Dissertations / Theses on the topic 'Ribonuclease III'

Dissertation presented to obtain the Ph.D degree in Biology.
Ribonucleases (RNases) are key factors in the control of all biological processes, since they modulate the stability of RNA transcripts, allowing rapid changes in gene expression. Some RNases are up-regulated under stress situations and are involved in virulence processes in pathogenic microorganisms. RNases also control the levels of regulatory RNAs, which play very important roles in cell physiology.(...)

Chemistry
Ph.D.
Ribonuclease III is a highly-conserved bacterial enzyme that cleaves double-stranded (ds) RNA structures, and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, those crystals involved complexes containing either cleaved RNA, or a mutant RNase III that is catalytically inactive. In addition, neither the biochemical properties of A. aeolicus (Aa)-RNase III, nor the reactivity epitopes of its cognate substrates are known. The goal of this project is to use Aa-RNase III, for which there is atomic-level structural information, to determine how RNase III recognizes its substrates and selects the target site. I first purified recombinant Aa-RNase III and defined the conditions that support its optimal in vitro catalytic activity. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of 70-85°C, a pH optimum of 8.0, and with either Mg2+ or Mn2+ supports efficient catalysis. Cognate substrates for Aa-RNase III were identified and their reactivity epitopes were characterized, including the specific bp sequence elements that determine processing reactivity and selectivity. Small RNA hairpins, based on the double-stranded structures associated with the Aquifex 16S and 23S rRNA precursors, are cleaved in vitro at sites that are consistent with production of the immediate precursors to the mature rRNAs. Third, the role of the dsRBD in scissile bond selection was examined by a mutational analysis of the conserved interactions of RNA binding motif 1 (RBM1) with the substrate proximal box (pb). The individual contributions towards substrate recognition were determined for conserved amino acid side chains in the RBM1. It also was shown that the dsRBD plays key dual roles in both binding energy and selectivity, through RBM1 responsiveness to proximal box bp sequence. The dsRBD is specifically responsive to an antideterminant (AD) bp in pb position 2. The relative structural rigidity of both dsRNA and dsRBD rationalizes the strong effect of an inhibitory bp at pb position 2: disruption of one RBM1 side chain interaction can effectively disrupt the other RBM1 side chain interactions. Finally, a cis-acting model was developed for subunit involvement in substrate recognition by RNase III. Structurally asymmetric mutant heterodimers of Escherichia coli (Ec)-RNase III were constructed, and asymmetric substrates were employed to reveal how RNase III can bind and deliver hairpin substrates to the active site cleft in a pathway that requires specific binding configurations of both enzyme and substrate.
Temple University--Theses

Chemistry
Ph.D.
The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates.
Temple University--Theses

Chemistry
Ph.D.
The bacteriophage T7 protein kinase enhances T7 growth under suboptimal growth conditions, including elevated temperature or limiting carbon source. T7PK phosphorylates numerous E. coli proteins, and it has been proposed that phosphorylation of these proteins is responsible for supporting T7 replication under stressful growth conditions. How the phosphorylation of host proteins supports T7 growth is not understood. Escherichia coli (Ec) RNase III is phosphorylated on serine in bacteriophage T7-infected cells. Phosphorylation of Ec-RNase III induces a ~4-fold increase in catalytic activity in vitro. Ec-RNase III is involved in the maturation of several T7 mRNAs, and it has been shown that RNase III processing controls the translational activity and stability of the T7 mRNAs. Perhaps T7PK phosphorylation of Ec- RNase III ensures optimal processing of T7 mRNAs under suboptimal growth conditions. In this study a biochemical analysis was performed on the N-terminal portion of the 0.7 gene (T7PK), exhibiting only the protein kinase activity. In addition to phosphotransferase activity, T7PK also undergoes self-phosphorylation on serine, which down-regulates catalytic activity by an unknown mechanism. Mass spectral analysis revealed that Ser216 is the autophosphorylation site in T7PK. The serine residue is highly conserved, which in turn suggests that autophosphorylation is a conserved reaction with functional importance. Phosphorylated T7PK exhibits reduced phosphotransferase activity, compared to its dephosphorylated counterpart (dT7PK). The dT7PK exhibits enhanced ability to phosphorylate proteins, as well as undergo autophosphorylation. The mechanism by which autophosphorylation inhibits T7PK activity is unknown. An in vitro phosphorylation assay revealed that T7PK directly phosphorylates RNase III. Ec-RNase III processing activity is stimulated from two to ten-fold upon phosphorylation by the T7PK. The primary site of phosphorylation in RNase III is found to be Ser33, and Ser34 may act as the recognition determinant for T7PK. This was established by Ser →Ala mutations at the concerned site. The enhancement of catalytic activity is primarily due to a larger turnover number (kcat), with some additional contribution from a greater substrate binding affinity, as revealed by lower Km and K‟D values. Substrate cleavage assays under single turn over conditions established that the product release is the rate limiting step. Since there is no significant increase in the kcat as measured under single-turnover (enzyme excess) conditions, the increase in the kcat in the steady-state is due to enhancement of the product release step, and not due to an enhancement of the hydrolysis (chemical) step.
Temple University--Theses

Les cancers du sein sont la première cause de mortalité chez les femmes occidentales. Ils présentent une grande hétérogénéité associée à une résistance aux traitements et un taux de rechute important. Ainsi, les cliniciens doivent personnaliser la prise en charge des patientes et ce par une meilleure connaissance des causes moléculaires qui participent à l’apparition et à la progression des tumeurs mammaires. Pour ce projet, nous avons choisi d’étudier les éventuelles valeurs pronostique et diagnostique de dicer, gène clef du mécanisme d’interférence ARN. Il a été montré une implication de Dicer dans la mise en place de l’hétérochromatine de novo des péricentromeres. Les données disponibles nous ont permis d’envisager que Dicer pourrait être impliquée dans la régulation de la stabilité chromosomique. Nous avons donc testé les valeurs pronostique et diagnostique de dicer (en PCR quantitative et en Tissue Microarray) dans une centaine d’échantillons de tumeurs, dans des lignées et modèles cellulaires. Parallèlement, nous avons étudié les conséquences d’une inhibition de l’expression de dicer sur le cycle cellulaire et sur la réponse à un stress réplicatif. Nos résultats ont montré que l’expression de dicer est un facteur pronostique indépendant de rechutes métastatiques. Par ailleurs, les cellules n’exprimant pas dicer ont montré des dérégulations du cycle cellulaire et de la voie de réponse aux cassures de l’ADN. En conclusion, l’altération de l’expression de dicer pourrait jouer un rôle dans l’apparition de l’instabilité chromosomique des cancers du sein et son analyse pourrait permettre une meilleure prise en charge des patientes à risque pour une rechute métastatique
Breast cancers are the first cause of mortality in occidental women population. Breast tumours can show various forms which are frequently resistant to therapeutics and prone to late relapse. Thus, the current clinical challenge consists in refining individual therapeutics management by employing tools provided by the study of molecular basis of mammary tumorigenesis. Besides, chromosomal instability (CIN) is a hallmark of breast cancer and recent data showed that Dicer, a key ribonuclease of the RNA interference mechanism, could be a regulator of chromosomal stability in human cells. We thus hypothesized that alteration of this protein could be associated with mammary tumorigenesis. In order to test this hypothesis, we assessed dicer transcription and expression in breast cancer cell lines and tissues corresponding to different phases of tumor progression. We further investigated the consequences of dicer knock-down on cell cycle and response to replicative stress. Our results show that dicer expression has an independent prognostic value for metastatic relapse prediction and is correlated with hormonal receptors expression. Furthermore, cells harbouring dicer inactivation presented defects in cell cycle and DNA breaks response pathways. Altogether, our data indicate that dicer inactivation could favour CIN during mammary tumorigenesis and this feature could represent a useful tool in breast cancer management

Biology
Ph.D.
The Escherichia coli ymdB gene encodes a ~19 kDa protein that binds ADP-ribose (ADPR) and metabolites related to NAD+. As such, it has been termed a macrodomain protein, referring to a conserved fold that binds ADPR. YmdB can catalyze the hydrolysis of O-acetyl-ADP-ribose (OAADPR), forming acetate and ADPR. OAADPR is a product of sirtuin action on lysine-acetylated proteins, which involves NAD+ as a cosubstrate. There is evidence that YmdB interacts with other proteins, including the conserved enzyme, ribonuclease III. Ribonuclease III (RNase III) is a double-strand(ds)-specific enzyme that processes diverse RNA precursors in bacterial cells to form the mature, functional forms that participate in protein synthesis and gene regulation. RNase III is involved in the maturation, turnover, and action of small noncoding RNAs (sRNAs), which play key roles in regulating bacterial gene expression in response to environmental inputs and changes in growth conditions. A mass-spectroscopy-based analysis of the E. coli proteome has shown that YmdB and RNase III interact in vivo. However, the functional importance of this interaction is not known. There is preliminary evidence that YmdB regulates RNase III activity during specific stress inputs. Thus, during cellular entry into stationary phase (nutrient limitation), or during the cold shock response, YmdB levels increase, which is correlated with a downregulation of RNase III activity. Inhibition of RNase III may alter the maturation and turnover of sRNAs, as well as other RNAs, during the adaptive response to stress. However, it is unclear whether the inhibition is a direct or indirect effect of YmdB on RNase III activity. Moreover, since YmdB binds ADPR, this (or related) metabolite may influence RNase III activity in an YmdB-dependent manner. If the YmdB-RNase III interaction in fact regulates RNase III, this interaction may connect post-transcriptional regulatory pathways with the cellular metabolic state, as reflected by NAD+ and ADPR levels. The goal of this project is to characterize the YmdB interaction with RNase III, with the long-range goal of understanding the mechanism and role of YmdB regulation of RNase III. Since both YmdB and RNase III are conserved bacterial proteins, characterization of YmdB and its influence on RNase III activity would provide insight on a conserved interaction in bacterial cells in general as well as reveal a potentially novel mechanism of post-transcriptional gene regulation.
Temple University--Theses

Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.

Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.

Les microARN sont de petits ARN non-codants qui jouent des rôles importants dans l’extinction de l’expression de gènes cibles. Ils sont impliqués dans la régulation de divers processus cellulaires, tel que la différenciation, la prolifération et l’apoptose. Jusqu'à présent, leur implication dans la biologie de la glande mammaire a été suggérée par quelques études portant principalement sur des situations pathologiques permettant la caractérisation des microARN comme marqueurs de différents types de tumeurs du sein. L'implication des microARN dans la régulation de la biologie normale de la glande mammaire reste à découvrir. Pour comprendre la fonction des microARN dans les différents stades physiologiques de la glande mammaire, nous avons développé trois approches : (i) L’identification de microARN spécifiques de la glande mammaire. Ces microARN ont été recherchés par la réalisation d’une banque de petits ARN. Au total 24 nouveaux microARN ont été clonés dont 6 sont spécifiques de la souris (Sdassi et al. , 2009). (ii) L’invalidation conditionnelle (système Cre-loxP) de Dicer, l'une des enzymes clés impliquées dans la maturation des microARN. L'inactivation a été principalement réalisée dans les cellules épithéliales mammaires par l'utilisation de deux lignées transgéniques MMTV-Cre et WAP-Cre croisées avec les souris Dicerfl/fl. Les souris Dicerfl/+/MMTV-Cre hétérozygotes présentent un défaut de lactation. Les observations histologiques montrent un défaut de développement de la glande mammaire détectable à partir de 6 jours de gestation. Les études transcriptomiques ont été effectuées afin de caractériser les voies de signalisation affectées. Les souris Dicerfl/fl/WAP-Cre présentent également un défaut de la lactation. Les études histologiques montrent des anomalies de la glande mammaire de ces souris en fin de lactation. (iii) La caractérisation de l’expression des microARN à différents stades physiologiques de la glande mammaire de souris (Silveri et al. , 2006; Sdassi et al. , 2009). Le rôle de l'un d'entre eux (miR-30b) a été recherché à partir de l’analyse de souris transgéniques qui surexpriment ce microARN dans les cellules épithéliales mammaires. Les femelles présentent un défaut de la lactation. Les analyses histologiques n’ont pas montré d’anomalies de développement pendant la gestation ou la lactation ; en revanche, des défauts du remodelage de la glande mammaire au Les microARN sont de petits ARN non-codants qui jouent des rôles importants dans l’extinction de l’expression de gènes cibles. Ils sont impliqués dans la régulation de divers processus cellulaires, tel que la différenciation, la prolifération et l’apoptose. Jusqu'à présent, leur implication dans la biologie de la glande mammaire a été suggérée par quelques études portant principalement sur des situations pathologiques permettant la caractérisation des microARN comme marqueurs de différents types de tumeurs du sein. L'implication des microARN dans la régulation de la biologie normale de la glande mammaire reste à découvrir. Pour comprendre la fonction des microARN dans les différents stades physiologiques de la glande mammaire, nous avons développé trois approches : (i) L’identification de microARN spécifiques de la glande mammaire. Ces microARN ont été recherchés par la réalisation d’une banque de petits ARN. Au total 24 nouveaux microARN ont été clonés dont 6 sont spécifiques de la souris (Sdassi et al. , 2009). (ii) L’invalidation conditionnelle (système Cre-loxP) de Dicer, l'une des enzymes clés impliquées dans la maturation des microARN. L'inactivation a été principalement réalisée dans les cellules épithéliales mammaires par l'utilisation de deux lignées transgéniques MMTV-Cre et WAP-Cre croisées avec les souris Dicerfl/fl. Les souris Dicerfl/+/MMTV-Cre hétérozygotes présentent un défaut de lactation. Les observations histologiques montrent un défaut de développement de la glande mammaire détectable à partir de 6 jours de gestation. Les études transcriptomiquesont été effectuées afin de caractériser les voies de signalisation affectées. Les souris Dicerfl/fl/WAP-Cre présentent également un défaut de la lactation. Les études histologiques montrent des anomalies de la glande mammaire de ces souris en fin de lactation. (iii) La caractérisation de l’expression des microARN à différents stades physiologiques de la glande mammaire de souris (Silveri et al. , 2006; Sdassi et al. , 2009). Le rôle de l'un d'entre eux (miR-30b) a été recherché à partir de l’analyse de souris transgéniques qui surexpriment ce microARN dans les cellules épithéliales mammaires. Les femelles présentent un défaut de la lactation. Les analyses histologiques n’ont pas montré d’anomalies de développement pendant la gestation ou la lactation ; en revanche, des défauts du remodelage de la glande mammaire au Les microARN sont de petits ARN non-codants qui jouent des rôles importants dans l’extinction de l’expression de gènes cibles. Ils sont impliqués dans la régulation de divers processus cellulaires, tel que la différenciation, la prolifération et l’apoptose. Jusqu'à présent, leur implication dans la biologie de la glande mammaire a été suggérée par quelques études portant principalement sur des situations pathologiques permettant la caractérisation des microARN comme marqueurs de différents types de tumeurs du sein. L'implication des microARN dans la régulation de la biologie normale de la glande mammaire reste à découvrir. Pour comprendre la fonction des microARN dans les différents stades physiologiques de la glande mammaire, nous avons développé trois approches :(i) L’identification de microARN spécifiques de la glande mammaire. Ces microARN ont été recherchés par la réalisation d’une banque de petits ARN. Au total 24 nouveaux microARN ont été clonés dont 6 sont spécifiques de la souris (Sdassi et al. , 2009). (ii) L’invalidation conditionnelle (système Cre-loxP) de Dicer, l'une des enzymes clés impliquées dans la maturation des microARN. L'inactivation a été principalement réalisée dans les cellules épithéliales mammaires par l'utilisation de deux lignées transgéniques MMTV-Cre et WAP-Cre croisées avec les souris Dicerfl/fl. Les souris Dicerfl/+/MMTV-Cre hétérozygotes présentent un défaut de lactation. Les observations histologiques montrent un défaut de développement de la glande mammaire détectable à partir de 6 jours de gestation. Les études transcriptomiques ont été effectuées afin de caractériser les voies de signalisation affectées. Les souris Dicerfl/fl/WAP-Cre présentent également un défaut de la lactation. Les études histologiques montrent des anomalies de la glande mammaire de ces sourisen fin de lactation. (iii) La caractérisation de l’expression des microARN à différents stades physiologiques de la glande mammaire de souris (Silveri et al. , 2006; Sdassi et al. ,2009). Le rôle de l'un d'entre eux (miR-30b) a été recherché à partir de l’analyse de souris transgéniques qui surexpriment ce microARN dans les cellules épithéliales mammaires. Les femelles présentent un défaut de la lactation. Les analyseshistologiques n’ont pas montré d’anomalies de développement pendant la gestation ou la lactation ; en revanche, des défauts du remodelage de la glande mammaire au cours de l’involution sont détectés chez ces souris. Les études du transcriptome ont permis d’identifier des gènes potentiellement impliqués dans ce phénotype. Nos résultats montrent pour la première fois l'implication des microARN dans la biologie de la glande mammaire normale. Au cours de ces études, nous avons produit plusieurs modèles animaux qui permettent d’identifier la fonction des microARN et les cibles de l’un d’entre eux (miR-30b) dans cet organe
MicroRNA are small non-coding RNA that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Few studies have described their implication in mammary gland biology, mainly focusing on pathological situations allowing the characterization of microRNA as markers of tumour class in breast cancer. The involvement of microRNA in the regulation of normal mammary gland biology remains to be uncovered. To understand the function of microRNA in the different steps of mammary gland biology we developed three approaches: 1/ Identification of organ- and tissue- (testicles) specific microRNA suggest the existence of specific microRNA in the mammary gland. These microRNA have been investigated by creating a bank of small RNA. Twenty four new microRNA were cloned, of which 6 are specific for the mouse (Sdassi et al. , 2009). The expression profiles of these new microRNA were analysed by qRT-PCR, to allow for better characterization. 2/ Conditional invalidation (system Cre-loxP) of Dicer, one of the key enzymes involved in the microRNA maturation. The inactivation is achieved mainly in the mammary epithelial cells by the use of an MMTV-Cre and WAP-Cre transgenic lines crossed with Dicerfl/fl mice. The heterozygote Dicerfl/+/MMTV-Cre mice present a defect of lactation. Histological observations show a default of mammary gland development detectable from 6 days of gestation onwards. Transcriptomic studies will be conducted to further characterize the affected signaling pathways. The Dicerfl/fl/WAP-Cre KO mice also exhibit a defect of lactation. The histological studies show abnormalities in mammary gland development at 18 days lactation. The genes regulated by microRNA in this model will be characterized by transcriptomic studies. 3/ The characterization of microRNA expression patterns at different physiological stages of the mammary gland development in mice has been described (Silveri et al. , 2006; Sdassi et al. , 2009). The role of one of these microRNA (miR-30b) is currently being analyzed by studying the phenotype of transgenic mice which over express this microRNA in mammary epithelial cells. The females present a defect of lactation associated with a default of this tissue morphology that is observed from the end of gestation onwards. Transcriptomic studies are underway to identify signaling pathways involved in this phenotype as well as the targets of this microRNA in the mammary gland. However, histological analysis did not show any developmental abnormalities associated withdefects of lactation. A remodeling defect of the mammary gland was found in these mice during involution. Transcriptome analysis has identified genes potentially involved in this phenotype. Our results demonstrate for the first time the involvement of microRNA in normal mammary gland biology and have generated animal tools that will help the understanding of microRNA function and targets in this organ

Le Post-Transcriptional Gene Silencing (PTGS) est un mécanisme dirigé contre les acides nucléiques invasifs endogènes (transposons) et exogènes (pathogènes, transgènes). Dans le cas des virus, le PTGS peut s’attaquer aux ARNs double-brin (dsRNAs) intermédiaires de la réplication virale et aux ARNs simple-brin viraux, mais il est souvent inhibé par des protéines virales appelées Viral Suppressor of RNAi (VSR). Chez la plante modèle Arabidopsis thaliana, une enzyme appelée RNase THREE-LIKE 1 (RTL1) est induite en réponse à l'infection virale et détruit les dsRNAs de manière non sélective. Cette enzyme devrait assurer à la plante une seconde ligne de défense en clivant les dsRNAs viraux, mais les VSR qui inhibent le PTGS inhibent généralement RTL1, indiquant que les virus ont mis en place des outils capables de combattre simultanément ces deux mécanismes de défense. Toutefois, un virus, le Turnip yellow mosaic virus (TYMV), est incapable d’inhiber RTL1 et semble même tirer profit de RTL1 pour réussir à infecter A. thaliana (Shamandi et al., 2015).Au cours de cette thèse, nous avons approfondi l’étude de l’interaction Arabidopsis-TYMV. Nous avons montré que le TYMV est incapable d’inhiber l’exécution du PTGS, mais est toutefois capable d’inhiber l’étape d’amplification du PTGS. Cette action est due à la protéine virale P69, et nous avons montré que P69 est retrouvée dans des corpuscules cytoplasmiques appelés siRNA-bodies qui sont le siège de l’amplification du PTGS. Par ailleurs, nous avons généré des mutants rtl1 et montré que l’absence de RTL1 retarde l’infection par le TYMV et augmente la quantité de siRNAs dirigés contre le virus, tandis que la surexpression de RTL1 favorise l’infection et inhibe la production des siRNAs anti-viraux. Nous avons observé que RTL1 était retrouvée dans les siRNA-bodies, et nous avons montré que RTL1 était capable de détruire non seulement les dsRNAs mais également les siRNAs. Ces résultats indiquent donc que le TYMV réussit à infecter A. thaliana en : i) se répliquant dans des invaginations de la membrane chloroplastique (Prod’homme et al., 2003) qui mettent vraisemblablement les dsRNAs intermédiaires de la réplication à l’abri du PTGS et de RTL1, ii) en induisant l’expression de RTL1 qui s’attaque aux dsRNAs et siRNAs induits par le PTGS dans les siRNA-bodies en réponse à l’infection, et iii) en exprimant la protéine P69 qui complète l’action de RTL1 en inhibant l’amplification du PTGS résiduel.Malgré un effet neutre ou négatif pour la plante vis-à-vis des virus, RTL1 est conservé dans toutes les accessions d’Arabidopsis, et l’étude du ratio des mutations synonymes et non synonymes dans les gènes RTL1 de 42 Eucotylédones suggère que RTL1 subit une pression de sélection de conservation, suggérant un rôle essentiel. Chez A. thaliana, RTL1 est exprimé faiblement dans la racine, les tissus en sénescence, et au cours du développement de la graine. Le phénotypage des plantes sauvages et des mutants rtl1 n’a pas révélé de différences morphologiques notables, mais nous avons observé que le poids des graines était supérieur chez les mutants rtl1. Par ailleurs, nous avons observé une senescence accrue chez le mutant rtl1, en particulier dans l’accession Ler. Cette différence entre Ler et Col nous a poussé à examiner si RTL1 pouvait contribuer à la variabilité naturelle du PTGS des transgènes entre les accessions Ler (PTGS peu efficace) et Col (PTGS très efficace). Nous avons observé que la mutation rtl1 n’affectait pas sensiblement l’efficacité du PTGS chez Col mais augmentait celle de Ler au niveau de Col, ce qui pourrait s’expliquer par une plus forte expression de RTL1 chez Ler que chez Col. L’effet de l’absence de RTL1 devra donc être précisé en condition normale et en condition d’infection en privilégiant Ler plutôt que Col
Post-Transcriptional Gene Silencing (PTGS) is a defense mechanism that targets invading nucleic acids of endogenous (transposons) or exogenous (pathogens, transgenes) origins. During virus infection, PTGS theoretically targets double-stranded (ds)RNA intermediates of viral replication and viral single-stranded RNAs; however, most viruses encode proteins, referred to as viral suppressor of RNAi (VSR), which inhibit PTGS. In the model plant Arabidopsis thaliana, an enzyme referred to as RNase THREE-LIKE 1 (RTL1) is induced in response to viral infection and cleaves dsRNAs in a non-specific manner. This enzyme should provide a second line of defense by cleaving viral dsRNAs, but VSR that inhibit PTGS generally inhibit RTL1, indicating that viruses had put in place tools that simultaneously counteract these two defense mechanisms. Nevertheless, at least one virus, Turnip yellow mosaic virus (TYMV), is not able to inhibit RTL1 and in fact seems to take advantage of RTL1 to successfully infect A. thaliana (Shamandi et al., 2015).In this thesis, we deepened the study of Arabidopsis-TYMV interaction. We show that TYMV is not able to inhibit PTGS execution but is able to inhibit PTGS amplification. This effect is due to the viral protein P69, and we show that P69 localizes in cytoplasmic foci called siRNA-bodies, where PTGS amplification takes place. Furthermore, using in house-generated rtl1 mutants, we show that the lack of RTL1 delays TYMV infection and promotes the production of siRNAs directed against the virus, whereas RTL1 overexpression enhances viral symptoms and suppresses the production of anti-viral siRNAs. We show that RTL1 is found in siRNA-bodies, and we show that RTL1 attacks not only dsRNAs but also siRNAs. These results indicate that, TYMV successfully infect A. thaliana by : i) replicating in chloroplast membrane invaginations (Prod’homme et al., 2003), which likely shelter dsRNAs intermediates of replication from PTGS and RTL1, ii) inducing RTL1 expression, which promotes the destruction of dsRNAs and siRNAs produced by PTGS in siRNA-bodies in response to TYMV infection, and iii) expressing the P69 protein to inhibit residual PTGS amplification.Despite a neutral or detrimental effect on plant anti-viral PTGS, RTL1 is conserved in all Arabidopsis accessions, and the study of synonymous and non-synonymous substitutions ratios in RTL1 genes from 42 dicotyledonous plant reveals that RTL1 is under the control of a conservative selection, suggesting an essential role. In A. thaliana, RTL1 is weakly expressed in roots, in senescent tissues and during seed development. Phenotyping wild-type plants and rtl1 mutants did not revealed any significant morphological differences, but we observed that seeds weight is enhanced in rtl1 mutants. Moreover, we observed an increased senescence in rtl1 mutants, in particular in the Ler accession. This difference between Ler and Col prompted us to determine if RTL1 could participate in the natural variability of transgene PTGS efficiency between Ler (weak PTGS) and Col (strong PTGS). We observed that rtl1 mutations have no significant effect on PTGS efficiency in Col, but enhances PTGS efficiency in Ler, up to the level of Col, which could be explained by a strongest RTL1 expression in Ler compared to Col. These results indicate that the effect of RTL1 impairment should be further examined in normal and infectious contexts by focusing on Ler rather than Col

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from premicroRNA. My thesis focuses on the functional characteristics of two Drosophila Dicers that makes them specific for their biological substrates. We found that RNA binding protein partners of Dicers and two small molecules, ATP and phosphate are key in regulating Drosophila Dicers’ specificity. Without any additional factor, recombinant Dicer-2 cleaves pre-miRNA, but its product is shorter than the authentic miRNA. However, the protein R2D2 and inorganic phosphate block pre-miRNA processing by Dicer-2. In contrast, Dicer-1 is inherently capable of processing the substrates of Dicer, long dsRNAs. Yet, partner protein of Dicer-1, Loqs-PB and ATP increase the efficiency of miRNA production from pre-miRNAs by Dicer-1, therefore enhance substrate specificity of Dicer-1. Our data highlight the role of ATP and regulatory dsRNA-binding partner proteins to achieve substrate specificity in Drosophila RNA silencing. Our study also sheds light onto the function of the helicase domain in Drosophila Dicers. Although Dicer-1 doesn’t hydrolyze ATP, ATP enhances miRNA production by increasing Dicer-1’s substrate specificity through lowering its KM. On the other hand, Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP, and ATP hydrolysis is required for Dicer-2 to process long dsRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, is processive; generating siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate. Piwi-dependent small RNAs, namely piRNAs, are a third class of small RNAs that are distinct from miRNAs and siRNAs. Their primary function is to repress transposons in the animal germline. piRNAs are Dicer-independent, and require Piwi family proteins for their biogenesis and function. Recently in addition to their presence in animal germlines, the presence and function of piRNA-like RNAs in the somatic tissues have been suggested (Yan et al. 2011; Morazzani et al. 2012; Rajasethupathy et al. 2012). We have investigated whether the piRNA-like reads in our many Drosophila head libraries come from the germline as a contaminant or are soma-specific. Most of the piRNA reads in our published head libraries show high similarity to germline piRNAs. However, piRNA-like reads from manually dissected heads are distinct from germline piRNAs, proving the presence of somatic piRNA-like small RNAs. We are currently asking the question whether these distinct piRNA-like reads in the heads are dependent on the Piwi family proteins, like the germline piRNAs.

Je me suis intéressé au système de défense anti-viral majeur de Drosophila melanogaster qui est la voie du RNA silencing (siRNA). A ce jour, le seul senseur d’acide nucléique viral et activateur de la voie siRNA est Dicer-2. Ainsi, le travail que j’ai effectué́ a permis d’apporter de nouvelles informations concernant la détection des ARNs viraux par Dicer-2. L’utilisation de méthodes de séquençage à haut débit (HTS) des petits ARNs dans des cellules S2 infectées par le Drosophila C Virus (DCV) à des temps précoces m’a permis de proposer un point d’entrée précis et interne de Dicer-2 sur l’ARN double brin de ce dicistrovirus. La validation de ce point faible dans la défense du virus a été effectuée en réalisant un HTS des petits ARNs dans des mouches de différents génotypes infectées avec DCV. J’ai ensuite caractérisé plus en profondeur cette région du génome virale en déterminant tout d’abord sa structure 2D puis sa sensibilité à des clivages médiés par des extraits embryonnaires de mouches. Finalement, l’utilisation de différents variants de Dicer-2 présentant des mutations du domaine DRA m’a permis de proposer un nouveau mécanisme de fonctionnement de cette protéine
My Ph.D revolved around the study of the major antiviral defense system of Drosophila melanogaster: the siRNA pathway. To date, the only viral nucleic acid sensor and siRNA pathway activator in drosophila is Dicer-2. Thus, the work I have done has provided new information regarding the detection of viral RNAs by Dicer-2. The use of high throughput sequencing (HTS) methods of small RNAs in S2 cells infected with Drosophila C Virus (DCV) at early time points has allowed me to propose a precise and internal entry point for Dicer-2 on the double-stranded RNA of this dicistrovirus. The validation of this weak point in the defence of the virus was carried out by performing an HTS of small RNAs in flies of different genotypes infected with DCV. I then characterized this region of the viral genome in more depth by first determining its 2D structure and then its sensitivity to cleavages mediated by embryonic fly extracts. Finally, the use of different variants of Dicer-2 with mutations in the DRA domain allowed me to propose a new mechanism of action for this protein

Les mélanocytes protègent lʼorganisme contre les effets délétères des rayonnements UV, en synthétisant de la mélanine. Leur transformation maligne entraine le développement de mélanomes, cancers particulièrement agressifs et résistants aux traitements. De nombreuses protéines, telles que BRN2, β-caténine et Dicer, impliquées dans divers processus cellulaires, sont dérégulées dans le mélanome. Cette thèse est consacrée à lʼimportance du statut de phosphorylation de BRN2 pour la prolifération et la migration du lignage mélanocytaire et à lʼimplication de BRN2, β-caténine et Dicer dans la réponse des mélanocytes à lʼexposition aux rayonnements UV. Premièrement, nous avons montré que les formes non phosphorylées (BRN2AA) et phosphorylées (BRN2TS) de BRN2 conduisent à une régulation différente de la prolifération et de la migration des mélanocytes. De plus, nous avons étudié la liaison et la transactivation de BNRN2AA et BRN2TS à MITF et PAX3, deux cibles de BRN2 impliquées dans la mise en place et la transformation du lignage mélanocytaire. La transcription de MITF est réprimée de façon similaire par les deux formes de BRN2, cependant, la transcription de PAX3 est induite par BRN2TS mais réprimée par BRN2AA. La migration et la prolifération des mélanocytes sont donc contrôlées par le statut de phosphorylation de BRN2 via PAX3 et par la quantité totale de BRN2 via MITF. Deuxièmement, nous avons évalué la possible régulation de BRN2 suite à une stimulation UV des mélanocytes. Nous avons démontré que l'expression de BRN2 est régulée au niveau transcriptionnel mais aussi au niveau du transcrit après irradiation UV. Par ailleurs, nous avons montré que BRN2 contrôle la transcription de Dicer et que BRN2 et Dicer créent une boucle de rétroaction positive qui régule l'expression des deux gènes. Troisièmement, nous nous sommes intéressés à lʼimportance de β-caténine dans la régulation de lʼexpression de Dicer lors de la réponse des mélanocytes à lʼexposition aux rayons UV. Nous avons montré que lʼexposition des mélanocytes aux rayons UV conduit à (i) lʼinhibition de lʼexpression de Dicer associé (ii) à la stabilisation et lʼaccumulation de β-caténine dans le noyau et (iii) lʼinduction de la pigmentation due à la répression de Dicer. Tous ces événements représenteraient dʼimportants processus de melanogenèse suite à une exposition aux rayonnements UV
Melanocytes protect the organism against the deleterious effects of UV rays by synthesizing melanin. The malignant transformation of these cells leads to melanoma, a particularly aggressive cancer refractory to treatment. Several proteins, such as BRN2, β-catenin and Dicer, involved in various cellular processes, were shown to be misregulated in melanoma. This PhD thesis is focused on the importance of BRN2 phosphorylation status on the proliferation and migration of the melanocyte lineage and on the implication of BRN2, β-catenin and Dicer in the response of melanocytes to UV-exposure. First, we showed that the non-phosphorylable (BRN2AA) and phosphorylable (BRN2TS) form of BRN2 leads to differential control of proliferation and migration of melanocyte lineage. Furthermore, we investigated the binding and transactivation of BRN2AA and BRN2TS on MITF and PAX3, two targets of BRN2 involved in the establishment and transformation of the melanocyte lineage. Both BRN2 forms similarly repress MITF transcription, whereas PAX3 transcription is induced by BRN2TS but repressed by BRN2AA. Altogether, melanocyte migration and proliferation are controlled by the BRN2 phosphorylation status through PAX3 and by the total BRN2 level through MITF. Second, we evaluated the possibility of BRN2 regulation following UV-stimulation of melanocytes. We brought evidence that BRN2 expression is regulated at transcriptional and transcript level following UVB irradiation. Furthermore, we showed that BRN2 controls Dicer transcription and that BRN2 and Dicer create a feedback loop that regulates both the expression of the BRN2 and Dicer genes. Third, we considered the importance of β-catenin on the regulation of Dicer expression in the response of melanocytes to UV exposure. We showed that the exposure of melanocytes to UV light leads to (i) the inhibition of Dicer expression associated with (ii) β-catenin stabilization and accumulation into the nucleus, and (iii) induction of pigmentation as aresult of Dicer repression. Altogether these events may represent important melanogenesis processes after UV light induction

博士
國立臺灣大學
生化科學研究所
90
Proteins possess the ultimate roles of executing biological processes, and more research efforts are directed toward functional and structural analysis of these macromolecules. Among a myriad of laboratory techniques, biophysical methods are mainly used for dissecting the properties and structures of proteins. From the structural information, structural-functional relationship can then be established. Studies on three different types of macromolecules, fusion peptide, snake toxin, and cytotoxic ribonucleases, using combinations of various fundamental or advanced biophysical techniques, have been carried out. The results are presented in this thesis. Part I Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point-mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu2-Glu11 and Trp14-Ile18, form amphipathic helical conformations, with Gly12-Gly13 forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp14-Ile18 reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion-peptide analog and explains how the analog interacts with the lipid bilayer at different pH values. Part II Bungarus fasciatus IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of Bungarus fasciatus by ion exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10-8 M with a-chymotrypsin as measured by a Langmuir binding isotherm using BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We have further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The 3D NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculation. The final set of 10 NMR structures was exceptionally well defined with average RMSD of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A in residues 3-58. Based on the 3D structure, we identified that the unusual chemical shifts observed for BF9 resulted from an aromatic ring current effect. The side chains of Phe23, Ty24, Tyr25, Phe35 and Phe47 exhibited many long-range NOEs and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among the proteins in the BPTI-like superfamily, we compared the 3D structure of BF9 with three BPTI-like proteins that possess distinct biological functions. We found that these proteins possessed similar secondary structure elements, but the loop regions and b-turn were different from one another. Based on residues at the functional site of each protein, we suggested that the flexibility, the rigidity, and the variations of the amino acid residues in both the loop and the b-turn regions are related to their biological functions. Part III Cytotoxic ribonucleases, isolated from oocyte and liver of bullfrog Rana catesbeiana, possess different base specificities, ribonucleolytic activities and cytotoxicities. To gain insight into the structure/function relationships on these ribonucleases, we cloned, expressed and purified recombinant as well as mutant proteins for several biophysical studies. CD experiments are used to check conformational stability, secondary structure and structural change at different pH values. Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) techniques are used for binding studies. NMR studies, including structure determination, protein folding, and dynamics, are carried out using native and 15N and/or 13C-labeled proteins. The following results will be presented. (1) Three dimensional solution structures of recombinant RC-RNase 2 and RC-RNase 4 have been determined. (2) Based on the biophysical data, we concluded that the reduction in catalytic and cytotoxic activities for the recombinant protein, which contains an extra Met residue and has a Gln instead of pyroglutamate at the N-terminus, are mainly due to the loss of two H-bonds in the N-terminal Gln residue. (3) Using RC-RNase 4''s mutant proteins, we confirmed that Trp15 plays an important role in its high thermostability and causes a unique characteristic of CD data with an additional ellipticity minimum at 228 nm. (4) The substrate-related residues in the base specificity of CpG vs. UpG among RC-RNase, RC-RNase 2 and RC-RNase 4 are derived using the NMR chemical shift mapping between free- and complex- structures. In addition, the binding characteristic based on SPR and ITC experiments were performed. (5) The complex model of ribonuclease inhibitor (RI) and RC-RNases model revealed that RC-RNases evade RI and catalyze cleavage of cellular RNA, which leads to cell death.