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1
Wadaan, Mohammad A. M. "Genetic and cellular studies of apogamic plasmodium development in Physarum polycephalum." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391038.
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Dickinson, P. "Fibronectin gene expression in higher eukaryotic cells." Thesis, University of Manchester, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378322.
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Huang, George T. J. "Molecular cloning and characterization of multiple transcripts of the hamster ALG7 gene." Thesis, Boston University, 1992. https://hdl.handle.net/2144/31297.
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Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1992 (Oral Biology).Includes bibliographical references (leaves 70-84).
The ALG7 gene encodes the tunicamycin-sensitive, dolichol-P-dependent Nacetylglucosamine- 1-phosphate transferase, GPT, that catalyzes the synthesis of the first dolichollinked sugar, Dol-PP-GlcNAc, in the N-glycosylation pathway. ALG7 has been evQlutionarily conserved and is essential for growth in all eukaryotes. The ALG7 gene expression in yeast is known to be regulated in part by the 3' untranslated regions (UTR) of the ALG7 multiple transcripts at the posttranscriptional level. To examine the regulatory features of the mammalian ALG7 gene, cloning and characterization of the hamster ALG7 mRNAs were undertaken. Polymerase chain reaction (PCR) using a single ALG7 gene-specific primer was performed to clone the cDNAs corresponding to the 3' and 5' ends of the ALG7 mRNAs from the Chinese hamster ovary (CHO) cells. The initial Northern blot analysis using a hamster ALG7 genomic DNA as a probe has shown that in the CHO cells the ALG7 gene is transcribed into three major messages, approximately 1.5, 1.9, and 2.2 kb in size. The 1.9 kb transcripts were cloned and sequenced. There is one consensus polyadenylation signal AAUAAA located 12 nucleotides (nt) upstream to the major poly(A) site. Three additional minor poly(A) sites are located at 18, 21 and 29 nt downstream from the AAUAAA sequence in this 1.9 kb class of mRNAs. [TRUNCATED]
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Tsolou, Avgi. "Cellular responses to uncapped telomeres in eukaryotic cells." Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442349.
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Johnston, Kelly L. "The interaction of Wolbachia bacteria with eukaryotic cells." Thesis, University of Liverpool, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420296.
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Boudarène, Lydia. "Transcription factor target search in live eukaryotic cells." Paris 7, 2013. http://www.theses.fr/2013PA077004.
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La régulation de l'expression des gènes est un mécanisme étroitement régulé. Au coeur de ce système, les facteurs de transcription jouent un rôle majeur dans l'initiation de la transcription, en induisant le recrutement de la machinerie transcriptionelle sur les séquences régulatrices des gènes. Pour ce faire, les facteurs de transcription doivent trouver et se fixer sur leur séquence cible de -10 paires de bases, au sein d'un génome composé de plusieurs milliards de paires de bases, et ce, dans un laps de temps compatible avec celui des processus biologique. Le mécanisme de recherche de cible a été largement étudié au cours des quarante dernières années, de manière théorique, in vitro et in vivo chez les bactéries et les levures. Au cours de l'étude présentée dans cette thèse, l'introduction au sein du génome de cellules eucaryotes du système modèle bactérien Répresseur Tétracycline-Opérateur Tétracycline, représentant respectivement le chercheur et la cible, a permis de révéler que le chercheur explore le noyau en combinant un processus de diffusion libre en trois dimensions, des fixations non-spécifiques ainsi qu'une diffusion en une dimension le long de l’ADN. L'efficacité de fixation sur la séquence cible de notre système modèle s'est avérée être plus faible que prévue, impliquant la prise en compte d'autres paramètres, tels que la concentration locale de facteurs de transcription ou l'organisation spatiale de la chromatine afin d'avoir une compréhension globale du mécanisme de régulation de l'expression géniqueGene regulation is a tightly controlled mechanism throughout evolution. At the core of this process, transcription factors play a major role in transcription initiation by binding and inducing transcriptional machinery recruitment at gene regulatory sequences. To execute their function, transcription factors have to find and bind on their -10 base pair target sequence within a genome of billions of base pairs in timing consistent with biological processes. In the last 40 years, target search process has been widely studied theoretically as well as in vitro and in vivo in bacteria and yeast, but not in high eukaryotes. In the work presented in this thesis, we show the eukaryotic live cell observations of an exogenously introduced bacterial Tetracycline Repressor System binding on an artificial gene array of Tetracycline Operator target sites. The target search mechanism of the Tetracycline Repressor exhibits diffusion in the nucleus by combining a free three-dimensional local and global diffusion, non-specific binding and one-dimensional sliding on the DNA. The binding efficiency on the target was found to be orders of magnitude lower than expected, suggesting that parameters such as local protein concentration or chromatin organization have to be considered for a global understanding of gene expression regulation
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Ren, Pei-Hsien. "Infection of eukaryotic cells by fibrillar polyglutamine aggregates /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Lucas, Paul. "Cationic polypeptides for gene delivery to eukaryotic cells." Thesis, University of Bath, 1995. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307110.
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Clark, Francis. "A computational study of gene structure and splicing in model eukaryote organisms /." St. Lucia, Qld, 2003. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17395.pdf.
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Jones, Emma. "Localizing RNA polymerase subunits in human cells." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299096.
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Prudden, Giulia. "Responses to a site-specific DNA double-stranded break in Schizosaccharomyces pombe." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249323.
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Sasse, Rosemary. "Post-translational modifications of #alpha#-tubulin in Trypanosoma brucei and Physarum polycephalum." Thesis, University of Kent, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329400.
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Holstein, Eva-Maria. "Mechanisms for maintaining the telomere cap in eukaryotic cells." Thesis, University of Newcastle Upon Tyne, 2012. http://hdl.handle.net/10443/1550.
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Telomeres are found at the end of linear eukaryotic chromosome ends and contribute to genetic stability. It is crucial that telomeres are not perceived and treated as doublestrand breaks by checkpoint and DNA repair proteins in order to prevent potentially lethal consequences, such as cellular ageing and cancer formation in mammalian cells (Murnane, 2006; Shin et al., 2006; Gilley et al., 2005). Hence, eukaryotic chromosome ends are masked by various telomere-binding proteins. Two crucial telomere capping proteins in Saccharomyces cerevisiae are Cdc13 and Yku70. Cdc13 forms a complex with Stn1 and Ten1 and binds specifically to single-stranded telomeric DNA (Grandin et al., 2001). Yku70 is a subunit of the Yku complex that is found at double-stranded telomeric DNA (Tuteja and Tuteja, 2000). A temperature-sensitive allele of CDC13, known as cdc13-1, or a null-mutation of YKU70 is used in this study to induce telomere uncapping and to study its consequences in vivo. This work looks into the puzzling observation that components of the nonsensemediated mRNA decay pathway (Upf1, Upf2, Upf3) suppress one form of telomere capping defect (yku70Δ), but enhance another (cdc13-1) (Addinall et al., 2011). The same effect can be seen in Ebs1, which has previously been linked to NMD (Azzalin et al., 2007). Here it is shown that Ebs1, like components of nonsense-mediated decay (NMD), regulate transcript levels of the two telomere binding proteins Stn1 and Ten1. Interestingly, increased levels of Stn1, but not Ten1, suppress the yku70Δ capping defect, but enhance the cdc13-1 capping defect, indicating that NMD and Ebs1 influence Cdc13- and Yku70-dependent telomere capping through modification of Stn1 levels. It is also shown that increase in Stn1 levels alters stochiometry of the Cdc13/Stn1/Ten1 (CST) complex. Cdc13 association to telomeres is significantly reduced at the presence of increased levels of Stn1. Inefficient telomere protection allows resection of telomeric DNA and the generation of ssDNA tracts triggers cell cycle arrest (Booth et al., 2001; Garvik et al., 1995; Lydall and Weinert, 1995; Weinert and Hartwell, 1993). It is shown that Upf2 and Ebs1 do not affect checkpoint activation in response to cdc13-1 uncapped telomeres, but are required for efficient G1 to S phase progression. Furthermore, Upf2 contributes to ssDNA generation at cdc13-1 uncapped telomeres in a parallel pathway to the exonuclease Exo1 and the checkpoint protein Rad24. Remarkably, elimination of Upf2 and Exo1 or Upf2 and Rad24 allows viability of cells in the absence of the otherwise essential telomere capping protein Cdc13.
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Ferrell, James R. ""Effects of nonthermal plasma on prokaryotic and eukaryotic cells"." Kent State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=kent1365781078.
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Oxhamre, Camilla. "Novel signaling pathways induced by bacterial toxins in eukaryotic cells /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-495-3/.
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Rozen, Florence. "Identification and role of cap binding proteins in eukaryotic cells." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74247.
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All eukaryotic cellular mRNAs (except for organelle mRNAs) are blocked at their 5$ sp prime$ end by the cap structure m$ sp7$GpppX (where X = any nucleotide). The cap structure plays a multifunctional role in both the nucleus and the cytoplasm. Two polypeptides of molecular mass of 20 and 115 kDa in HeLa nuclear extracts were shown to recognize and be crosslinked to the cap structure of eukaryotic mRNAs in a cap-dependent fashion. Their cap binding properties have been characterized, although their function in the nucleus is unknown. Previous studies have shown that cap function in the cytoplasm is mediated by eukaryotic initiation factors (eIF)-4A, -4B and -4F (termed cap binding proteins) which interact with the cap structure and are involved in 40S ribosomal subunit binding to mRNA during translation initiation. eIF-4A contains an ATP binding consensus sequence AXXGXGKT, consistent with its ATP binding and hydrolyzing activity. The amino proximal glycine and lysine were mutated to isoleucine and asparagine, respectively, in order to determine the importance of these amino acids in ATP binding as examined by UV-induced crosslinking to ($ alpha sp{32}$P) dATP. Mutation of the lysine, but not the glycine, abolished ($ alpha sp{32}$P) dATP crosslinking to eIF-4A. There has been indirect evidence to suggest that eIF-4A, -4B and -4F interact with the cap structure and use the energy derived from ATP hydrolysis to unwind 5$ sp prime$ proximal secondary structure in mRNA, to facilitate ribosome binding. Using a novel RNA unwinding assay, direct evidence was obtained indicating that combinations of eIF-4A and eIF-4B, or eIF-4F and eIF-4B, exhibit helicase activity. A unique aspect of this activity is that it functions in a bidirectional fashion. The data indicate that cap binding proteins in the cytoplasm, and possibly the nucleus, play a key role in the regulation of gene expression.
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ARMADA, Ana Maria Buttle de Mendonça Mourão Possidónio de. "ABC efflux pumps in eukaryotic cells from function to regulation." Doctoral thesis, Instituto de Higiene e Medicina Tropical, 2019. http://hdl.handle.net/10362/66659.
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A resistência à quimioterapia é um problema grave no tratamento de doenças infeciosas e parasitárias e oncológicas. Microrganismos procariotas (bactérias), agentes patogénicos eucariotas (fungos, helmintas e protozoários) e células eucariotas, como é o caso das células tumorais, desenvolvem frequentemente resistência aos agentes quimioterápicos. A resistência a múltiplos fármacos (MDR) está relacionada com o aumento da expressão e atividade do sistema membranar de transportadores de efluxo (bombas ABC). Estas bombas são responsáveis por falhas terapêuticas tanto no cancro como nas doenças infeciosas.
Embora existam diversos moduladores de bombas ABC, estes transportadores são ubiquamente expressos nos tecidos epiteliais humanos, pelo que a aplicação clinica de inibidores de bombas de efluxo causa grave toxicidade sistémica. Permanece, portanto, a necessidade de encontrar moduladores menos tóxicos capazes de reverter o fenótipo MDR, resensibilizando as células resistentes à quimioterapia. O reconhecimento da existência de fitoquímicos capazes de modular os transportadores ABC com menor toxicidade tem gerado novas linhas de investigação.
Este estudo teve como objetivo principal avaliar a influência de bombas de efluxo ABCB1 no desenvolvimento de quimioresistência e analisar a atividade pleiotrópica de moduladores sintéticos e naturais de bombas de efluxo em diversos modelos biológicos: parasita multicelular (Schistosoma mansoni), linha macrofágica de murganho infetada por um protozoário intracelular (Leishmania infantum) e linhas celulares malignas de origem humana.
Os métodos regularmente utilizados no estudo dos transportadores ABC não permitem determinar a sua eficácia em tempo real. Neste estudo foram aplicadas metodologias baseadas no transporte celular de substratos fluorescentes capazes de avaliar em tempo real a funcionalidade das bombas de efluxo por fluorimetria e por citometria de fluxo. A microscopia de fluorescência e a técnica de PCR quantitativo em tempo real (RT-qPCR) foram também aplicadas.
Nos ensaios ex vivo foi possível demonstrar que as bombas ABCB1 de S. mansoni estavam envolvidas na resistência ao prazinquantel e que verapamil (VP), modulador sintético de bombas ABCB1, reverte a resistência. Nos macrófagos modificados pela infeção por L. infantum foi observado um expressivo incremento de bombas ABCB1 na membrana celular. Inibidores sintéticos de canais iónicos e de bombas de efluxo, como VP, ouabaína, tioridazina e cloropromazina, e o composto natural 6-gingerol (6G) reduziram a atividade de efluxo dos macrófagos não infetados. Complementarmente a atividade destes moduladores foi também relacionada com o stress oxidativo dos macrófagos. O tratamento com inibidores sintéticos de efluxo e com o 6G diminuiu a sobrevivência dos parasitas intracelulares e a sua capacidade de se multiplicar, sugerindo que estes moduladores e o VP, em particular, podem ter um efeito leishmanicida direto ou indireto. Em células de carcinoma mamário resistentes à doxorrubicina e com sobreexpressão de bombas ABCB1, o VP reverteu a resistência ao paclitaxel e o 6G mostrou ser citotóxico para as células resistentes, o que poderá estar relacionado com oincremento das espécies reativas de oxigénio, e consequentemente com o desequilíbrio homeostático. Nestas células foi possível demonstrar correlação direta entre a atividade de efluxo e a expressão das bombas ABCB1 e que os microRNAs podem modular o fenótipo resistente, interferindo na expressão génica de transportadores ABCB1.Resistance to chemotherapy is a serious problem in the treatment of infectious and parasitic and oncological diseases. Prokaryotic microorganisms (bacteria), eukaryotic pathogens (fungi, helminths and protozoa) and eukaryotic cells, such as tumor cells, often develop resistance to chemotherapeutic agents. Multiple drug resistance (MDR) is related to an increased expression and activity of the membrane efflux transporter systems (ABC pumps). These pumps are responsible for therapeutic failures in both cancer and infectious diseases. Although there are several ABC pump modulators, these transporters are ubiquitously expressed in human epithelial tissues, so the clinical application of efflux pump inhibitors causes severe systemic toxicity. Therefore, there is a need to find less toxic modulators capable of reversing the MDR phenotype and resensitize resistant cells to chemotherapy. The recognition of the existence of phytochemicals capable of modulating ABC transporters with lower toxicity has generated new lines of investigation. The objective of this study was to evaluate the influence of ABCB1 efflux pumps on the development of chemoresistance and to analyze the pleiotropic activity of synthetic and natural modulators of efflux pumps in several biological models: multicellular parasite (Schistosoma mansoni), murine macrophage cell lines infected by an intracellular protozoan (Leishmania infantum) and malignant cell lines of human origin. The methods regularly used in the study of ABC transporters are not able to determine their effectiveness in real-time. In this study, methodologies based on the cellular transport of fluorescent substrates capable of evaluating in real time the functionality of the efflux pumps by fluorometry and by flow cytometry were used. Fluorescence microscopy and the quantitative real-time PCR technique (RT-qPCR) were also applied. In the ex vivo assays it was possible to demonstrate that S. mansoni ABCB1 pumps were involved in praziquantel resistance and that verapamil (VP), synthetic pump modulator ABCB1, reverses the resistance. In macrophages modified by infection by L. infantum an expressive increase of ABCB1 pumps in the cell membrane was observed. Synthetic ion channel inhibitors and efflux pumps, such as VP, ouabain, thioridazine and chlorpromazine, and natural compound 6-gingerol (6G) reduced the efflux activity of uninfected macrophages. In addition, the activity of these modulators was also related to the oxidative stress of the macrophages. Treatment with synthetic inhibitors of efflux and with 6G decreased the survival of intracellular parasites and their ability to multiply, suggesting that these modulators and VP in particular may have a direct or indirect leishmanicidal effect. In breast carcinoma cells resistant to doxorubicin and with overexpression of ABCB1 pumps, VP reversed resistance to paclitaxel and 6G was cytotoxic to resistant cells, which may be related to the increase of reactive oxygen species, and consequently to the homeostatic imbalance. In these cells it was possible to demonstrate a direct correlation between efflux activity and ABCB1 pump expression and that microRNAs can modulate the resistant phenotype by interfering with the gene expression of ABCB1 transporters.
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Cridge, Andrew Graham, and n/a. "Characterization of the eukaryotic translation termination sequence element." University of Otago. Department of Biochemistry, 2005. http://adt.otago.ac.nz./public/adt-NZDU20060804.095722.
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Termination of protein synthesis occurs in response to the translocation of a stop codon (UAA, UAG or UGA) into the A site of the ribosome. Unlike sense codons, stop signals in the mRNA are recognized by two classes of specialized proteins called release factors (RFs): the class I or decoding RF, which recognizes the stop codon and promotes peptidyl-tRNA hydrolysis and class II RF, a G-protein that promotes the dissociation of the decoding RF from the ribosome. The discovery that stop codons are decoded by a protein factor rather than a specific tRNA opened up the possibility that the signal for termination of protein synthesis might extend beyond the stop codon itself. Biochemical and genetic experiments in prokaryotes confirmed that bias in nucleotide usage around stop codons correlates with translation termination efficiency. The objective of the current investigation was to define the eukaryotic termination signal by determining the bias in the nucleotide sequence surrounding eukaryotic stop codons and to identify whether this was a determinant of translation termination efficiency.
Bioinformatic analysis of five diverse eukaryotic genomes was undertaken to identify potential eukaryotic translation termination signal elements. Significant nucleotide bias was identified both 5� and 3� of the stop codon in all the genomes investigated. Correlations were identified between nucleotide bias and gene expression levels, and between nucleotide bias and natural recoding sites predicting that nucleotides 5� and 3� of the stop codon affect termination efficiency. These correlations were common to all organisms investigated and suggested the existence of a eukaryotic termination signal.
Termination signals identified from the bioinformatic analysis were assayed to determine the efficiency of termination in an in vitro dual luciferase reporter assay. Results indicated that nucleotides both 5� and 3� of the stop codon could significantly alter termination signal efficiency, although readthrough did not vary by greater than 1%. The effect of nucleotides 3� to the stop codon on termination efficiency was investigated further in mammalian cultured cells using the dual luciferase reporter assay. Results showed a significant relationship between the identity of these nucleotides and observed termination efficiencies with nucleotides at positions +4 and +8 giving the strongest correlation. Termination sequence elements of the form UGA CUN NCN mediated up to 5% readthrough in cultured cells.
Investigations into the underlying mechanisms that were responsible for the variation in termination efficiency were also undertaken. Co-transfection of specific suppressor tRNAs enhanced but did not change the pattern of observed termination efficiency, indicating that the mechanisms mediated by the termination signal element was not mediated through suppressor tRNA binding. Alignments of 18S rRNA sequences indicated potential extensive interactions between the rRNA and the mRNA termination signal element. Experiments that assessed the effect of eRF1 levels on termination at inefficient termination signals in vitro revealed that increased levels of eRF1 could improve termination efficiency.
These results indicate that, as in prokaryotes, specific nucleotides beyond the stop codon modulate translation termination efficiency in eukaryotes, and that the translation termination signal should be considered a sequence element.
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Bermudez, Vladimir Paredes. "Role of transcription factors in eukaryotic DNA replication /." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9924864.
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Malik, Shehre-Banoo. "The early evolution of meiotic genes." Diss., University of Iowa, 2007. http://ir.uiowa.edu/etd/275/.
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Clayton, Alison Louise. "Core histone acetylation of active genes." Thesis, University of Portsmouth, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240358.
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Metz, Anneke Maria. "Function of the wheat eukaryotic initiation factors eIF(ISO)4G and eIF4B in translation /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Tang, Terry, and University of Lethbridge Faculty of Arts and Science. "Mathematical modeling of eukaryotic gene expression." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2010, 2010. http://hdl.handle.net/10133/2567.
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Using the Gillespie algorithm, the export of the mRNA molecules from their transcription
site to the nuclear pore complex is simulated. The effect of various structures in the nu-
cleus on the efficiency of export is discussed. The results show that having some of the
space filled by chromatin near the mRNA synthesis site shortens the transport time. Next, the complete eukaryotic gene expression including transcription, splicing, mRNA export, translation, and mRNA degradation is modeled using delay stochastic simulation. This allows for the study of stochastic effects during the process and on the protein production rate patterns. Various protein production patterns can be produced by adjusting the poly-A tail length of the mRNA and the promoter efficiency of the gene. After that, the opposing effects of the chromatin density on the seeking time of the transcription factors for the promoter and the exit time of the mRNA product are discussed.xi, 102 leaves ; 28 cm
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Lea, Nicholas. "Processing and trafficking of Shiga-like toxin 1 in eukaryotic cells." Thesis, University of Warwick, 1996. http://wrap.warwick.ac.uk/79995/.
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Shiga toxin (ST) and the Escherichia coli Shiga-like toxins (SLTs) are type 11 ribosome inactivating proteins (RIPs). All members of this group exhibit specific RNA N-glycosidase activity, the prototype being the plant toxin ricin. ST and the SL Ts are bipartite toxins composed of a catalytic A subunit and a pentamer of cell binding B subunits. These toxins show overall structural similarities to ricin, which is also a bipartite toxin with a catalytic A chain and a single cell binding B chain. The A chains of ST and SL T 1 show homology to ricin A chain, particularly in the active site region, and appear identical in their enzymatic mechanism. The respective B chains however are structurally very different and interact with quite different cellular components. In this study, the role of intracellular proteolytic activation of SLT 1 is addressed using a molecular biological approach. The biological characteristics of several mutant SL Ts has been investigated both in vitro, by addition of exogenous protease, and in vivo by comparing the relative cytotoxicities of mutant and wild type proteins in Vero cells. The intracellular processing of these mutant toxins has also been examined. In parallel, the biological properties of a ricin A chain SL T 1 chimeric protein has been investigated. The ultimate aim of this study was to extend our knowledge of the proteolytic processing requirements of SL T 1 and it has led to the conclusion that proteolytic removal ofthe A2 portion of SL T 1 is not an essential prerequisite for intoxication of Vero cells with SLT 1.
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Ariza, Maria Eugenia. "Molecular mechanisms of Pb2+ and Hg2+-induced mutagenesis in eukaryotic cells /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487940665436736.
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Theodoropoulos, Christina. "Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro." Thesis, Queensland University of Technology, 2003. https://eprints.qut.edu.au/37160/6/37160_Digitised%20Thesis.pdf.
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Diarrhoea is one of the leading causes of morbidity and mortality in
populations in developing countries and is a significant health issue
throughout the world. Despite the frequency and the severity of the
diarrhoeal disease, mechanisms of pathogenesis for many of the causative
agents have been poorly characterised. Although implicated in a number of
intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides
generally has been dismissed as an enteropathogen due to the lack of clearly
demonstrated virulence-associated properties such as production of
cytotoxins and enterotoxins or invasive abilities. However, evidence from a
number of sources has indicated that this species may be the cause of a
number of clinical infections. The work described in this thesis seeks to
resolve this discrepancy by investigating the pathogenic potential of
P. shigelloides using in vitro cell models. The focus of this research centres
on how this organism interacts with human host cells in an experimental
model.
Very little is known about the pathogenic potential of P. shigel/oides and its
mechanisms in human infections and disease. However, disease
manifestations mimic those of other related microorganisms. Chapter 2
reviews microbial pathogenesis in general, with an emphasis on
understanding the mechanisms resulting from infection with bacterial
pathogens and the alterations in host cell biology. In addition, this review
analyses the pathogenic status of a poorly-defined enteropathogen,
P. shigelloides.
Key stages of pathogenicity must occur in order for a bacterial pathogen to
cause disease. Such stages include bacterial adherence to host tissue,
bacterial entry into host tissues (usually required), multiplication within host
tissues, evasion of host defence mechanisms and the causation of damage.
In this study, these key strategies in infection and disease were sought to
help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve
isolates of P. shigelloides, obtained from clinical cases of gastroenteritis,
were used to infect monolayers of human intestinal epithelial cells in vitro.
Ultrastructural analysis demonstrated that P. shigelloides was able to adhere
to the microvilli at the apical surface of the epithelial cells and also to the
plasma membranes of both apical and basal surfaces. Furthermore, it was
demonstrated that these isolates were able to enter intestinal epithelial cells.
Internalised bacteria often were confined within vacuoles surrounded by
single or multiple membranes. Observation of bacteria within membranebound
vacuoles suggests that uptake of P. shigelloides into intestinal
epithelial cells occurs via a process morphologically comparable to
phagocytosis. Bacterial cells also were observed free in the host cell
cytoplasm, indicating that P. shige/loides is able to escape from the
surrounding vacuolar membrane and exist within the cytosol of the host.
Plesiomonas shigelloides has not only been implicated in gastrointestinal
infections, but also in a range of non-intestinal infections such as
cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by
which P. shigelloides causes these infections are not understood. Previous
research was unable to ascertain the pathogenic potential of P. shigel/oides
using cells of non-intestinal origin (HEp-2 cells derived from a human larynx
carcinoma and Hela cells derived from a cervical carcinoma). However, with
the recent findings (from this study) that P. shigelloides can adhere to and
enter intestinal cells, it was hypothesised, that P. shigel/oides would be able
to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which
previously have been shown to be invasive to intestinally derived Caco-2
cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells
(Chapter 4). These isolates were shown to adhere to and enter both nonintestinal
host cell lines. Plesiomonas shigelloides were observed within
vacuoles surrounded by single and multiple membranes, as well as free in
the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells.
Comparisons of the number of bacteria adhered to and present intracellularly
within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides
for Caco-2 cells. This study conclusively showed for the first time that
P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the
potential ability to cause an infection and/or disease of extra-intestinal sites in
humans.
Further high resolution ultrastructural analysis of the mechanisms involved in
P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed
numerous prominent surface features which appeared to be involved in the
binding of P. shige/loides to host cells. These surface structures varied in
morphology from small bumps across the bacterial cell surface to much
longer filaments. Evidence that flagella might play a role in bacterial
adherence also was found. The hypothesis that filamentous appendages are
morphologically expressed when in contact with host cells also was tested.
Observations of bacteria free in the host cell cytosol suggests that
P. shigelloides is able to lyse free from the initial vacuolar compartment. The
vacuoles containing P. shigel/oides within host cells have not been
characterised and the point at which P. shigelloides escapes from the
surrounding vacuolar compartment has not been determined. A cytochemical
detection assay for acid phosphatase, an enzymatic marker for lysosomes,
was used to analyse the co-localisation of bacteria-containing vacuoles and
acid phosphatase activity (Chapter 6). Acid phosphatase activity was not
detected in these bacteria-containing vacuoles. However, the surface of
many intracellular and extracellular bacteria demonstrated high levels of acid
phosphatase activity, leading to the proposal of a new virulence factor for
P. shigelloides.
For many pathogens, the efficiency with which they adhere to and enter host
cells is dependant upon the bacterial phase of growth. Such dependency
reflects the timing of expression of particular virulence factors important for
bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an
overnight culture of P. shigelloides was used to investigate a number of
interactions, however, it was unknown whether this allowed expression of
bacterial factors to permit efficient P. shigelloides attachment and entry into
human cells. In this study (Chapter 7), a number of clinical and
environmental P. shigelloides isolates were investigated to determine
whether adherence and entry into host cells in vitro was more efficient during
exponential-phase or stationary-phase bacterial growth. An increase in the
number of adherent and intracellular bacteria was demonstrated when
bacteria were inoculated into host cell cultures in exponential phase cultures.
This was demonstrated clearly for 3 out of 4 isolates examined. In addition,
an increase in the morphological expression of filamentous appendages, a
suggested virulence factor for P. shigel/oides, was observed for bacteria in
exponential growth phase. These observations suggest that virulence
determinants for P. shigel/oides may be more efficiently expressed when
bacteria are in exponential growth phase. This study demonstrated also, for
the first time, that environmental water isolates of P. shigelloides were able to
adhere to and enter human intestinal cells in vitro. These isolates were seen
to enter Caco-2 host cells through a process comparable to the clinical
isolates examined. These findings support the hypothesis of a water
transmission route for P. shigelloides infections.
The results presented in this thesis contribute significantly to our
understanding of the pathogenic mechanisms involved in P. shigelloides
infections and disease. Several of the factors involved in P. shigelloides
pathogenesis have homologues in other pathogens of the human intestine,
namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated
strains of Escherichia coli. This study emphasises the relevance
of research into Plesiomonas as a means of furthering our understanding of
bacterial virulence in general. As well it provides tantalising clues on normal
and pathogenic host cell mechanisms.
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Sturm, Richard Alan. "Control mechanisms of higher eukaryotic gene transcription--divergent histone genes /." Title page, contents and abstract only, 1985. http://web4.library.adelaide.edu.au/theses/09PH/09phs936.pdf.
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Narayanan, Vidhya. "Inverted repeats as a source of eukaryotic genome instability." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24774.
Full textAbstract:
Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2009.Committee Chair: Lobachev, Kirill; Committee Co-Chair: Chernoff, Yury; Committee Member: Crouse, Gray; Committee Member: Goodisman, Michael; Committee Member: Streelman, Todd.
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Lai, Wai-lung. "The role of unfolded protein response in the cytotoxicity mechanism of N-(4-hydroxyphenyl)retinamide." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B39793928.
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Lai, Wai-lung, and 黎威龍. "The role of unfolded protein response in the cytotoxicity mechanism ofN-(4-hydroxyphenyl)retinamide." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B39793928.
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Hays, Shan Mitchell. "Characterization of the histone genes, the N-terminus of H4, and the methylation mutant DIM-3 of Neurospora crassa /." view abstract or download file of text, 2001. http://wwwlib.umi.com/cr/uoregon/fullcit?p3024514.
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Thesis (Ph. D.)--University of Oregon, 2001.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 161-174). Also available for download via the World Wide Web; free to University of Oregon users.
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Weinhausen, Britta [Verfasser]. "Scanning X-ray nano-diffraction on eukaryotic cells : From freeze-dried to living cells / Britta Weinhausen." Göttingen : Universitätsverlag Göttingen, 2014. http://d-nb.info/1154360733/34.
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Cao, Yuan. "Investigating the roles of translation elongation factor 1B in mammalian cells." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/8089.
Full textAbstract:
Eukaryotic protein translation elongation is tightly controlled by several regulation factors. Eukaryotic translation elongation factor 1B (eEF1B) is the GTP exchange factor for eukaryotic translation elongation factor 1A (eEF1A), which is a G-protein transporting aminoacyl-tRNA to the A site of the ribosome in a GTP dependent manner. The structure of the heavy complex composed of eEF1B and eEF1A (eEF1H) has been widely studied and several models have been proposed, but it is yet not clear how the subunits of the two proteins interact with each other. eEF1B is made up of three subunits, eEF1Bα, eEF1Bδ and eEF1Bγ, and each subunit has been found to be over expressed in different types of cancer. A copy number variant near the eEF1Bδ gene is associated with amyotrophic lateral sclerosis. The two isoforms of eEF1A, eEF1A1 and eEF1A2, are 92% identical, but only eEF1A1 was found to interact with eEF1B subunits in yeast two hybrid (Y2H) experiments. The aims of this PhD project are to investigate the potential involvement of eEF1B in disease, as well as the relationship between eEF1B and eEF1A2. All three eEF1B subunits were present in almost all the cell types and mouse tissues tested. eEF1Bδ showed different variants, the heaviest of which is tissue specific and expressed only in brain and spinal cord. eEF1Bα and eEF1Bδ showed certain abnormalities in transformed cell lines, although in the breast cancer tissues tested no apparent change in eEF1B expression was found. Knockdown of eEF1B did not significantly affect NSC34 cell viability over short periods. In spinal cord sections from motor neurone disease (MND) patients, half of the cases showed a change of eEF1B protein expression compared to normal spinal cord, with either a higher level in glial cells, or a lower level in motor neurones. eEF1B and eEF1A2 were found to be co-expressed in mouse motor neurones, and proximity ligation assay also detected physical interactions between both eEF1A isoforms and eEF1B subunits in mammalian cells, contrary to the previous Y2H study. Experiments in a mouse model with no eEF1A2 expression also support this finding. In heart and skeletal muscle from wasted mice where eEF1A is absent the expression of eEF1Bα and eEF1Bδ was down regulated at both protein and mRNA level, suggesting that eEF1A2 and eEF1B not only physically interact, but also show an interdependence in expression. Overall the results from cultured cells, mouse and human tissues in this study demonstrate the potential involvement of eEF1B in MND, and its interaction with eEF1A, which contributes to the understanding of the non-canonical functions of eEF1B and the structure of eEF1H.
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Polak, Monica E. "The development of biosensing systems incorporating eukaryotic cells for rapid toxicity assessment." Thesis, University of Bedfordshire, 1997. http://hdl.handle.net/10547/621818.
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This thesis describes the development of biosensing systems incorporating eukaryotic cells. The ultimate objective of this work was to design devices capable of rapidly assessing the toxicity of effluents and environmental pollutants. Although much work remains to be done in order to achieve this goal, the work reported here demonstrates, in principle, the approaches adopted. The first approach exploited the reducing nature of healthy biological cells. So called 'redox mediated whole cell biosensors' have been described before. In this work, an algal toxicity test of short duration was developed and sensors incorporating cultured fish cells were described for the first time. The sensitivity of biosensors incorporating the green alga Selenastrum capricornutum, to diuron and pentachlorophenol, was found to compare favourably with that from other standard ecotoxicological tests. However, although the sensitivity of biosensors incorporating immobilised BF-2 fish cells was found to compare well with that of other fish cell-based toxicity tests, it appeared that whole organism tests were much more sensitive to the compounlds tested. The second approach involved the genetic manipulation of fish cells in order to incorporate luminescent reporter genes. Although this work is less well advanced, it demonstrates that the luc reporter gene can be successfully inserted into BF -2 fish cells and that these transformed cells can produce a luminescent response when incubated with luciferin substrate. Preliminary investigations have indicated that the sensitivity of luc-transformed BF-2 cells to 4-chlorophenol is comparable to that of some standard whole organism ecotoxicological tests and although much work is still required to validate this approach, it could eventually provide a simple, sensitive and rapid route to toxicity assessment.
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Du, Plessis Michelle. "The role of carnitine in eukaryotic cells : Using yeast as a model." Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/97946.
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Thesis (MSc)--Stellenbosch University, 2015.ENGLISH ABSTRACT: Previous studies in yeast in this laboratory have found carnitine to be both protective against oxidative stress induced by hydrogen peroxide and to increase the detrimental effect of dithiothreitol. These phenotypes were found to be independent of the role of carnitine within the carnitine shuttle. A screen for suppressor mutations for these carnitine-dependent phenotypes identified, among others, Δcho2 and Δopi3. Cho2p and Opi3p catalyse the sequential methylation reactions in the formation of phosphatidylcholine from phosphatidylethanolamine. Therefore, this study aimed to investigate the relationship between choline, phosphatidylcholine and the carnitine phenotypes. Liquid growth assays of Δcho2 and Δopi3 cultures revealed that addition of choline can restore the protective effects of carnitine against hydrogen peroxide. The connection between the cellular phospholipid composition and the carnitine-dependent shuttleindependent phenotypes was also investigated. Analysis of the lipid composition of cells by LCMS showed that Δcho2 and Δopi3 had a largely different lipid composition compared with the wild type, most notably, a reduction in phosphatidylcholine and an increase in triacylglycerol content were observed for both mutants. These changes were reversed by supplementation with choline. However, no effects on the lipid composition of cells in response to carnitine treatment were observed, either when supplemented alone or in combination with DTT and hydrogen peroxide. Carnitine has also been investigated in mammalian systems for its potential to protect cells from oxidative stress, an effect which would be of benefit in various neurodegenerative disorders. Several studies have documented the positive effects of carnitine against oxidative stress in mammalian cells however the mechanism behind this action remains unknown. It is therefore thought that, provided similar effects for carnitine can be shown in mammalian cells as was observed in yeast, it would be beneficial to use yeast as a model system for the study of the molecular changes induced by carnitine. In view of this, the effects of carnitine on toxicity induced by oxidative stress in mammalian neural cells were compared to that which has been observed in yeast. For this purpose the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, a measure of reductive capacity of cells, was used. However, no effects for carnitine were observed in the MTT assay in combination with either dithiothreitol or paraquat.
AFRIKAANSE OPSOMMING: Vorige studies op gis in hierdie laboratorium het bevind dat karnitien beskermend is teenoor oksidatiewe stres wat deur waterstofperoksied geïnduseer word en ook die nadelige effek van ditiotreitol verhoog. Hierdie fenotipes is gevind om onafhanklik te wees van die rol van karnitien binne die karnitien-pendel. Die sifting vir onderdrukker-mutasies van hierdie karnitienafhanklike fenotipes het onder andere Δcho2 en Δopi3 geïdentifiseer. Cho2p en Opi3p kataliseer die opvolgende metileringsreaksies tydens die vorming van fosfatidielcholien vanaf fosfatidieletanolamien. Hierdie studie het dus gepoog om die verhouding tussen cholien, fosfatidielcholien en die karnitienfenotipes te ondersoek. Vloeistofanalises van Δcho2- en Δopi3-kulture het aangedui dat die byvoeging van cholien die beskermende effekte van karnitien teenoor waterstofperoksied kan herstel. Die verband tussen die sellulêre fosfolipiedsamestelling en die karnitienafhanklike pendel-onafhanklike fenotipes is ook ondersoek. Die analise van die lipiedsamestelling van selle deur middel van LCMS het getoon dat Δcho2 en Δopi3 ‘n grootliks verskillende samestelling het in vergelyking met die wilde tipe, en daar is veral ‘n afname in fosfatidielcholien en ‘n verhoging in triasielgliserol-inhoud vir beide mutante waargeneem. Hierdie veranderinge is omgekeer deur aanvulling met cholien. Geen effekte op die lipiedsamestelling van die selle is egter in reaksie op die karnitienbehandelings waargeneem nie, hetsy toe dit alleen aangevul is of in kombinasie met ditiotreitol en waterstofperoksied. Karnitien is ook in soogdierstelsels ondersoek vir sy potensiaal om selle teen oksidatiewe stres te beskerm, ‘n effek wat groot voordeel sal inhou vir verskeie neurodegeneratiewe steurings. Verskeie studies het reeds die positiewe effekte van karnitien teen oksidatiewe stres in soogdierselle opgeteken, hoewel die meganisme agter hierdie werking nog onbekend is. Daar word dus vermoed dat, gegewe dat soortgelyke effekte vir karnitien in soogdierselle getoon kan word as wat in gis waargeneem is, dit voordelig sou wees om gis as ‘n modelsisteem vir die studie van die molekulêre veranderinge wat deur karnitien geïnduseer word, te gebruik. In die lig hiervan is die effekte van karnitien op giftigheid wat deur oksidatiewe stres in soogdiersenuselle geïnduseer is, vergelyk met dít wat in gis waargeneem is. Om hierdie rede is die 3-[4,5-dimetieltiasool-2-iel]-2,5-difeniel tetrasoliumbromied (MTT) essaiëring, ‘n meting van die verminderende kapasiteit van selle, gebruik. Geen effekte vir karnitien is egter met die MTT essaiëring in kombinasie met óf ditiotreitol óf parakwat waargeneem nie.
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Murphey, Roberta Jean. "Synthesis of deoxyhypusine in eukaryotic initiation factor 4D in rat hepatoma cells." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184691.
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The aim of this research was to study the mechanism involved in the synthesis of deoxyhypusine, the intermediate step in the synthesis of the amino acid hypusine. Oeoxyhypusine is derived from the butylamine moiety of a spermidine molecule which is added to the famino group of one lysine in the eukaryotic initiation factor 40 (eIF-4D). Initially, a hepatoma tissue cell (HTC) lysate with a pH of 9.5 in glycine buffer and with a depleted spermidine pool supported deoxyhypusine synthesis in protein. Since CHES buffer was as efficient as glycine buffer, the synthesis of deoxyhypusine was pH dependent (optimum ∼9.2) and not buffer dependent. Next, several inhibitors were used in the cell-free system to block deoxyhypusine synthesis. Only guazatine, a plant amine oxidase inhibitor, completely inhibited deoxyhypusine synthesis. This suggested that an oxidase was involved in deoxyhypusine synthesis. In addition factors were investigated as possible allosteric stimulators of deoxyhypusine formation. NAD⁺, NADH, FAD⁺, FMN⁺, and as nicotinamide were tested for effects on deoxyhypusine formation. NAD⁺ was the most efficient stimulator, but NAOH and nicotinamide also stimulated deoxyhypusine formation. Although these factors increased the synthesis of deoxyhypusine, these assays were done in buffer with low concentrations of spermidine. When the spermidine pool was replenished, these effects were diminished. Thus, it appeared that NAD⁺ may lower the apparent K(m) for spermidine without affecting the V(max) of deoxyhypusine synthesis. The inhibition of deoxyhypusine synthesis by guazatine implied the involvement of a polyamine oxidase. Therefore, the effect of oxygen depletion on deoxyhypusine formation was investigated. The depletion of oxygen reduced the level of deoxyhypusine synthesis to 12% of the control. This activity could be restored to 85% by reoxygenation of the lysate. Thus in support of the suggestion made by the guazatine data, a spermidine oxidase in involved in deoxyhypusine formation. The most significant contribution of this work was the development of a cell free system to study deoxyhypusine. This synthesis required an unusually high pH in vitro and required polyamine depletion (Chapter 2). In addition, synthesis requires a unique spermidine oxidase that is blocked by a guazatine and is conditionally stimulated by NAD⁺ (Chapter 3).
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Zheng, Qun. "Analysis of the Caenorhabditis elegans rpc-1 gene." Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/4129.
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Thesis (Ph.D.)--University of Missouri-Columbia, 2005.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 25, 2007) Vita. Includes bibliographical references.
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Goff, Randal D. "Structure-activity studies of glycosphingolipids as antigens of natural killer T cells /." Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1519.pdf.
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Lange, Anja. "Structural characterization of the interaction of the Stam2's ubiquitin binding domains with ubiquitin chains by NMR : Cooperativity or not, that is the question !" Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10308.
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Résumé en anglais uniquementFrom the discovery of ubiquitin and its function as signal for proteasomal degradation over 20 years ago to this days, it became evident that ubiquitin is a universal signal in eukaryotic cells. Ubiquitin in its different forms is involved in many versatile cellular processes. Knowing that the ubiquitin signal is differently translated, depending on its occurrences as mono-ubiquitin or poly-ubiquitin, raises the question: how do cells distinguish between the different occurrences of ubiquitin and translate it into the proper response? Proteins interacting with ubiquitin contain so called ubiquitin binding domains (UBDs), whereas the affinities to ubiquitin vary from a few _M to mM. So far only three (K63, K48 and linear chains) out of the eight possible chain-linkages can be produced in sufficient amounts to characterize their interaction with UBDs. K48- and K63- linked ubiquitin chains regulate different cellular events and need to be recognized by different proteins. Thus, it is of prime importance to characterize the binding of different UBDs to these two kinds of ubiquitin chains, as it can give important clues related to the general mechanism of chain discrimination by ubiquitin adapter proteins. Some isolated UBDs exhibit a preference for one chain linkage type over the other, whereas others do not discriminate between mono-ubiquitin or K63- and K48-linked chains. Interestingly, many ubiquitin adapter proteins harbor more than one UBD. STAM2 is a ubiquitin adapter protein, that is involved in endosomal receptor sorting and supposed to preferentially bind mono-ubiquitin and K63- over K48-linked ubiquitin. STAM2 contains two UBDs (a VHS and UIM domain) that were shown to bind to ubiquitin . The current manuscript shows that STAM2’s SH3 domain binds ubiquitin as well. To understand the function of the sequential arrangement of three UBDs in one protein, first binding of the individual VHS and UIM domains to monoubiquitin as well as K48- and K63-linked di-ubiquitin was investigated. This work shows, that the VHS domain displays a different mode of binding for K63- and K48-linked diubiquitin. In spite of the fact, that the apparent Kd for both chains is the same, only one VHS domain can bind to K48-linked di-ubiquitin chains (with a preference for the distal domain), whereas K63-linked di-ubiquitin can accommodate two VHS domains at a time. Since no conclusion can be drawn with respect to the apparent Kds, the different binding modes might gain more impact in consideration of the ensemble of three UBDs. Results presented in this manuscript, based on a construct containing the VHS and UIM domain, show that binding to K63- but not K48-linked di-ubiquitin is cooperative
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Linka, Marc. "Understanding the origin and function of organellar metabolite transport proteins in photosynthetic eukaryotes Galdieria sulphuraria and Arabidopsis thaliana as model systems /." Diss., Connect to online resource - MSU authorized users, 2008.
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Ter-Hovhannisyan, Vardges. "Unsupervised and semi-supervised training methods for eukaryotic gene prediction." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26645.
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Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009.Committee Chair: Mark Borodovky; Committee Member: Jung H. Choi; Committee Member: King Jordan; Committee Member: Leonid Bunimovich; Committee Member: Yury Chernoff. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Stephens, Matthew Jon Craig. "DNA aptamers that selectively label eukaryotic cells depending on the expression of the cell surface protein, P2X7." Thesis, University of Portsmouth, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618284.
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The ATP gated cation channel P2X7, is a cell surface protein whose function and activation is linked with several human diseases. The aim of this project was to raise DNA Aptamers targeting specifically the extracellular domain of the P2X7 protein with the hope to use these new ligands as a method for the study of the biological functions of the P2X7 protein and its potential as a therapeutic target. The strategy involved isolating Aptamers that would bind to human em~ryonic kidney cells transformed with a construct which forced the cells to express native mouse P2X7 on its cell surface (POSITIVE SELECTION), whilst not binding to untransformed non-P2X7 expressing cells (NEGATIVE SELECTION). Two candidate sequences from this selection emerged as, what we called, "winners" which were the most likely sequences to putatively bind mP2X7 (work ongoing). Interestingly using the procedure in reverse, that is to create Aptamers that specifically bind to non-transformed cells and not transformed cells, also produced viable putative Aptamers, which, once labelled, showed selective binding. These results are discussed in relation to future applications and the potential insights and implications for the study of cell surface protein expression patterns. 3
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Amasino, Audra Leigh. "Keep the ORCs at bay : how eukaryotic cells ensure one round of DNA replication per cell cycle." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/128988.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2020Cataloged from student-submitted PDF of thesis.
Includes bibliographical references.
During each cell cycle, eukaryotic cells must faithfully replicate their genome, ensuring exactly one full copy is made. Both under-replicating or over-replicating the genome can have deleterious consequences including cell death, genome instability and cancer. Thus, this process is tightly regulated. The major mechanism to ensure that DNA is replicated once per cell cycle entails the temporal separation of two key replication events: helicase loading and helicase activation. Helicase loading occurs during the G1 phase of the cell cycle. In S. cerevisiae cells, Cyclin-Dependent Kinases (CDKs) prevent helicase loading outside of G1 by phosphorylating three of the four helicase-loading proteins: Mcm2-7, Cdc6, and the Origin Recognition Complex (ORC). Phosphorylation of free Mcm2-7 and Cdc6 leads to their removal from the nucleus (Mcm2-7 by nuclear export and Cdc6 by protein degradation). However, phosphorylated ORC remains in the nucleus bound to origins.
ORC phosphorylation intrinsically inhibits the helicase loading reaction. In in vitro reconstituted helicase loading reactions, CDK phosphorylation of ORC is sufficient to completely inhibit helicase loading. However, the precise event(s) during helicase loading that are affected by ORC phosphorylation were not known prior to this study. To identify the steps of helicase loading that are inhibited by ORC phosphorylation, we used single-molecule microscopy to compare the progression of helicase loading with phosphorylated versus unphosphorylated ORC. Successful helicase loading results in two head-to-head Mcm2-7 helicases encircling DNA. We show that ORC phosphorylation prevents loading of both the first and second Mcm2-7 complexes. An initial intermediate in helicase loading containing origin DNA and all four proteins (the OCCM) still forms when ORC is phosphorylated, albeit slower.
Focusing on events after OCCM formation, we found that ORC phosphorylation alters Cdt1 dissociation kinetics and inhibits successful Mcm2-7 ring closing. ORC is phosphorylated on both the Orc2 and Orc6 subunits in vivo; we find that in vitro phosphorylation of either single subunit leads to nearly identical effects as phosphorylation of both subunits. My studies suggest a model in which ORC directly controls Mcm2-7 ring closing through physical interactions with both Cdt1 and Mcm2-7 and these interactions, and thus ring closing, are inhibited by ORC phosphorylation.
by Audra Leigh Amasino.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Hoch, Duane A. "Thermodynamic studies of the Escherichia coli factor for inversion stimulation and the eukaryotic nucleosome core particle." Online access for everyone, 2006. http://www.dissertations.wsu.edu/Dissertations/Fall2006/D_Hoch_112006.pdf.
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Chen, Lu-hua. "Evaluation of eIF-2 alpha phosphorylation in patients with Alzheimer's disease /." View the Table of Contents & Abstract, 2007. http://sunzi.lib.hku.hk/hkuto/record/B38284273.
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Sweet, Deborah Jane. "The SEC20-TIP1 complex." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307092.
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Ruud, Kelley Astrid. "Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana and of the wheat eukaryotic initiation factor eIF4G /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
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Tam, Chun-yee. "A novel role of the E3 ubiquitin ligase as a transcription regulation in eukaryotic cell nucleus." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43278528.
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Gustafsson, Oskar. "Novel and efficient delivery of CRISPR/CAS9 for genome engineering in eukaryotic cells." Thesis, KTH, Skolan för bioteknologi (BIO), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-215307.
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Abdulkarim, Baroj. "The eukaryotic translation initiation factor 2, a hero turned villain in β cells." Doctoral thesis, Universite Libre de Bruxelles, 2017. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/251713.
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The prevalence of type 2 diabetes is increasing dramatically worldwide. Type 2 diabetes is a major health and socio-economic burden. Genetic predisposition and the obesity epidemic, due to sedentary life style and high caloric food intake, are associated with development of type 2 diabetes. Circulating free fatty acids (FFAs), in particular saturated FFAs, are linked with insulin resistance and β cell dysfunction. Following this background we performed RNA sequencing of human pancreatic islets treated with the saturated FFA palmitate to acquire a global image of the islet response to this insult. We identified several stress pathways induced by palmitate with a major induction of the endoplasmic reticulum (ER) stress response. The ER stress response, in particular the PKR-like ER kinase (PERK) branch, has been shown to be induced by saturated FFA. It leads to increased β cell apoptosis both in fluorescence activated cell sorter (FACS) purified rat β cells and human islets. We further clarified the role of this pathway by studying the involvement of the constitutive repressor of eIF2α phosphorylation (CReP) in a monogenic form of diabetes. CReP is a repressor of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation. A direct target of PERK, eIF2α is involved in translational attenuation and induction of apoptosis. We have shown that CReP loss-of-function leads to a new syndrome of young onset diabetes, intellectual disability and microcephaly. The identified R658C mutation abrogated CReP activity leading to increased eIF2α phosphorylation and β cell apoptosis. To further demonstrate the importance of eIF2α dysregulation in β cell demise, we used guanabenz, a chemical inhibitor of growth arrest DNA damage inducible 34 (GADD34). GADD34 is an ER stress-induced repressor of eIF2α phosphorylation. Guanabenz potentiated FFA-mediated ER stress and apoptosis in clonal and primary rat β cells and in human islets through the activation of CCAAT/enhancer binding protein homologous protein (CHOP), downstream of eIF2α. Guanabenz administration in mice impaired glucose tolerance and led to β cell dysfunction. In ex vivo experiments guanabenz also induced β cell dysfunction in mouse and rat islets.In conclusion our data demonstrate that the dysregulation of signaling in the PERK/eIF2α pathway is crucial for β cell demise. Together with previously reported monogenic diabetes caused by loss-of-function mutations in PERK in man and the eIF2αS51A mutation in mice, our findings suggest that a narrow regulation of PERK/eIF2α signaling is central for proper β cell function and survival.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished