Investigation of I -compounds (newly discovered indigenous DNA modifications) by different approaches

I-compounds are newly discovered covalent DNA modifications detected by the $\sp{32}$P-postlabeling assay. They are age-dependent, tissue-specific and sex-different. The origin(s), chemistry and function(s) of I-compounds are unknown. The total level of I-compounds in 8-10 month old rat liver is 1 adduct in 10$\sp7$ nucleotides, which is not neglectable. It is proposed that I-compounds may play a role in spontaneous tumorigenesis and aging.^ In the present project, I-compounds were investigated by several different approaches. (1) Dietary modulation of I-compounds. (2) Comparison of I-compounds with persistent carcinogen DNA adducts and 5-methylcytosine. (3) Strain differences of I-compounds in relation to organ site spontaneous tumorigenesis. (4) Effects of nongenotoxic hepatocarcinogenes on I-compounds.^ It was demonstrated that the formation of I-compounds is diet-related. Rats fed natural ingredient diet exhibited more complex I-spot patterns and much higher levels than rats fed purified diet. Variation of major nutrients (carbohydrate, protein and fat) in the diet, produced quantitative differences in I-compounds of rat liver and kidney DNAs. Physiological level of vitamin E in the diet reduced intensity of one I-spot compared with vitamin E deficient diet. However, extremely high level of vitamin E in the diet gave extra spot and enhanced the intensities of some I-spots.^ In regenerating rat liver, I-compounds levels were reduced, as carcinogen DNA adducts, but not 5-methylcytosine, i.e. a normal DNA modification.^ Animals with higher incidences of spontaneous tumor or degenerative diseases tended to have a lower level of I-compounds.^ Choline devoid diet induced a drastic reduction of I-compound level in rat liver compared with choline supplemented diet. I-compound levels were reduced after multi-doses of carbon tetrachloride (CCl$\sb4$) exposure in rats and single dose exposure in mice. An inverse relationship was observed between I-compound level and DNA replication rate. CCl$\sb4$-related DNA adduct was detected in mice liver and intensities of some I-spots were enhanced 24 h after a single dose exposure.^ The mechanisms and explanations of these observations will be discussed. I-compounds are potentially useful indicators in carcinogenesis studies. ^

Control of {\it recA\/} expression in {\it Escherichia coli}

The recA gene is essential for SOS response induction, for inducible DNA repair and for homologous recombination in E. coli. The level of recA expression is significant for these functions. A basal level of about 1000 molecules of RecA protein is sufficient for homologous recombination of the cell and is essential for the induction of the SOS response. Based on previous observations, two models regarding the origin of the basal RecA protein were postulated. One was that it comes from the leaky expression of the LexA repressed promoter. The other was that it is from another weak but constitutive promoter. The first part of this thesis is to study these possibilities. An $\Omega$ cartridge containing the transcription terminator of gene 32 of T4 phage was exploited to define a second promoter for recA expression. Insertion of this $\Omega$ cartridge downstream of the known promoter gave rise to only minor expression. Purification and N-terminus sequencing of the RecA protein from the insertion mutant did not support the existence of a second promoter. To determine whether the basal RecA is due to the leaky expression of the known LexA repressed promoter, recA expression of a SOS induction minus strain (basal level expression of recA) was compared with that of a recA promoter down mutation recA1270. The result demonstrated that there is leaky expression from the LexA repressed promoter. All the evidence supports the conclusion that there is only one promoter for both basal and induced expression levels of recA.^ Several translation enhancer sequences which are complementary to different regions of the 16S rRNA were found to exist in recA mRNA. The leader sequence of recA mRNA is highly complementary to a region of the 16S rRNA. Thus it appeared that recA expression could be regulated at post-transcriptional levels. The second part of this thesis is focused on the study of the post-transcriptional control of recA expression. Deletions of the complementary regions were created to examine their effect on recA expression. The results indicated that all of the complementary regions were important for the normal expression of recA and their effects were post-transcriptional. RNA secondary structures of wild type recA mRNA was inspected and a stem-loop structure was revealed. The expression down mutations at codon 10 and 11 were found to stabilize this structure. The conclusions of the second part of this thesis are that there is post-transcriptional control for recA expression and the leader sequence of recA mRNA plays more than one role in the control of recA expression. ^

ATP- and carbachol -stimulated 1,2-diacylglycerol production during sensitization of adenylyl cyclase

Stimulation of LM5 cells with the phorbol ester 4$\beta$-phorbol 12-myristate 13-acetate (PMA), causes a 2-4 fold sensitization of hormonally-stimulated adenylyl cyclase (AC) activity. This effect is thought to be due to protein kinase C (PKC)-mediated phosphorylation of either G$\sb{\rm i}$ or the catalytic subunit of AC. PKC are components of the phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP$\sb2$-PLC) pathway. The currently accepted model of this pathway is that its activation by an agonist results in the production of inositol 1,4,5-triphosphate (IP$\sb3$) which causes Ca$\sp{++}$ mobilization, and 1,2-diacylglycerols (DAG) which activate PKC. Based on this model, we predicted that stimulation of purinergic and muscarinic receptors with the agonists ATP and carbachol (CCh), respectively in the LM5 cells, should sensitize AC. Surprisingly we found that only stimulation of the purinergic receptors in these cells caused a sensitization of PGE$\sb1$-stimulated AC measured in cell-free assays.^ We hypothesized that ATP-and CCh-stimulated differential DAG production contributes to the effectiveness of these two agonists to sensitize PGE$\sb1$-stimulated AC activity. To test this hypothesis directly, we performed a combined high-performance liquid chromatography and gas-liquid chromatography analysis of the DAG produced in the LM5 cells in response to stimulation with ATP and CCh.^ We found that both ATP and CCh increased levels of 23 species of DAG. Relative to the control levels (0.261 nmol DAG/100 nmol phospholipid) the CCh-induced increase in DAG levels was 280% (0.738 $\pm$ 0.051 nmol DAG/100 nmol phospholipid) whereas the ATP-induced levels increased 180% (0.441 t 0.006 nmol DAG/100 nmol phospholipid). Neither agonist created new species or eliminated the existing ones. The major species which comprised $\approx$50% of the total cellular DAG in all of the groups were 16:0-18:1, 18:0-18:1, 18:1-18:1, and 18:0-20:4. CCh was more effective than ATP at stimulating these major DAG species.^ It is concluded that factor(s) other than DAG contribute(s) to the differences between ATP-and CCh-sensitization of PGE$\sb1$-stimulated AC activity in the LM5 cells. ^

The cDNA cloning of murine HIP /L29 and the functional analysis of HIP/L29 domains

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is a heparin/heparan sulfate (Hp/HS) binding protein found in many adult human tissues. Potential functions of this protein are promotion of embryo adhesion, modulation of blood coagulation, and control of cell growth. While these activities are diverse, the ability of human HIP/L29 to interact with Hp/HS at the cell surface may be a unifying mechanism of action since Hp/HS influences all of these processes. A murine ortholog has been identified that has 78.8% homology over the entire sequence and identity over the N-terminal 64 amino acids when compared to human HIP/L29. Northern, Western, and immunohistochemical analysis shows that murine HIP/L29 mRNA and protein are expressed in a tissue specific manner. Murine HIP/L29 is enriched in the membrane fraction of NmuMG cells where it is eluted with high salt, suggesting that it is a peripheral membrane protein. The ability of murine HIP/L29 to bind Hp is verified by studies using native and recombinant forms of murine HIP/L29. A synthetic peptide (HIP peptide-2) derived from the identical N-terminal region of HIP/L29 proteins was tested for the ability to bind Hp and support cell adhesion. This peptide was chosen because it conforms to a proposed consensus sequence for Hp/HS binding peptides. HIP peptide-2 binds Hp in a dose-dependent, saturable, and selective manner and supports Hp-dependent cell adhesion. However, a scrambled form of this peptide displayed similar activities indicating a lack of peptide sequence specificity required for activity. Lastly, an unbiased approach was used to identify sequences within human and mouse HIP/L29 proteins necessary for Hp/HS binding. A panel of recombinant proteins was made that collectively are deficient in every human HIP/L29 domain. The activities of these deletion mutants and recombinant murine HIP/L29 were compared to the activity of recombinant human HIP/L29 in a number of assays designed to look at differences in the ability to bind Hp/HS. These studies suggest that each domain within human HIP/L29 is important for binding to Hp/HS and divergences in the C-terminus of human and mouse HIP/L29 account for a decrease in murine HIP/L29 affinity for Hp/HS. It is apparent that multiple domains within human and mouse HIP/L29 contribute to the function of Hp/HS binding. The interaction of multiple HIP/L29 domains with Hp/HS will influence the biological activity of HIP/L29 proteins. ^

Regulation of p53 antiproliferative function by transactivation domain phosphorylation at threonine 18 and serine 20

We investigated the induction and physiological role of Thr18 and Ser20 phosphorylation of p53 in response to DNA damage caused by treatment with ionizing (IR) or ultraviolet (UV) radiation. Polyclonal antibodies specifically recognizing phospho-Thr18 and phospho-Ser20 were used to detect p53 phosphorylation in vivo. Analyses of five wild-type (wt) p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 and Ser20 after treatment with IR or UV. Importantly, the phosphorylation of p53 at Thr18 and Ser20 correlated with induction of the p53 downstream targets p21Waf1/Cip1 (p21) and Mdm-2, suggesting a transactivation enhancing role for Thr18 and Ser20 phosphorylation. Whereas Thr18 phosphorylation appears to abolish side-chain hydrogen bonding between Thr18 and Asp21, Ser20 phosphorylation may introduce charge attraction between Ser20 and Lys24. Both of these interactions could contribute to stabilizing α-helical conformation within the p53 transactivation domain. Mutagenesis-derived phosphorylation mimicry of p53 at Thr18 and Ser20 by Asp substitution (p53T18D/S20D) altered transactivation domain conformation and significantly reduced the interaction of p53 with the transactivation repressor Mdm-2. Mdm-2 interaction was also reduced with p53 containing a single site Asp substitution at Ser20 (p53S20D) and with the Thr18/Asp21 hydrogen bond disrupting p53 mutants p53T18A, p53T18D and p53D21A. In contrast, no direct effect was observed on the interaction of p53T18A, p53T18D and p53D21A with the basal transcription factor TAF II31. However, prior incubation of p53T18A, p53T18D and p53D21A with Mdm-2 modulated TAFII31 interaction, suggesting Mdm-2 blocks the accessibility of p53 to TAFII31. Consistently, p53-null cells transfected with p53S20D and p53T18A, p53T18D and p53D21A demonstrated enhanced endogenous p21 expression; transfection with p53T18D/S20D most significantly enhanced p21 and fas/APO-1 (fas ) expression. Expression of p53T18A, p53T18D and p53D21A in p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. Cell proliferation was also significantly curtailed in p53-null cells transfected with p53T18D/S20D relative to cells transfected with wt p53. We conclude the irradiation-induced phosphorylation of p53 at Thr18 and Ser20 alters the α-helical conformation of its transactivation domain. Altered conformation reduces direct interaction with the transrepressor Mdm-2, enhancing indirect recruitment of the basal transcription factor TAFII31, facilitating sequence-specific transactivation function resulting in proliferative arrest. ^

Sequence-related structural DNA anomalies affect restriction enzyme activity and DNA polymerase I binding

DNA serves as a target molecule for several types of enzymes and may assume a wide variety of structural motifs depending upon the local sequence. The BssHII restriction site (GC)3 resides in a 9bp region of alternating pyrimidine and purine residues within the &phis;X174 genome. Such sequences are known to demonstrate non-canonical helical behavior under the appropriate conditions. The kinetics of BssHII cleavage was investigated in supercoiled and linear plasmid DNA, and in a 323bp DNA fragment obtained via amplification of &phis;X174. The rate of enzyme cleavage was enhanced in the supercoiled form and in the presence of 50μM cobalt hexamine. Similarly, cobalt hexamine was also found to enhance TaqI activity directly adjacent to the (GC)3 region. ^ Initial DNA polymerase I binding studies (including a gel mobility shift assay and a protection assay) indicated a notable interaction between DNA polymerase I and the BssHII site. An in-depth study revealed that equilibrium binding of DNA polymerase I to the T7 RNA polymerase promoter was comparable to that of the (GC)3 site, however the strongest interaction was observed with a cruciform containing region. Increasing the ionic strength of the solution environment, including the addition of DNA polymerase I reaction buffer significantly decreased the equilibrium dissociation constant values. ^ It is suggested that the region within or around the BssHII site experiences a conformational change generating a novel structure under the influence of supercoiled tension or 50μM cobalt hexamine. It is proposed that this transition may enhance enzyme activity and binding by providing an initial enzyme-docking site—the rate-limiting step in restriction enzyme kinetics. The high binding potential of DNA polymerase I for each of the motifs described, is hypothesized to be due to recognition of the structural DNA anomalies by the 3′–5′ exonuclease domain. ^

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by Subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)(3)L(3) (Vinogradov model) plus 144 heme groups, a Value of M for HbGp oligomer of 3560 kDa can be predicted. This Value is nearly 500 kDa higher than the unique HbGp M Value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s(20,w)(0), values of 58.1 +/- 0.2 S and 59.6 +/- 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 100 and 3700 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V(bar), for HbGp was performed based on density measurements, providing a value of 0.764 +/- 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model. (c) 2008 Elsevier Inc. All rights reserved.

Emerging trends at the interface of chemistry and biology: Applications to the design of human therapeutics

This article describes recent developments in the design and implementation of various strategies towards the development of novel therapeutics using first principles from biology and chemistry. Strategies for multi-target therapeutics and network analysis with a focus on cancer and HIV are discussed. Methods for gene and siRNA delivery are presented along with challenges and opportunities for siRNA therapeutics. Advances in protein design methodology and screening are described, with a focus on their application to the design of antibody based therapeutics. Future advances in this area relevant to vaccine design are also mentioned.

Molecular characterization of the contribution of autophagy to antigen presentation using quantitative proteomics

L’autophagie est une voie hautement conservée de dégradation lysosomale des constituants cellulaires qui est essentiel à l’homéostasie cellulaire et contribue à l’apprêtement et à la présentation des antigènes. Les rôles relativement récents de l'autophagie dans l'immunité innée et acquise sous-tendent de nouveaux paradigmes immunologiques pouvant faciliter le développement de nouvelles thérapies où la dérégulation de l’autophagie est associée à des maladies auto-immunes. Cependant, l'étude in vivo de la réponse autophagique est difficile en raison du nombre limité de méthodes d'analyse pouvant fournir une définition dynamique des protéines clés impliquées dans cette voie. En conséquence, nous avons développé un programme de recherche en protéomique intégrée afin d’identifier et de quantifier les proteines associées à l'autophagie et de déterminer les mécanismes moléculaires régissant les fonctions de l’autophagosome dans la présentation antigénique en utilisant une approche de biologie des systèmes. Pour étudier comment l'autophagie et la présentation antigénique sont activement régulés dans les macrophages, nous avons d'abord procédé à une étude protéomique à grande échelle sous différentes conditions connues pour stimuler l'autophagie, tels l’activation par les cytokines et l’infection virale. La cytokine tumor necrosis factor-alpha (TNF-alpha) est l'une des principales cytokines pro-inflammatoires qui intervient dans les réactions locales et systémiques afin de développer une réponse immune adaptative. La protéomique quantitative d'extraits membranaires de macrophages contrôles et stimulés avec le TNF-alpha a révélé que l'activation des macrophages a entrainé la dégradation de protéines mitochondriales et des changements d’abondance de plusieurs protéines impliquées dans le trafic vésiculaire et la réponse immunitaire. Nous avons constaté que la dégradation des protéines mitochondriales était sous le contrôle de la voie ATG5, et était spécifique au TNF-alpha. En outre, l’utilisation d’un nouveau système de présentation antigènique, nous a permi de constater que l'induction de la mitophagie par le TNF-alpha a entrainée l’apprêtement et la présentation d’antigènes mitochondriaux par des molécules du CMH de classe I, contribuant ainsi la variation du répertoire immunopeptidomique à la surface cellulaire. Ces résultats mettent en évidence un rôle insoupçonné du TNF-alpha dans la mitophagie et permet une meilleure compréhension des mécanismes responsables de la présentation d’auto-antigènes par les molécules du CMH de classe I. Une interaction complexe existe également entre infection virale et l'autophagie. Récemment, notre laboratoire a fourni une première preuve suggérant que la macroautophagie peut contribuer à la présentation de protéines virales par les molécules du CMH de classe I lors de l’infection virale par l'herpès simplex virus de type 1 (HSV-1). Le virus HSV1 fait parti des virus humains les plus complexes et les plus répandues. Bien que la composition des particules virales a été étudiée précédemment, on connaît moins bien l'expression de l'ensemble du protéome viral lors de l’infection des cellules hôtes. Afin de caractériser les changements dynamiques de l’expression des protéines virales lors de l’infection, nous avons analysé par LC-MS/MS le protéome du HSV1 dans les macrophages infectés. Ces analyses nous ont permis d’identifier un total de 67 protéines virales structurales et non structurales (82% du protéome HSV1) en utilisant le spectromètre de masse LTQ-Orbitrap. Nous avons également identifié 90 nouveaux sites de phosphorylation et de dix nouveaux sites d’ubiquitylation sur différentes protéines virales. Suite à l’ubiquitylation, les protéines virales peuvent se localiser au noyau ou participer à des événements de fusion avec la membrane nucléaire, suggérant ainsi que cette modification pourrait influer le trafic vésiculaire des protéines virales. Le traitement avec des inhibiteurs de la réplication de l'ADN induit des changements sur l'abondance et la modification des protéines virales, mettant en évidence l'interdépendance des protéines virales au cours du cycle de vie du virus. Compte tenu de l'importance de la dynamique d'expression, de l’ubiquitylation et la phosphorylation sur la fonction des proteines virales, ces résultats ouvriront la voie vers de nouvelles études sur la biologie des virus de l'herpès. Fait intéressant, l'infection HSV1 dans les macrophages déclenche une nouvelle forme d'autophagie qui diffère remarquablement de la macroautophagie. Ce processus, appelé autophagie associée à l’enveloppe nucléaire (nuclear envelope derived autophagy, NEDA), conduit à la formation de vésicules membranaires contenant 4 couches lipidiques provenant de l'enveloppe nucléaire où on retrouve une grande proportion de certaines protéines virales, telle la glycoprotéine B. Les mécanismes régissant NEDA et leur importance lors de l’infection virale sont encore méconnus. En utilisant un essai de présentation antigénique, nous avons pu montrer que la voie NEDA est indépendante d’ATG5 et participe à l’apprêtement et la présentation d’antigènes viraux par le CMH de classe I. Pour comprendre l'implication de NEDA dans la présentation des antigènes, il est essentiel de caractériser le protéome des autophagosomes isolés à partir de macrophages infectés par HSV1. Aussi, nous avons développé une nouvelle approche de fractionnement basé sur l’isolation de lysosomes chargés de billes de latex, nous permettant ainsi d’obtenir des extraits cellulaires enrichis en autophagosomes. Le transfert des antigènes HSV1 dans les autophagosomes a été determine par protéomique quantitative. Les protéines provenant de l’enveloppe nucléaire ont été préférentiellement transférées dans les autophagosome lors de l'infection des macrophages par le HSV1. Les analyses protéomiques d’autophagosomes impliquant NEDA ou la macroautophagie ont permis de decouvrir des mécanismes jouant un rôle clé dans l’immunodominance de la glycoprotéine B lors de l'infection HSV1. Ces analyses ont également révélées que diverses voies autophagiques peuvent être induites pour favoriser la capture sélective de protéines virales, façonnant de façon dynamique la nature de la réponse immunitaire lors d'une infection. En conclusion, l'application des méthodes de protéomique quantitative a joué un rôle clé dans l'identification et la quantification des protéines ayant des rôles importants dans la régulation de l'autophagie chez les macrophages, et nous a permis d'identifier les changements qui se produisent lors de la formation des autophagosomes lors de maladies inflammatoires ou d’infection virale. En outre, notre approche de biologie des systèmes, qui combine la protéomique quantitative basée sur la spectrométrie de masse avec des essais fonctionnels tels la présentation antigénique, nous a permis d’acquérir de nouvelles connaissances sur les mécanismes moléculaires régissant les fonctions de l'autophagie lors de la présentation antigénique. Une meilleure compréhension de ces mécanismes permettra de réduire les effets nuisibles de l'immunodominance suite à l'infection virale ou lors du développement du cancer en mettant en place une réponse immunitaire appropriée.

Analytical method for determination of nitric oxide in zebrafish larvae: Toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coil lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae. (C) 2011 Elsevier Inc. All rights reserved.

An environmentally friendly analytical procedure for nickel determination by atomic and molecular spectrometry after cloud point extraction in different samples

Cloud point extraction (CPE) was employed for separation and preconcentration prior to the determination of nickel by graphite furnace atomic absorption spectrometry (GFAAS), flame atomic absorption spectrometry (FAAS) or UV-Vis spectrophotometry. Di-2-pyridyl ketone salicyloylhydrazone (DPKSH) was used for the first time as a complexing agent in CPE. The nickel complex was extracted from the aqueous phase using the Triton X-114 surfactant. Under optimized conditions, limits of detection obtained with GFAAS, FAAS and UV-Vis spectrophotometry were 0.14, 0.76 and 1.5 mu g L-1, respectively. The extraction was quantitative and the enrichment factor was estimated to be 27. The method was applied to natural waters, hemodialysis concentrates, urine and honey samples. Accuracy was evaluated by analysis of the NIST 1643e Water standard reference material.

Revealing the elusive molecular biology of the vault RNA

Recently several novel and previously reported non-protein-coding RNAs (ncRNAs) have been identified to be upregulated upon Epstein-Barr virus (EBV) infection in human B-lymphocytes. A group of these significantly upregulated ncRNAs are called vault RNAs (vtRNAs). ,b Only about 5% of the total cellular vtRNAs are connected to the vault particle, the largest known ribonucleoprotein particle (RNP) in eukaryotic cells. However the function of this ncRNA family and moreover of the vault particle remains still rather unclear. Our previous findings suggest a link between EBV infection and vtRNA expression. Consequently we are interested which part of the viral genome is responsible for the upregulation and moreover which function the vtRNAs might possess during virus propagation. To address this question we have separately overexpressed specific EBV-encoded, latently expressed proteins in BL2-cells to determine the influence on the vault RNA levels. Thereby we identified one EBV-encoded protein, called Latent Membrane Protein 1 (LMP1), which significantly contributes to the vtRNA upregulation. We used LMP1 mutants to characterize the region of the protein and the responsible pathway for triggering the elevated vtRNA expression. Our results suggest that the NFkB- pathway might be involved in this process. To investigate a possible functional connection between the vtRNA and EBV infection, we have overexpressed vtRNA1-1 in BL41, a cell line usually not expressing this vault RNA. We show that overexpression of vtRNA1-1 leads to a better viral establishment and markedly protects cells from undergoing apoptosis. Knock-down of the major vault protein, the main component of the vault particle, had no effect on EBV infection and apoptosis resistance. Thus these results support the view that the observed phenotype is caused by the vtRNA rather than the vault particle.

Highlights in Analytical Chemistry in Switzerland: OMA & OPA - A Software Tool for Mass Spectrometric Sequencing of Nucleic Acids

Oligonucleotides comprising unnatural building blocks, which interfere with the translation machinery, have gained increased attention for the treatment of gene-related diseases (e.g. antisense, RNAi). Due to structural modifications, synthetic oligonucleotides exhibit increased biostability and bioavailability upon administration. Consequently, classical enzyme-based sequencing methods are not applicable to their sequence elucidation and verification. Tandem mass spectrometry is the method of choice for performing such tasks, since gas-phase dissociation is not restricted to natural nucleic acids. However, tandem mass spectrometric analysis can generate product ion spectra of tremendous complexity, as the number of possible fragments grows rapidly with increasing sequence length. The fact that structural modifications affect the dissociation pathways greatly increases the variety of analytically valuable fragment ions. The gas-phase dissociation of oligonucleotides is characterized by the cleavage of one of the four bonds along the phosphodiester chain, by the accompanying loss of nucleases, and by the generation of internal fragments due to secondary backbone cleavage. For example, an 18-mer oligonucleotide yields a total number of 272’920 theoretical fragment ions. In contrast to the processing of peptide product ion spectra, which nowadays is highly automated, there is a lack of tools assisting the interpretation of oligonucleotide data. The existing web-based and stand-alone software applications are primarily designed for the sequence analysis of natural nucleic acids, but do not account for chemical modifications and adducts. Consequently, we developed a software to support the interpretation of mass spectrometric data of natural and modified nucleic acids and their adducts with chemotherapeutic agents.

Novel analytical and numerical methods for solving fractional dynamical systems

During the past three decades, the subject of fractional calculus (that is, calculus of integrals and derivatives of arbitrary order) has gained considerable popularity and importance, mainly due to its demonstrated applications in numerous diverse and widespread fields in science and engineering. For example, fractional calculus has been successfully applied to problems in system biology, physics, chemistry and biochemistry, hydrology, medicine, and finance. In many cases these new fractional-order models are more adequate than the previously used integer-order models, because fractional derivatives and integrals enable the description of the memory and hereditary properties inherent in various materials and processes that are governed by anomalous diffusion. Hence, there is a growing need to find the solution behaviour of these fractional differential equations. However, the analytic solutions of most fractional differential equations generally cannot be obtained. As a consequence, approximate and numerical techniques are playing an important role in identifying the solution behaviour of such fractional equations and exploring their applications. The main objective of this thesis is to develop new effective numerical methods and supporting analysis, based on the finite difference and finite element methods, for solving time, space and time-space fractional dynamical systems involving fractional derivatives in one and two spatial dimensions. A series of five published papers and one manuscript in preparation will be presented on the solution of the space fractional diffusion equation, space fractional advectiondispersion equation, time and space fractional diffusion equation, time and space fractional Fokker-Planck equation with a linear or non-linear source term, and fractional cable equation involving two time fractional derivatives, respectively. One important contribution of this thesis is the demonstration of how to choose different approximation techniques for different fractional derivatives. Special attention has been paid to the Riesz space fractional derivative, due to its important application in the field of groundwater flow, system biology and finance. We present three numerical methods to approximate the Riesz space fractional derivative, namely the L1/ L2-approximation method, the standard/shifted Gr¨unwald method, and the matrix transform method (MTM). The first two methods are based on the finite difference method, while the MTM allows discretisation in space using either the finite difference or finite element methods. Furthermore, we prove the equivalence of the Riesz fractional derivative and the fractional Laplacian operator under homogeneous Dirichlet boundary conditions – a result that had not previously been established. This result justifies the aforementioned use of the MTM to approximate the Riesz fractional derivative. After spatial discretisation, the time-space fractional partial differential equation is transformed into a system of fractional-in-time differential equations. We then investigate numerical methods to handle time fractional derivatives, be they Caputo type or Riemann-Liouville type. This leads to new methods utilising either finite difference strategies or the Laplace transform method for advancing the solution in time. The stability and convergence of our proposed numerical methods are also investigated. Numerical experiments are carried out in support of our theoretical analysis. We also emphasise that the numerical methods we develop are applicable for many other types of fractional partial differential equations.

Addressing accuracy and precision issues in iTRAQ quantitation

iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.