Le virus de l’hépatite C (VHC) est, encore aujourd’hui, un problème de santé mondiale majeur entraînant dans certains cas des cirrhoses et des hépatocarcinomes. De nombreux efforts ont été fournis depuis les années 80 afin de développer un traitement efficace et sûr de cette infection touchant les hépatocytes. Le traitement interféron/ribavirine, utilisé dans les années 2000, a aujourd’hui été remplacé par des thérapies utilisant des agents antiviraux directs, beaucoup plus efficaces. Ces traitements restent cependant perfectibles notamment du fait de certains effets secondaires, de leur coût élevé et de potentielles interactions médicamenteuses avec d’autres composés thérapeutiques. L’équipe de glycochimie du Laboratoire de Chimie Biologique s’est intéressée à la synthèse de C-nucléosides analogues de la ribavirine. Parmi-eux, un composé, le SRO91, s’est révélé efficace contre des réplicons du VHC et présente une faible toxicité. Dans le but d’améliorer l’activité anti-VHC du SRO91, deux axes ont été développés dans ce projet : l’adressage vers les cellules du foie, et l’amélioration de la pénétration cellulaire. Un premier conjugué entre un galactoside et SRO91 a ainsi été synthétisé, afin de profiter de la forte interaction du galactose avec les récepteurs aux asialoglycoprotéines, principalement exprimés à la surface des hépatocytes. Afin d’améliorer sa pénétration cellulaire, le nucléoside a également été conjugué à des peptides de fusion du VHC. Ces séquences peptidiques très hydrophobes sont capables de s’insérer dans la membrane cellulaire et de permettre la fusion. Trois peptides ont été sélectionnés en se basant sur la littérature : HCV3 (VFLVG), HCV6 (YVGDLSGSVFL) et HCV7 (SWHINRTALNSNDS), synthétisés par SPPS puis conjugués au nucléoside ou à un fluorophore. L’activité membranotropique des peptides sur des liposomes a alors été étudiée par calorimétrie (DSC et ITC), spectrofluorescence et microscopie à épifluorescence. Ces études ont ainsi permis de montrer que, parmi les séquences sélectionnées, HCV7 semble montrer la meilleure activité en pénétration membranaire alors que HCV6 s’est révélé être la séquence la plus fusogénique. / Hepatitis C virus (HCV) is a global healthcare issue responsible for cirrhosis and hepatocarcinoma. Strong efforts have been made since the 80’s to develop efficient and safe treatments for this liver infection. Hence, the treatment based on interferon/ribavirin, developed in 2002, has been replaced by much more efficient therapies involving direct-acting antivirals. However, the different side-effects, high cost and possible drug-drug interactions make room for improvements to this treatment. In the Laboratoire de Chimie Biologique, several C-nucleosides, analogs of ribavirin have been developed. Among them, one compound, named SRO91, seems effective against HCV replicons with low toxicity. This thesis work focused on improving SRO91 anti-HCV activity by implementing a targeting strategy and enhancing cell-penetration. We built our targeting strategy on the strong interaction between galactose and asialoglycoprotein receptors. Thus, a SRO91-galactose conjugate was synthesized, in order to address the antiviral to hepatocytes. To enhance cell-penetration we conjugated our nucleoside to HCV fusion peptides, since these highly hydrophobic sequences are able to anchor in cell membranes, leading to fusion. Three peptides were selected based on the literature: HCV3 (VFLVG), HCV6 (YVGDLSGSVFL) and HCV7 (SWHINRTALNSNDS), synthesized by SPPS and conjugated to SRO91 or a fluorescent tag. Several techniques were used to study the membranotropic activity of theses sequences on liposomes as membrane models, including calorimetry (DSC and ITC), spectrofluorescence and epifluorescence microscopy. Thus, among the selected peptides, HCV7 seems to be the more potent as a membrane-penetrating agent but HCV6 shows the best fusogenic activity.

Transcriptional regulation of cell cycle controlled genes is fundamental to cell division in eukaryotes and a broad spectrum of physiological processes directly related to cell proliferation. Expression of the cell cycle dependent human H4, H3 and H1 histone genes is coordinately regulated at both the transcriptional and posttranscriptional levels. We have systematically analyzed the protein/DNA interactions of the immediate 5'regions of three prototypical cell cycle controlled histone genes, designated H4-F0108, H3-ST519 and H1-FNC16, to define components of the cellular mechanisms mediating transcriptional regulation. Multiple biochemically distinct protein/DNA interactions were characterized for each of these genes, and the binding sites of several promoter-specific nuclear DNA binding activities were delineated at single nucleotide resolution using a variety of techniques. These findings were integrated with results obtained by others and revealed that the in vitro factor binding sites in H4, H3 and H1 histone promoters coincide with genomic protein/DNA interaction sites defined in vivofor the H4-F0108 and H3-STS19 genes, and with evolutionarily conserved cis-acting sequences shown to affect the efficiency of histone gene transcription. Specifically, we have defined binding sites for Sp1, ATF, CP1/NF-Y, HiNF-D, HiNF-M, HiNF-P and HMG-I related factors. Based on sequence-similarities and cross-competition experiments, we postulate that most of these protein/DNA interaction elements are associated with more than one class of histone genes. Thus, the protein/DNA interactions characterized in this study may represent components of a cellular mechanism that couples transcription rates of the various histone gene classes. Regulation of the protein/DNA interactions involved in transcriptional control of these H4, H3 and H1 histone genes was investigated in a spectrum of cell types using several distinct in vitro cell culture models for the onset of differentiation and quiescence, as well as cell cycle progression. Moreover, we studied control of histone gene associated DNA binding activities during hepatic development from fetus to adult in transgenic mice reflecting the onset of differentiation and quiescence in vivo. We show that the H4 histone promoter protein/DNA interaction mediated by factor HiNF-D is selectivelymodulated, and directly at the level of DNA binding activity, during the entry into, progress through and exit from the cell cycle in normal diploid cells, as well as during hepatic development. The regulation of this protein/DNA interaction occurs in parallel with analogous interactions occurring in H3 and H1 histone genes. Moreover, these proliferation-specific protein/DNA interactions are collectively deregulated during the cell cycle in four distinct cell types displaying properties of the transformed phenotype. Hence, the cellular competency to coordinately transcribe distinct classes of histone genes during the cell cycle may be mediated by the intricate interplay of constitutively expressed general transcription factors and temporally regulated, cell growth controlled nuclear factors interacting specifically with cell cycle dependent histone genes. Finally, we show that HiNF-D is represented by two electrophoretically distinct species. The ratio of these forms of HiNF-D fluctuates dramatically during the cell cycle of normal diploid cells, but remains relatively constant in tumor cells. Total HiNF-D binding activity embodied by both HiNF-D species is negatively influenced in vitro by incubation with exogenous phosphatase activity. These observations provide a first indication for the hypothesis that HiNF-D may exist in distinct post-translationally modified forms that are subject to a stringent cell growth control mechanism involving protein kinases and phosphatases. Such a cellular post-translational modification mechanism, which directly impinges on (or activates) the DNA binding activity of a key factor controlling histone genes, would provide a highly efficient means by which to influence the rate of transcription in rapid response to intra-cellular requirements for histone mRNA and extra-cellular cues signalling the onset and cessation of cell proliferation.

Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. HIV-1 expresses multiple genes from a single provirus by alternative splicing. Early in viral expression, fully spliced 2-kb viral RNA is exported from the nucleus and encodes the viral regulatory protein, Rev, which is essential for nuclear transport of partially spliced and unspliced genomic-length RNA. Rev binds to an RNA structural element called the Rev response element (RRE) and mediates nuclear export through the leucine-rich nuclear export signal (NES) pathway. The human Rev Interacting Protein (hRIP) interacts specifically with the Rev NES. Rev NES mutants that are unable to export Rev-dependent RNAs are also unable to bind to hRIP. The hRIP cDNA encodes a 562 amino acid protein containing an N-terminal zinc finger with homology to Arf GAP domains, a central serine and threonine rich region, and C-terminal phenylalanine-glycine (FG) repeats characteristic of nucleoporins. To identify an hRIP ortholog in a genetically tractable organism, we performed database searches using the N-terminal zinc finger of hRIP. Using this approach, we identified a novel gene in Schizosaccharomyces pombe. Alignment of the entire reading frame of the putative ortholog with hRIP indicates similarity with the serine/threonine rich region and with the FG repeats, suggesting that S. pombecould be a good model system to study the cellular function of hRIP. We find that the S. pombe ORF is an essential gene, which encodes a 483 amino acid protein that is also able to interact with the NES of HIV-1 Rev. Based on being an essential gene, and the presence of a putative Arf GAP domain, the ORF was named an Arf GAP essential for viability, agv1+. We show that Agv1 is not directly involved in the nuclear export of poly(A+) RNA or 5S rRNA, nuclear export of leucine-rich NES-containing proteins, or nuclear import of nuclear localization signal (NLS)-containing proteins. However, Agv1 does appear to play a role in the cytoplasmic localization of 5S rRNA. We demonstrate that loss of Agv1 alters the localization of endoplasmic reticulum (ER) membrane and Golgi membrane resident proteins, accumulates intracellular membrane, and blocks processing of carboxypeptidase Y. Furthermore, the S. cerevisiae ADP-ribosylation factor (Arf) GTPase activating protein (GAP) Glo3, but not a catalytically inactive Glo3 mutant [R59K], is able to partially compensate for the loss of Agv1 function in temperature sensitive strains, indicating that Agv1 is an S. pombe Arf GAP with some functional features similar to S. cerevisiae Glo3.

<p>U ovom radu prikazana je sinteza 11 tiazolnih izostera goniofufurona (1-11),<br />4 konformaciono kruta analoga goniofufurona (12-15) i jednog butadiolnog<br />derivata&nbsp; tiazofurina&nbsp; (16).&nbsp; Takođe,&nbsp; izvr&scaron;eno&nbsp; je&nbsp; ispitivanje&nbsp; i&nbsp; poređenje<br />biolo&scaron;kih&nbsp; aktivnosti&nbsp; sintetisanih&nbsp; analoga&nbsp; sa&nbsp; sa&nbsp; aktivno&scaron;ću&nbsp; i&nbsp; selektivno&scaron;ću<br />kako GF i TF tako i doksorubicina, jedinjenja &scaron;irokog spektra dejstva (DOX).<br />Hiralni&nbsp; prekursor&nbsp; novosintetisanih&nbsp; jedinjenja&nbsp; 1-15&nbsp; bila&nbsp; je&nbsp; D-glukoza,&nbsp; a<br />jedinjenja 16 D-arabinoza.</p> / <p>A multistep synthesis of 11 novel thiazole isosteres of goniofufurone (1-11), 4 novel conformationally constrained isosteres of goniofufurone (12-15), as well as&nbsp; one&nbsp; butanediole&nbsp; derivative&nbsp; of&nbsp; tiazofurin&nbsp; (16)&nbsp; has&nbsp; been&nbsp; achieved.&nbsp; In&nbsp; vitro cytotoxicity of newly synthetized derivatives has been evaluated and compared with the cytotoxicities of goniofufurone, tiazofurin and doxorubicin.</p>

This thesis deals with the preparation of acyclic nucleosides and nucleoside phosphonates of compounds T-705 (6-fluoro-3-hydroxypyrazine-2-carboxamide) and T-1105 (3-hydroxypyrazine-2-carboxamide). Acyclic nucleoside phosphonates are substances that can terminate viral RNA or DNA replication, and some of them are used in the treatment of viral diseases. T-705 and T-1105 have shown activity against the influenza virus, and T-705 has already been approved for its treatment in Japan. Since both compounds mimic natural nucleobases in the body, their acyclic nucleosides and nucleoside phosphonates also have the potential to be biologically active. Methods for the synthesis of 3-fluoro-2-(phosphonomethoxy)propyl and 3-hydroxy-2-(phosphonomethoxy)propyl derivatives of T-705 and T-1105, their prodrugs containing lipophilic groups for the improvement of the pharmacokinetic properties and also their phosphonate diphosphates, suitable for the biological activity measurements, have been proposed. Some of these derivatives were subsequently prepared. Key words: acyclic nucleosides, acyclic nucleoside phosphonates, T-705, T-1105, favipiravir, antiviral activity, influenza

The development of methods and techniques is the driving force of scientific research. In this work, we described two large-scale screens in studying transcriptional activation and tumor suppression. In Part I, we studied transcriptional activation mechanisms by deriving and characterizing activation defective mutants. Promoter-specific transcriptional activators stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the “target.” The identification of direct in vivo targets of activators has been a major challenge. We perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo. Utilizing these mutants, we demonstrated that Tra is an essential target for Gal4 activation, Gal4 and Tra1 bind cooperatively at the promoter and the Gal4–Tra1 interaction occurs predominantly on the promoter. In addition, we demonstrated that the Gal4-interaction site on Tra1 is highly selective. In Part II, we described a functional genomics approach to discover new tumor suppressor genes. A goal of contemporary cancer research is to identify the genes responsible for neoplastic transformation. Cells that are immortalized but non-tumorigenic were stably transduced with pools of short hairpin RNAs (shRNAs) and tested for their ability to form tumors in mice. ShRNAs in any resulting tumors were identified by sequencing to reveal candidate TSGs, which were then validated both experimentally and clinically by analysis of human tumor samples. Using this approach, we identified and validated 33 candidate TSGs. We found that most candidate TSGs were down-regulated in >70% of human lung squamous cell carcinoma (hLSCC) samples, and 17 candidate TSGs negatively regulate FGFR signalling pathway, and their ectopic expression inhibited growth of hLSCC xenografts. Furthermore, we suggest that by examining at the expression level of TSGs in lung cancer patients, we can predict their drug responsiveness to FGFR inhibitors. In conclusion, we have identified many new lung squamous cell cancer TSGs, using an experimental strategy that can be broadly applied to find TSGs in other tumor types.

Background To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice. Methods Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters. Results We distinguished numerous, unique plasma miRNAs both up- and down-regulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and down-regulated miRNAs, included, but were not limited to, 574-5p, 466g, 466f-3p, 375, 29c, and 148a. There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point ( P < 0.001). There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point ( P = 0.011). Conclusion We identified unique plasma miRNAs both up- and down-regulated in lethally dosed APAP poisoned mice.

The proper workings of an organism rely on the accurate expression of genes throughout its lifetime. An important determinant for protein production is the availability of template mRNA molecules, the net effect of which is governed by their rates of synthesis vs. their rates of degradation. Normal mRNAs are proposed to be relatively stable in the cytoplasm while present in a protective, circularized conformation – the closed loop – through eIF4G-bridged interactions with 3’-bound poly(A) binding protein (Pab1p) and 5’-bound eIF4E. Introduction of a premature nonsense codon into an otherwise normal mRNA results in its rapid destabilization in cells, suggesting that not all stop codons behave the same, and events at premature termination events that lead to accelerated degradation of nonsense-containing mRNAs likely differ from those at normal termination, in which normal decay rates are maintained. The enhanced degradation observed for nonsense-containing mRNAs occurs through an evolutionarily conserved pathway involving the products of the UPF1, UPF2/NMD2, and UPF3 genes, the precise biochemical roles of which have remained elusive. We have developed a yeast cell-free translation system that allows us to assay biochemical events occurring at premature termination codons, compare them to those occurring at normal terminators, and study the role of Upf1p in these events. We find that premature termination is an inefficient process compared to normal termination and that one outcome of termination at a premature termination codon (PTC) is reinitiation at a nearby start codon. This in vitro post-termination reinitiation phenotype is dependent on the presence of Upf1p, a finding we have recapitulated in vivo. We also developed biochemical assays to define a role for Upf1p in translation following premature termination in vitro and find that Upf1p is involved in post-termination ribosome dissociation and reutilization. Supporting this idea are our findings that Upf1p predominantly cosediments with purified 40S ribosomal subunits. Finally, using our in vitro translation/toeprinting system, we have further characterized events leading to the formation of the mRNA closed loop structure and find that two states of the closed loop exist. The first requires the preinitiation 48S complex and includes Pab1p, eIF4G, eIF4E, and eIF3, whereas the second is formed after 60S joining and additionally requires the translation termination factors eRF1 and eRF3.

Deutsch: Die vorliegende Arbeit beinhaltet die kombinatorische Synthese sowie biomedizinische Aspekte von neuen, lipophilisierten Nucleosiden (Nucleolipiden) als small molecules. Für die Synthesen wurden sowohl Nucleosid-Metabolite als auch -Antimetabolite lipophilisiert. Als Lipidreste wurden natürlich vorkommende Verbindungen, wie azyklische Terpene und (a)symmetrische Ketone verwendet. Diese wurden am O-2‘,3‘-cis-glycosidischen Rest oder an der N(3)-Position von β-D-Pyrimidinen oder an der N(1)-Position von β-D-Purinen eingeführt. Die Einführung der Reste erfolgte durch Ketalisierung der glyconischen Hydroxylgruppen oder durch direkte Alkylierung sowie durch Dimroth-Umlagerung des Aglycons. Zusätzlich wurden in weiteren Reaktionen ausgewählte Nucleolipide in 2-(Cyanoethyl)phosphoramidit für die automatische DNA-Festphasensynthese von Oligo-nucleotiden umgewandelt. Diese wurden für eine Reihe von Penetrationsversuchen hinsichtlich ihres Einlagerungs-und Penetrationsverhaltens in eine künstliche Lipidmembran untersucht und untereinander verglichen. Die synthetisierten Nucleolipide wurden NMR-spektroskopisch im Hinblick auf die strukturellen Parameter (1) Zuckerpucker (3’T2‘⇌3’T2‘) und (2) die Konformation um die exozyklische C(4‘)-C(5‘)-Bindung (γ+(g)⇌γt⇌γ-(g)) charakterisiert. Außerdem wurden die Nucleolipide hinsichtlich ihrer biologischen Aktivität in in vitro-Tests auf humane, differenzierte THP-1-Makrophagen bezüglich des Immunoeffekts und auf eine Rattengliom- sowie einer humanen Gliom-Zellline bezüglich der Antitumoraktivität getestet. English: The thesis comprises the combinatorial synthesis and biomedicinal aspects of novel lipophilized nucleosides (Nucleolipids) as small molecules. Nucleoside-metabolites, as well as -antimetabolites, were used for the lipophilization. The chemical structure of the lipid residues resembles naturally-occurring compounds, namely acyclic terpenes, and (a)symmetric ketones. They are positioned either at the O-2’,3’-cis-glyconic moiety or at the N(3) of β-D-pyrimidines or N(1) of β-D-purines. The introduction of the lipophilic residues was performed either by ketalization of the glyconic hydroxyls or by direct alkylation as well as by Dimroth rearrangement at the N-alkylated aglycone. Additionally, selected nucleolipids were further converted to 2-(cyanoethyl) phosphoramidites as building blocks for automated solid phase nucleic acid synthesis. The latters were used for the preparation of a series of lipo-oligonucleotides which were studied with respect to their immobilization within artificial lipid bilayers and compared concerning immobilization rate and stability. The resulting nucleolipids were characterized with respect to the structural parameters (1) the sugar pucker (3’T2‘⇌3’T2‘) as well as (2) the conformation around the exocyclic C(4’)-C(5’)-bond (γ+(g)⇌γt⇌γ-(g)) by 1H-NMR-spectroscopy. Moreover, the biological activity of the nucleolipids was tested in-vitro on human, differentiated THP-1-macrophages for the immunoeffect and towards the rat gliom cell line BT4Ca as well as a human gliom (GOS-3) for anticancer activity.

Actuellement, les nucléosides représentent une classe majeure de composés dans les différentes thérapies anti-virales. Leur développement au cours des 50 dernières années a contribué à la mise sur le marché d'une quarantaine de composés, notamment dans la lutte des hépatites, des herpès et du VIH. Les infections virales représentent cependant toujours un large problème de santé publique, de par l'apparition de résistances aux médicaments existants ainsi que de nouvelles espèces virales. Il est donc nécessaire de développer de nouveaux antiviraux plus actifs et plus sûrs. Dans ce manuscrit, il est décrit la synthèse métallo-catalysée d'acyclonucléosides phosphonate innovants, modifiés sur la partie phosphonate, nucléobase et sur la chaîne acyclique. Pour ce faire, nous nous sommes appuyés sur des réactions de métathèses croisées et la synthèse convergente d'un large panel de nucléobases et phosphonates modifiés. Une importance toute particulière a été donnée à l'utilisation de nouvelles techniques d'activation comme le micro-onde ou les ultrasons. Ces divers procédés de synthèse ont permis la synthèse de trois séries d'alkényl phosphononucléosides, qui se sont révélés d'une activité antivirale remarquable sur certains virus à herpès, comme le VZV ou le CMV. / Nucleosides represents a major class of compounds in different antiviral chemotherapies. Their development from now 50 years has led to the emergence of nearly 40 compounds, to contain many epidemics and in the fight against viral infections as HIV, hepatitis or herpes. Viral infections still represent a tremendous problem in public health with the emergence of resistance to known drugs and the appearance of new viruses. In this context, it's essential to develop new antivirals with higher activity and safer. This manuscript describes organo-metallic syntheses of new modified acyclonucleoside phosphonates, on the nucleobase, the phosphonate or the acyclic spacer. Thus, the use of cross-metathesis and convergent syntheses of a broad panel of nucleic bases and modified phosphonates represents a cornerstone of our work. A particular importance was given to the use of new activations way, as ultrasound or microwave irradiation. Those processes allowed us to synthetize three new families of acyclic phosphononucleosides, which revealed a remarkable antiviral activity against some herpes viruses, as VZV or CMV.