Influenza H5N1 poses a significant threat to global health in both agricultural world and general populations due to the highly virulent nature of this virus. The potential of mutations or recombination’s of this virus with other Influenza strains could lead to the generation of pandemic strain of the virus capable of bridging the infectivity divide between avian species to human. Currently, for all Influenza infections, vaccination remains the primary strategy for prevention and control. Many vaccine strategies have been developed in an attempt to combat the threat from avian flu. Codon optimization has been used to improve vaccine efficacy for many vaccines, which rely on in vivo expression of a protein antigen. In this study, two types of vaccine platforms were used to evaluate the differential effect of codon optimization of the hemagglutinin (HA) gene of the highly pathogenic avian influenza H5N1 virus, A/Indonesia/5/05 (Ind05) on the level of protection and immune response provided against lethal challenge. Taking advantage of the degenerate nature of codon usage, a codon optimized gene was synthesized to enhance the use of codons represented by the most abundant tRNAs. The codon optimized HA gene produces a protein, which remains identical to the wild type protein regardless of codon changes but is theoretically expressed at higher level based on codon usage. The synthesized genes were cloned into a DNA plasmid based expression vector as well as a replication competent VSV viral vector. In vitro expression studies, using both a HA-expression plasmid and a recombinant VSV HA-virus, compared expression between optimized- and wild type-HA constructs. Vaccination with both optimized HA and wild type HA DNA vaccine platforms and recombinant VSV HA viruses, followed by lethal challenge with Ind05, was then used to determine the relative efficacy of each vaccine and subsequent immune response in a mouse model.

Influenza H5N1 poses a significant threat to global health in both agricultural world and general populations due to the highly virulent nature of this virus. The potential of mutations or recombination’s of this virus with other Influenza strains could lead to the generation of pandemic strain of the virus capable of bridging the infectivity divide between avian species to human. Currently, for all Influenza infections, vaccination remains the primary strategy for prevention and control. Many vaccine strategies have been developed in an attempt to combat the threat from avian flu. Codon optimization has been used to improve vaccine efficacy for many vaccines, which rely on in vivo expression of a protein antigen. In this study, two types of vaccine platforms were used to evaluate the differential effect of codon optimization of the hemagglutinin (HA) gene of the highly pathogenic avian influenza H5N1 virus, A/Indonesia/5/05 (Ind05) on the level of protection and immune response provided against lethal challenge. Taking advantage of the degenerate nature of codon usage, a codon optimized gene was synthesized to enhance the use of codons represented by the most abundant tRNAs. The codon optimized HA gene produces a protein, which remains identical to the wild type protein regardless of codon changes but is theoretically expressed at higher level based on codon usage. The synthesized genes were cloned into a DNA plasmid based expression vector as well as a replication competent VSV viral vector. In vitro expression studies, using both a HA-expression plasmid and a recombinant VSV HA-virus, compared expression between optimized- and wild type-HA constructs. Vaccination with both optimized HA and wild type HA DNA vaccine platforms and recombinant VSV HA viruses, followed by lethal challenge with Ind05, was then used to determine the relative efficacy of each vaccine and subsequent immune response in a mouse model.

No description available.

No description available.

Die Enzymsuperfamilie der löslichen Sulfotransferasen (SULT) spielt eine wichtige Rolle in der Phase II des Fremdstoffmetabolismus. Sie katalysieren den Transfer einer Sulfonylgruppe auf nucleophile Gruppen endogener und exogener Substrate. Die Sulfokonjugation von Fremdstoffen erhöht deren Wasserlöslichkeit und behindert die passive Permeation von Zellmembranen. Dadurch wird die Ausscheidung dieser konjugierten Substanzen erleichtert. In Abhängigkeit von der Struktur des Zielmoleküls kann die Sulfokonjugation aber auch zur metabolischen Aktivierung von Fremdstoffen durch die Bildung instabiler Metabolite führen. Die SULT-vermittelte Aktivierung promutagener Substanzen ist somit von toxikologischem Interesse. Für die Detektion SULT-vermittelter Mutagenität mittels bakterieller in-vitro Testsysteme ist die heterologe Expression der fremdstoffmetabolisierenden Enzyme direkt in den Indikatorzellen notwendig. S. typhimurium exprimieren selbst keine SULT, und externe Metabolisierungssysteme sind problematisch, weil die negativ geladenen, kurzlebigen Metabolite nur schlecht die Zellmembran penetrieren können. Die Expression humaner Enyme in Bakterien ist jedoch zum Teil sehr kritisch. So zeigen z.B. sehr ähnliche Enzyme (SULT1A2*1 und *2) deutliche Unterschiede im Expressionsniveau bei exakt gleichen äußeren Bedingungen. Dies erschwert den Vergleich der enzymatischen Aktivitäten dieser Enzyme im in-vitro Testsystem. Andere Enzyme (z.B. SULT2B1b) werden unter Verwendung ihrer Wildtyp-cDNA zum Teil nicht detektierbar exprimiert. Deshalb sollte in dieser Arbeit eine Methode zur Optimierung der heterologen Expression fremdstoffmetabolisierender Enzyme für Genotoxizitätsuntersuchungen etabliert werden. <br><br> Es wurde bereits gezeigt dass synonyme Codonaustausche am 5’-Ende der humanen SULT1A2-cDNA zu einer Erhöhung der Expression des entsprechenden Enzyms in S. typhimurium führten. Dementsprechend wurden in dieser Arbeit Codonaustausche am 5’-Ende der cDNA verschiedener SULT (1A1*1, 1A2*1, 2B1b) sowie der Ratten Glutathion-S-Transferase Theta 2 (rGSTT2) und dem Reportergen Luciferase durchgeführt. Die Expression der so generierten Konstrukte wurde in verschiedenen S. typhimurium und E. coli Stämmen quantifiziert und die Aktivität der überexprimierten Enzyme im Ames-Test bzw. im Enzym-Aktivitätsassay überprüft. Durch das Einführen seltener Codons in die cDNA konnte die Proteinexpression von SULT1A1*1, SULT1A2*1 und SULT2B1b maximal 7-fach, 18-fach und 100-fach im Vergleich zur Wildtyp-cDNA gesteigert werden. Die Expression der rGSTT2 wurde ebenfalls durch das Einführen seltener Codons erhöht (maximal 5-fach). Bei dem Reportergen Luciferase jedoch führte das Austauschen häufiger Codons gegen seltene Codons zu einer Reduktion der Proteinexpression um 80 %. Die Expression von Fusionsproteinen aus 2B1b (5’-Ende) und Luciferase (3’-Ende) wurde durch das Einführen seltener Codons ebenfalls um 50 % reduziert. Die S. typhimurium Stämme mit optimierter SULT 1A1*1- bzw. 1A2*1-Expression wurden im Ames-Test eingesetzt und zeigten im Vergleich zu den geringer exprimierenden Stämmen eine höhere Sensitivität. Für SULT2B1b konnte keine Mutagenaktivierung im Ames-Test nachgewiesen werden. Allerdings zeigte ein Enzym-Aktivitätsassay mit Dehydroepiandosteron, dass das bakteriell exprimierte Enzym funktionell war. Da in der Literatur der Effekt seltener Codons auf die Expression in Bakterien bisher fast ausschließlich als inhibitorisch beschrieben wurde, sollte die Wirkungsweise der hier beobachteten Expressionserhöhung durch seltene Codons genauer untersucht werden. Dazu wurden verschiedene Konstrukte der SULT1A2*1 und der SULT2B1b, die unterschiedlich viele seltene Codons in verschiedenen Kombinationen besaßen, hergestellt. Es konnten jedoch keine einzelnen Codons, die für die Expressionssteigerung allein verantwortlich waren, identifiziert werden. Die Plasmidkopienzahl in den verschiedenen SULT2B1b-Klonen war konstant und die SULT2B1b-mRNA-Konzentration zeigte nur moderate Schwankungen, die nicht als Ursache für die dramatische Erhöhung der SULT2B1b-Expression in Frage kommen. Die berechnete Stabilität der potentiellen mRNA-Sekundärstrukturen wurde durch die seltenen Codons häufig stark gesenkt und ist als eine mögliche Ursache für die Expressionssteigerung anzusehen. Zusätzlich erhöhten die seltenen Codons den Consensus der Downstream Box zur 16S rRNA, was ebenfalls eine Ursache für die Expressionssteigerung sein kann. <br><br> In dieser Arbeit konnte somit die Expression der humanen SULT1A1*1, 1A2*1 und der 2B1b sowie der rGSTT2 erfolgreich mittels synonymer Codonaustausche erhöht werden. Die so optimierten S. typhimurium Stämme zeigten im Ames-Test eine erhöhte Sensitivität gegenüber SULT aktivierten Promutagenen bzw. erhöhte Aktivität in spezifischen Enymaktivitätsassays. / The enzyme super familiy of human sulfotransferases (SULT) plays an important role in phase II metabolism of xenobiotics. They catalyze the transfer of a sulfonyl moiety to nucleophilic groups of endogenous and exogenous substrates. Sulfoconjugation of xenobiotics facilitates their excretion by increasing the water solubility and inhibiting passive permeation of cell membranes. Depending on the molecular structure of the substance, sulfonation can also lead to metabolic activation. Highly reactive resonance-stabilized carbenium- and nitrenium-ions that are able to covalently bind to cellular nucleophiles, e.g. DNA, can be formed. Thus, SULT-mediated activation of promutagenic compounds is of toxicological interest. The detection of SULT-mediated mutagenicity in bacterial in-vitro testsystems (e.g. S. typhimurium) requires the heterologous expression of xenobiotic-metabolizing enzymes directly in these indicator cells. S. typhimurium do not express endogenous SULT and external metabolic systems are problematic as penetration of cell membranes is hampered for charged and short-lived metabolites. But the expression of human enzymes in bacteria can be problematic too. SULT1A2*1 and *2 for instance are allelic variants that differ only in two amino acids. However, using the same experimental conditions strong differences in their expression level have been observed. This complicates the comparison of the mutagenic activities of the polymorphic enzymes in the in-vitro test system. Other enzymes (e.g. SULT2B1b) show no detectable expression in bacteria when their genuine cDNA obtained from human tissues is used. Therefore, the aim of this study was to to optimize protein levels of heterologously expressed xenobiotic-metabolizing enzymes in indicator cells for mutagenicity testing. <br><br> So far it has been shown that synonymous codon-exchanges at the 5’end of human SULT1A2-cDNA led to an enhanced expression of the corresponding enzyme in S. typhimurium. Accordingly, codon-exchanges at the 5’end of SULT1A1*1, -1A2*1, -2B1b, rat glutathione-S-transferase theta 2 (rGSTT2) and the reportergene luciferase were conducted. The expression of the resulting constructs was quantified in S. typhimurium and E. coli using specific antibodies and activity of the overexpressed enzymes was proved by Ames test and enzyme activity assays. The introduction of low-usage codons at the 5’end of SULT1A1*1, -1A2*1 and -2B1b cDNA led to a 7-, 18- and 100-fold increase of expression level, respectively. Expression of rGSTT2 was 5-fold enhanced after the introduction of low-usage codons. In contrast, the introduction of low-usage codons into the luciferase cDNA resulted in a decrease of protein expression up to 80 %. Fusionproteins of SULT2B1b (5’end) and luciferase (3’end) showed a reduction of protein expression about 50 % after the introduction of low-usage codons. S. typhimurium strains with optimized SULT1A1*1 and -1A2*1 expression were used in the Ames test and showed a higher sensitivity compared to the lower expressing strains. For SULT2B1b no mutagen-activation could be detected in the Ames test, but enzyme activity was proved through Dehydroepiandosterone sulfation in vitro. <br><br> Since an inhibitory effect of low-usage codons on expression in bacteria was described in literature, the enhancement of expression after the introduction of low-usage codons observed in this study was analyzed more in detail. Various constructs of SULT1A2*1 and -2B1b cDNAs containing different numbers and combinations of synonymous low-usage codons were generated. No single codon that was responsible for the enhanced expression could be identified. Plasmid copy number of different SULT2B1b constructs was unchanged and SULT2B1b-mRNA showed only moderate variations that could not explain the strong enhancement of SULT2B1b expression. Calculations suggested that the stability of potential mRNA secondary structures was reduced due to the introduction of low-usage codons. Moreover, the consensus of the downstream box and the 16S rRNA was increased. Both effects probably improved the efficiency of translation and thereby increased the yield of protein expression. <br><br> In this study the heterologous expression of SULT1A1*1, -1A2*1, -2B1b and rGSTT2 could be enhanced by the introduction of synonymous low-usage codons. The optimized S. typhimurium strains showed higher activities in enzyme assays with specific substrates and an increased sensitivity towards SULT-activated promutagens.

Although codon usage bias has been shown to persist through non-random mutations and selection, many avenues of research into the applications of codon usage bias have remained unexplored. In this dissertation, we present several new applications of codon usage bias and their practical uses in a phylogenetic construct. We first review the literature and provide background into other software applications of codon usage bias in Chapter 1. In Chapter 2, we show that in tetrapods, codon aversion in orthologs is phylogenetically conserved. We further this analysis in Chapter 3 by exploring codon use and aversion across the Tree of Life, providing frameworks for other researchers to analyze different species subsets. We present a novel algorithm to recover species relationships using codon aversion, without regard to orthologous relationships in Chapter 4. We present several other algorithms in Chapter 5 to also recover species relationships using biases in codon pairing. Chapter 6 analyzes the relationship between codon usage bias in viruses that infect humans and proteins found in tissues that they infect. In Chapter 7, we present our discovery of a conservation in coding sequence lengths in orthologous genes that allowed us to accurately recover orthologous gene relationships and reduce overall ortholog identification runtime by over 96%. In Chapter 8 we discuss a novel algorithm for extracting a ramp of slowly-translated codons located at the beginning of gene sequences, allowing researchers to quickly identify translational bottlenecks. Finally, Chapter 9 touches on future applications of codon usage bias in phylogenetics. This dissertation represents a major vertical leap in phylogenetics by providing a framework and paradigm shift toward utilizing codon usage and coding sequence length biases in future analyses.

No description available.

Messenger RNA transcripts of αs2- and к-casein are translated at 25% of the efficiency of αs1- and β-casein transcripts; however, the molecular mechanisms governing the difference are unknown. We hypothesized that the bovine casein translational efficiency is influenced by characteristics of the untranslated regions (UTRs) and coding regions. The main objective of this study was to identify molecular mechanisms that explain differential translational regulation between bovine β- and αs2-casein by assessing the role of each putative translational regulatory factor found throughout full-length sequences in both in cellular and cell-free translation systems. This dissertation begins with the cloning and initial characterization of bovine β- and αs2-casein. Transcript analysis indicates that the two genes share similar characteristics of nucleotide sequence around the coding region and secondary structure. It is confirmed that αs2-casein mRNA has a lower translational efficiency compared to that of β-casein in a cell-free system. The latter portion of this thesis investigates further the UTRs and codon usage effect on difference in translational efficiency between β- and αs2-casein. Overall, our data suggest that β-casein 3’ UTR and αs2-casein 5’ UTR exert stimulatory effects on translation yet their effectiveness depends on the upstream and downstream sequences with which they are associated. Replacement of the UTRs of αs2-casein mRNA with those of β-casein did not stimulate translation. A stronger effect on translational efficiency was found in the coding region of αs2-casein which displays unfavourable codons at the 3’ terminus. Deletion of a 28-codon fragment from the 3’ terminus of the αs2-casein coding region increased translation to a par with β-casein. We suggest that the last 28 codons of αs2-casein is the main regulatory sequence that attenuates its expression and is responsible for the different translational expression of β- and αs2-casein mRNAs. Identification of regulatory factors that are responsible for translation efficiency improves our understanding of the molecular mechanisms of control of milk protein prodiction in secretory cells of the bovine mammary glands. / NSERC canada

<p> Three pieces of work are contained in this thesis. OGRe is a relational database that stores mitochondrial genomes of animals. The database has been operational for approximately five years and the number of genomes in the database has expanded to over 1000 in this period. However, sometimes, new genomes can not be added to the database because of small errors in the source ffies. Several improvements to the update method and the organizational structure of OGRe have been done, which are presented in the first part of this thesis. </p> <p> The second part of this thesis is a study on codon usage in mitochondrial genomes of mammals and fish. Codon usage bias can be caused by mutation and translational selection. In this study, we use some statistical tests and likelihood-based tests to determine which factors are most important in causing codon bias in mitochondrial genomes of mammals and fish. It is found that codon usage patterns seem to be determined principally by complex context-dependent mutational effects. </p> <p> The third part of this thesis is a phylogenetic study of 159 avian species obtained using mitochondrial rRNA sequences that were provided by Dr. van Tuinen. In this study, two methods are used: one considers sites of sequences as independently evolving; the other includes the secondary structure of rRNAs. Unfortunately, the amount of information in the rRNA sequences seems to be insufficient to determine the whole phylogeny of birds. However, our results make it clear that several traditionally defined orders are polyphyletic and therefore need to be redefined. </p> / Thesis / Master of Science (MSc)

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22356号 / 医博第4597号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 岩田 想, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM