morphology. (Key words: morphology, ontogenetic trajectories, allometry, position coordinates, position vectors, translation trajectories, translation vectors, relative position vectors, and harbor porpoise).
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Yao, Mu. "Study of mammalian heart failure." Thesis, The University of Sydney, 1997. https://hdl.handle.net/2123/27637.
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Heart failure is the end-stage of heart diseases. In this state the heart is unable to pump an adequate amount of blood to meet the metabolic requirement of the peripheral tissues. Heart failure is a complex clinical syndrome that can result from any heart disease. Clinically, it is manifested by cardiomegaly, breathlessness, and fluid retention. In spite of advances in
pharmacological treatment, it remains a highly disabling and lethal disorder. Until now, the mechanism underlying this syndrome remains obscure. The major focus of this study was to investigate this mechanism by analysis of both human and animal failing hearts.
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Egwuekwe, Ejike Roland. "Vitamin D Receptor Gene Polymorphisms Knowledge And Breast Cancer In Texas." ScholarWorks, 2019. https://scholarworks.waldenu.edu/dissertations/6199.
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Breast cancer is a world health problem and is a leading cause of cancer-related death among women in the United States. However, breast cancer risks were reported to be reduced through exposure to Vitamin D through its Receptors identified as the p53 target gene. The purpose of this study was to assess the associations between VDR gene polymorphisms knowledge/awareness and decisions to reduce breast cancer risks and likelihood of mammogram screening among women in Texas. Data from survey were used. Roy adaptation model was the theoretical framework that guided this quasi- experimental, quantitative research. The dependent variables were decisions to reduce breast cancer risks and likelihood of mammogram screening. The independent variables were knowledge about VDR gene polymorphisms and exposure to vitamin D. The covariates were level of education, awareness, lifestyle, breast self-exams, mammograms, age, early menarche, late menopause, and family history of breast cancer. The chi-square test and regression analysis were used to test the stated research hypotheses and to answer the research questions. Knowledge of VDR gene polymorphisms and exposure to vitamin D were not significantly associated with breast cancer risk, ï?£2 (3, N= 250) =3.84, p > 0.05. Also, awareness of the risk factors for breast cancer was not significantly associated with decisions to go for mammogram screenings or to enroll in breast cancer risk-reduction programs, ï?£2 (3, N= 250) =1.58, p > 0.05. To advocate for the promotion of awareness of the importance of pharmacogenetic testing for VDR gene polymorphisms for early detection of breast cancer, which would help to undertake appropriate therapeutic measures in a timely manner to prevent cancer metastasis, further research is warranted.
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Andersen, Nicholas John Yeaman Charles A. "Characterization of mammalian exocyst subunit Sec3." [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/327.
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Nelson, Greg. "Mammalian sweet and umami taste receptors /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3191985.
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Savolainen, Linda. "Transcription Associated Recombination in Mammalian Cells." Doctoral thesis, Stockholm : Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-38931.
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Diss. (sammanfattning) Stockholm : Stockholms universitet, 2010.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
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Oswald, Corina. "Mitochondrial copper homeostasis in mammalian cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-61580.
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Assembly of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the correct insertion of subunits, accessory proteins, cofactors and prosthetic groups. Most of the fundamental biological knowledge concerning mitochondrial copper homeostasis and insertion of copper into COX derives from investigations in the yeast Saccharomyces cerevisiae. In this organism, Cox17 was the first identified factor involved in this pathway. It is a low molecular weight protein containing highly conserved twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is essential for its function.
So far, the role of Cox17 in the mammalian mitochondrial copper metabolism has not been well elucidated. Homozygous disruption of the mouse COX17 gene leads to COX deficiency followed by embryonic death, which implies an indispensable role for Cox17 in cell survival.
In this thesis, the role of COX17 in the biogenesis of the respiratory chain in HeLa cells was explored by use of siRNA. The knockdown of COX17 results in a reduced steady-state concentration of the copper-bearing subunits of COX and affects growth of HeLa cells accompagnied by an accumulation of ROS and apoptotic cells. Furthermore, in accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper center of COX, COX17 siRNA knockdown affects COX-activity and -assembly. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so called supercomplexes. While the abundance of COX dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. Accumulation of a novel ~150 kDa complex containing Cox1, but not Cox2 could be observed. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. Data presented here suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. Instead an interdependent assembly scenario for the formation of supercomplexes is proposed that requires the coordinated synthesis and association of individual complexes.
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Elvers, Ingegerd. "Replication Fork Stability in Mammalian Cells." Doctoral thesis, Stockholms universitet, Institutionen för genetik, mikrobiologi och toxikologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-56697.
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Maintaining replication fork integrity is vital to preserve genomic stability and avoid cancer. Physical DNA damage and altered nucleotide or protein pools represent replication obstacles, generating replicative stress. Numerous cellular responses have evolved to ensure faithful DNA replication despite such challenges. Understanding those responses is essential to understand and prevent or treat replication-associated diseases, such as cancer. Re-priming is a mechanism to allow resumption of DNA synthesis past a fork-stalling lesion. This was recently suggested in yeast and explains the formation of gaps during DNA replication on damaged DNA. Using a combination of assays, we indicate the existence of re-priming also in human cells following UV irradiation. The gap left behind a re-primed fork must be stabilised to avoid replication-associated collapse. Our results show that the checkpoint signalling protein CHK1 is dispensable for stabilisation of replication forks after UV irradiation, despite its role in replication fork progression on UV-damaged DNA. It is not known what proteins are necessary for collapse of an unsealed gap or a stalled fork. We exclude one, previously suggested, endonuclease from this mechanism in UV-irradiated human fibroblasts. We also show that focus formation of repair protein RAD51 is not necessarily associated with cellular sensitivity to agents inducing replicative stress, in rad51d CHO mutant cells. Multiple factors are required for replication fork stability, also under unperturbed conditions. We identify the histone methyltransferase SET8 as an important player in the maintenance of replication fork stability. SET8 is required for replication fork progression, and depletion of SET8 led to the formation of replication-associated DNA damage. In summary, our results increase the knowledge about mechanisms and signalling at replication forks in unperturbed cells and after induction of replicative stress.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Submitted. Paper 3: Manuscript. Paper 5: Submitted.
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Asahara, Masakazu. "Variability and evolvability in mammalian dentition." 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/175150.
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Howarth, Frank Christopher. "Magnesium homeostasis in the mammalian heart." Thesis, University of Central Lancashire, 1994. http://clok.uclan.ac.uk/20059/.
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The magnesium ion (Mg2 ) is involved in a variety of physiological and biochemical processes including the activation of over 300 enzymes and the control of transmembrane movement of cations. In most mammalian cells intracellular Mg2+ is kept well below electrochemical equilibrium. In cardiac muscle cells recent studies suggest that the intracellular and extracellular concentrations of Mg2+ are around I and 0.5 mIs4, respectively. The equilibrium potential for Mg2 is approximately - 10 mV. In resting muscle the membrane potential is far more negative than this and M g2+ extrusion must take place aganist an electrochemical gradient. Factors which may be important in preserving intracellular Mg2+ include; changes in membrane potential which occur during the action potential, intracellular buffering, intracellular compartmental redistribution, the permeability characteristics of the plasma membrane and transport stystems in the plasma membrane. The aims of the study are to investigate the effects of extracellular Mg2+ on contractility and coronary flow and the effects of hormonal and extracellular cationic control of Mg2+ homeostasis in the rat heart. Subsequently, the effects of dietary magnesium on the magnesium, calcium, sodium and potassium content of the heart (and other tissues) in young rats is also investigated. Perfusion of the isolated heart with elevated extracellular Mg2+ (1.2 - 7.2 mM) caused a profound reduction in the force of contraction and an associated increase in the coronary flow rate. The selective 13-adrenoceptor agonist isoprenaline (10 M), evoked a large Mg2+ efflux and an associated increase in the force and rate of contraction in the isolated perfused heart. Quantitativley, similar increases in Mg2+ efflux were seen during stimulation of the isolated heart with the adenylate cyclase activator, forskolin (10 M). Stimulation of superfused electrically paced ventricle segments with either isoprenaline, noradrenaline or adrenaline (10-6 M) also evoked a large net Mg2+ efflux. The isoprenaline-evoked Mg2+ efflux was significantly reduced during treatment of the heart with either the 0- antagonist, propranolol (I o-5 M), or the Ca2 -channel blocker, verapamil (1 o-5 M) Stimulation of mag-ftira-2 AM loaded cardiac myocytes in suspension with isoprenaline did not result in any change in intracellular M g2+. Elevations of extracellular Na+ concentration (as NaCl or sodium isethionate), elevated chloride (as choline chloride) and sucrose (at concentrations osmotically equivalent to elevated Na+) all evoked large Mg2+ eftiuxes in the isolated perfused heart. The elevated Na (as NaCl) -evoked Mg 2 efflux was partially, though not significantly, inhibited by the Na channel blocker, amiloride (10 M) and propranolol (lOs M). During stimulation of the heart with elevated Na+ there was no increase in lactate dehydrogenase activity indicating that the release of Mg2+ was not due to cell damage. Treatment of the heart with verapamil (I o-6 M), which dramatically reduced the force of contraction, had little effect on the Mg2+ efflux evoked by elevated Na+ (as sodium sulphate). Acute perfusion of the isolated heart with nominally Mg2+ free physiological solution over 15 min caused a significant reduction in the magnesium content of the heart, thereafter, magnesium was well conserved. Magnesium deficient diets fed to young rats over a period of approximately one month caused reduction in food consumption, retardation of growth, reductions in some organ weights and various cationic disturbances in the heart and other tissues. In the heart there were no significant changes to sodium or potassium but there was a significant increase in calcium and reduction in magnesium. Collectively, these results suggest that magnesium is well conserved in the heart and that the catecholamines and changes in extracellular osmolarity may be important physiological regulators of magnesium homeostasis in the heart.
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Lapage, John M. J. "Polyclonal architecture of the mammalian head." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/77488/.
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While much of modern developmental biology has focussed upon molecularly defined cell populations, relatively little is understood about how clonal groups within these broad cell populations organise complex tissues. In this thesis, I explore the clonal architecture of the jaws and teeth, and of the dermal bones of the calvaria, revealing cryptic modules as novel developmental features in both. I combine Confetti multicolour genetic lineage labelling with novel analytical techniques in order to map clonal populations in 3D and provide quantitative parameters for clonal expansions. Tooth identity within the mandible is thought to be encoded by the an initial proximodistal position within the branchial arch, which implies that cells do not undergo migration. I observe distal Hand2-Cre labelled cells in the proximal territory, which necessitates migration. These distal cells give rise to the mandible, alveolar bone and a small proportion of odontoblasts, while unlabelled proximal cells were found in the distal territory of the incisors in different proportions. The clonal composition of teeth and jaw bones is dissected by novel analysis of mixed cell populations. I find odontogenic and alveolar bone populations to share a common lineage, comprising a cryptic developmental unit distinct from the mandible, a feature that I can also verify in another transgenic for the upper jaw. I also find that the initial tooth composition radically changes in ontogenetic time. Starting from similar compositions of distal and proximal cells I find that in incisors the distal population expands while the proximal wanes, while in molars the opposite occurs. This is the first evidence for a temporally changing cell population structure underlying the well defined heterodonty between incisors and molars and allows a reinterpretation of early tooth specification events. The dermal bones of the calvaria are thought to grow in thickness by static osteoblasts depositing matrix appositionally and growth is supposed to occur exclusively at sutures. Whole calvaria single-cell clonal lineage analysis of cranial neural crest cells with Wnt1-Cre and Confetti labelling reveals an extensively dynamic program of invasive growth distributed throughout all parts of the dermal bone. Cryptic clonal modules grow laterally, with invasion through and into the bone primarily organised around the centres of these `patches'. The process of bone maturation is revealed to consist of a series of invasions between the three layers of the bone, with the innermost compacta layer driving initial thickness growth by invasion into the middle spongy layer, and the outermost (dermis-adjacent) compacta layer driving later growth. Conversely there is no evidence to suggest that the sutures are principal generative regions, as they do not share a common clonal lineage with adjacent bone. I also investigate muscle attachment regions and find that the same clones traverse tissue boundaries from bone into muscle connective tissues, thus a clonal model predicated on joint 'attachment point precursor cells' can now explain patterns of skeletomuscular connectivity previously found at the population level by my supervisor. A novel generative model of bone growth from cryptic clonal patch modules is proposed, allowing us for the first time to understand thickness growth and the evolutionary transition from a micromeric to a macromeric dermal bone condition, events first visible in crown gnathostomes (placoderms).
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Byrne, Paula Catherine. "Molecular analysis of mammalian serine hydroxymethyltransferase." Thesis, University of Surrey, 1992. http://epubs.surrey.ac.uk/842904/.
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The overall objectives of this thesis were the isolation and expression of cDNA sequences coding for mammalian SHMT. A probe was developed using the available amino acid sequence of the rabbit cytosolic SHMT (Martini et al., 1987) as a basis for designing primers, which were then used to amplify a region of the sequences coding for rabbit cytosolic SHMT (chapter 3). This amplified region was subsequently used to screen a rabbit liver cDNA library. The screening proved successful, with the isolation of sixty-seven clones potentially coding for SHMT. Further analysis of one of these clones, pUS1203, has provided the nucleotide sequence of the complete coding sequences for cytosolic SHMT (chapter 4). Contained within this clone were the 5' and 3' UTRs of 155 and 653 nucleotides respectively. The complete SHMT cDNA was cloned into the expression vector pUS1000, to generate pUS1202, where transcription of the SHMT cDNA was driven by the human cytomegalovirus promoter. Expression of this recombinant SHMT in COS-1 cells was achieved (chapter 5). Site specific mutagenesis to remove an upstream ATG codon within the 5' UTR was found to dramatically increase the levels of SHMT activity, when the mutated SHMT cDNA (pUS1208) was transfected into COS-1 cells. The level of SHMT activity in the cells transfected with pUS1208 was 100-fold higher than the activity found in cells transfected with pUS1000 and 50-fold higher than in cells transfected with pUS1202. Subsequent screening of a human breast cancer cell line cDNA library with the complete rabbit SHMT cDNA insert in pUS1203, identified 20 possible clones containing SHMT sequence (chapter 6). Sequence analysis of the largest of these, pUS1206, revealed that it contained 930bp of SHMT coding sequences, 890bp of 3' UTR and 850bp of an intron located 5' to the coding sequences. Comparison of the human and rabbit SHMT cDNAs reveals a high degree of constraint for the coding sequences that extends to a region of the 3' UTR (chapter 7).
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Schuliga, Michael, and michael schuliga@deakin edu au. "Steroidogenesis in cultured mammalian glial cells." Deakin University. School of Biological and Chemical Sciences, 1998. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20061207.154152.
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A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control.
In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes.
Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*.
A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P.
The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times.
The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ã-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP.
Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).
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Schuh, Sonya Marie. "Signaling pathways of mammalian sperm capacitation /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/10547.
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Zotter, Angelika Monika. "Protein Dynamics in Mammalian Genome Maintenance." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/12292.
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Korsieporn, Pira. "Interaction of plasticizers with mammalian cells." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98982.
Full textAbstract:
This investigation was focused on the in vitro metabolism of DEHA, DEHP and other common plasticizers by mammalian cell lines. The metabolic products and cell viability of hepatocytes (mouse and human) and human umbilical endothelial cells exposed to plasticizers was investigated in static culture.Gas chromatography and mass spectrometry showed that all of the plasticizers investigated were partially degraded, but at differing rates, depending on the plasticizer and cell line. Solubility and stearic effects were found to play important roles in determining the rate of hydrolysis. The only metabolic product observed was 2-ethyl hexanol, which accumulated in culture. This was due to the lack of alcohol dehydrogenase production in the human hepatocyte cell line used.
Hepatocyte cell viability was not significantly affected at 4 days of exposure to DEHA. By 12 days, only 50% of the cells remained viable when compared to control experiments. These results suggest that the accumulation of plasticizers metabolites, specifically 2-ethyl hexanol, may have potentially toxic effects.