Thèse ou mémoire avec insertion d'articles. / Ce ne sont pas moins de 98% de notre ADN qui constituent la partie non-codante du génome. D'abord qualifiée d'ADN « camelote », il a plus tard été identifié que cette portion de notre patrimoine génétique est caractérisée par une structure particulière de la chromatine associée aux fonctions régulatrices à l'origine de l'expression des programmes génétiques nécessaires à l'établissement de la myriade des types cellulaires composant notre organisme. Ce code régulateur constitue l'épigénome qui est une composante indispensable à l'expression organisée de l'exome qui représente la portion codante de notre ADN; il a par exemple été identifié certaines régions régulatrices appelées « enhancers » qui possèdent la capacité d'amplifier l'expression de gènes codants localisés à distance via une courbure de la chromatine permettant le rapprochement géographique de régions éloignées du génome. De cette façon si chaque cellule qui nous compose présente au sein de leur noyau une séquence d'ADN rigoureusement identique, c'est la manière dont le génome s'organise qui définit l'identité et le destin cellulaire. Cette organisation obéit à une hiérarchie à l'origine de la régulation fine de l'expression des gènes et pour laquelle une quelconque perturbation peut avoir des conséquences néfastes sur l'homéostasie cellulaire ou tissulaire. Ces dernières années la réduction drastique des couts de séquençage du génome humain a permis de développer de nombreuses techniques nous offrant l'opportunité de déchiffrer les impacts des variations génétiques non-codantes sur les phénotypes : de cette façon l'investigation des polymorphismes d'un seul nucléotide comme source de variation (SNPs), la capture de l'organisation tridimensionnelle du génome, ou encore la mesure de l'expression de gènes codants ou non-codants à l'échelle tissulaire ou de la cellule unique sont autant d'outils qui permettent potentiellement de décrypter le langage non-codant associé à l'établissement des phénotypes. En revanche la complexité des données générées par ces types d'approches ainsi que les défis que représente une intégration multidimensionnelle nécessite de développer des algorithmes rigoureux aussi bien analytiques qu'expérimentaux pour extraire les informations clés qui permettront de comprendre les phénotypes. Ce travail de doctorat visait à déchiffrer les conséquences de certaines variations génétiques non-codantes associées à la longévité et au rétrécissement aortique calcifié pour comprendre les mécanismes reliés à ces phénotypes par le développement d'approches holistiques intégrant une dualité expérimentale et analytique. À partir de données d'associations génétiques pour la longévité et d'expression de gènes à l'échelle tissulaire et de la cellule unique et grâce à l'utilisation d'approches évaluant la causalité d'associations par randomisation Mendélienne, il a été ainsi démontré que le tissu sanguin joue un rôle important dans la longévité par le biais de processus reliés à l'immunité innée et adaptative, et également que l'expression de 16 gènes dans le sang est associée à l'établissement de la trajectoire de la longévité directement ainsi que par le biais de certaines maladies. En utilisant la cartographie génétique tridimensionnelle et des informations relatives à la structure de la chromatine, il a d'autre part été possible durant ce doctorat de comprendre la régulation génétique au locus 1p21.2 associé au rétrécissement aortique calcifié et à l'expression de la palmdelphine dans la valve aortique, puis en complément grâce à des expériences *in vitro* dans des cellules de valve aortique issues de patients d'identifier un rôle de la palmdelphine dans la fibrose, un processus important dans la pathogenèse de cette maladie. Enfin, l'exploitation conjointe de données relatives à l'organisation tridimensionnelle du génome, à la structure de la chromatine et à l'expression de gènes codants et non-codants dans la valve aortique a aussi permis d'identifier que l'ARN long non-codant LINC01013 dérive d'un « super-enhancer » et est impliqué dans le développement de fibrose dans le rétrécissement aortique calcifié en contrôlant l'expression de *CCN2*, un gène important pour le maintien de l'homéostasie de la matrice extracellulaire de nombreux tissus. Ce travail de doctorat a donc renforcé l'importance du rôle des régions non-codantes dans le maintien de l'homéostasie et de l'intégrité des phénotypes en déchiffrant les conséquences de variations génétiques non-codantes associées à la longévité et au rétrécissement aortique calcifié. D'autre part, celui-ci a souligné la puissance d'approches holistiques intégrant une dualité expérimentale et analytique par le développement de nouveaux algorithmes qui ont permis d'identifier des mécanismes reliés à l'acquisition de ces phénotypes qui demeuraient jusqu'alors inconnus.

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

Made available in DSpace on 2016-06-02T20:21:28Z (GMT). No. of bitstreams: 1 4218.pdf: 11765337 bytes, checksum: cdae8703c8d7792c7037dde48c22c345 (MD5) Previous issue date: 2012-04-04 / Financiadora de Estudos e Projetos / Plant cell wall confers to the cell plant, structural support as well as protection against pathogens and phytophagous. Among the cell wall polysaccharides includes pectic substances, which are composed of partially methyl-esterified galacturonic acid residues linked by α-1,4 glycosidic bonds. These enzymes are often synthesized by phytophatogenic micro-organisms for invasion of host plant or by own plant for modeling plant cell wall. The pectic substances are the major component of middle lamella and are natural degraded by pectinases action. Pectin methylesterase (PME) catalysis removes methyl-ester groups, and the Endo-polygalacturonase (Endo-PG) promoves the randomly hydrolysis reaction of α-1,4 bonds. One of the most importante agricultural pest species of the family Curculionidae (Coleoptera: Curculionidae) is Sphenophorus levis, the sugarcane weevil. The larvae of this insect penetrate into the rhizome and build galleries in the stem, decreasing productivity and causing the death of the plant. Large damages to the crop like that are significant in the costs of products derived from sugarcane. Considering the impact of this pest in the sugarcane crop and the absence of efficient method for control, new strategies for controlling are still necessary. The analysis of the cDNA library of S. levis larvaes shows the presence of one PME and one Endo-PG genes that we called Sl-PME and Sl-EndoPG respectively. Considering the importance of studies of insect pests and the extensively use of theses pectinases in different industry fields, we performed the characterization of genomic sequences coding for S. levis pectinases (Sl-Pectinases). It was also carried out the production and characterization of a Sl-PME and a Sl-EndoPG recombinant, expressed in heterologous system. We also accomplished analysis of gene expression by qRTPCR in different stages of development as well as different tissues, and phylogenetic studies between Sl-Pectinases and other pectinases from different kingdoms. The Sl- Pectinases sequences identified, show more similar to homologous insect genes deposited in the GenBank, especially with Sitophilus oryzae. The phylogenetic analysis indicates that the insect group is more correlated with bacteria group and fungi group respectively to PMEs and EndoPGs sequences. Pectinases genomic sequences revealed two introns for Sl-EndoPG gene with 53 and 166 bp, but no one for Sl-PME gene. Both of Sl-Pectinases Recombinant showed catalytic activity. The recombinant Sl-EndoPG shows optimal activity at pH 5,06 ± 0,27 and 49,74 ± 2,49 oC, but extremely low thermostability. For the polygalacturonic acid no-methylated as substract, the enzyme revealed Km = 3,88 mg.mL-1, Vmax = 21.96 μM.s-1 e Kcat = 3.137 s-1; for the citrus pectin partially methylated as substract, the enzyme presented Km = 4,98 mg.mL-1, Vmax = 17,19 μM.s-1 e Kcat = 2.456 s-1. Results in expression analysis suggest that S. levis pectinases have a digestive enzymes role, actting on the midgut. The present work represents the first pectinases of insect produced in Pichia pastoris heterologous system and characterized as optimal conditions of activity, thermostability and kinetic parameters. / A parede celular vegetal confere à célula vegetal suporte estrutural, proteção contra patógenos e fitófagos. Dentre os polissacarídeos da parede celular vegetal são inclusas as substâncias pécticas, as quais são compostas por resíduos de ácido galacturônico, parcialmente esterificados, ligados em série via ligações glicosídicas α- 1,4. As substâncias pécticas são o maior componente da lamela média e são naturalmente degradadas pela ação enzimática das pectinases. A Pectina metilesterase (PME) catalisa a remoção dos grupos metil-ester, e a Endo- Poligalacturonase (Endo-PG) promove a reação de hidrólise aleatória das ligações α- 1,4. Essas enzimas são comumente sintetizadas por micro-organismos fitopatógenos para invasão ao hospedeiro ou pelas próprias plantas para modelamento da parede celular vegetal. Na agricultura, uma das mais importantes espécies pragas da família Curculionidae (Coleoptera: Curculionidae) é o Sphenophorus levis, o bicudo da canade- açúcar. As larvas deste inseto penetram no rizoma e constroem galerias ao longo do colmo, causando queda na produtividade ou até mesmo a morte da planta. Grandes danos na cultura como esses são significativos nos custos de produtos derivados da cana-de-açúcar. Considerando o impacto dessa praga na cultura e a ausência de eficientes métodos de controle, novas estratégias ainda são necessárias no combate à praga. A análise de uma biblioteca de cDNA de larvas do inseto S. levis mostrou a presença de genes codificantes para uma PME e uma Endo-PG, os quais nomeamos de Sl-PME e Sl-EndoPG respectivamente. Devido a importância nos estudos de insetos pragas e a extensa aplicação das pectinases em diversos campos industriais, foi promovida a caracterização das sequências genômicas codificantes para as pectinases de S. levis (Sl-Pectinases). Também foi realizada a produção e caracterização de uma Sl-PME e uma Sl-EndoPG recombinantes, expressas em sistema heterólogo. Além disso, foram conduzidas análises de expressão gênica por qRT-PCR em diferentes estágios de desenvolvimento e diferentes tecidos; e estudos filogenéticos entre as Sl-Pectinases e outras pectinases de diferentes reinos. As sequências das Sl-Pectinases identificadas apresentaram maior similaridade com genes homólogos de insetos depositados no GenBank, principalmente como o Sitophilus oryzae. A análise filogenética indicou que o grupo dos insetos é mais correlacionado com o grupo das bactérias e com o grupo de fungos, respectivamente para as sequências PMEs e Endo-PGs. As sequências genômicas das pectinases revelaram dois introns para Sl-EndoPG com 53 e 166 pb, mas nenhum para o gene Sl- PME. Ambas as Sl-Pectinases Recombinantes apresentaram atividade catalítica. A Sl- EndoPG recombinante mostrou maior atividade em pH 5,06 ± 0,27 e 49,74 ± 2,49 oC, mas baixa termoestabilidade. Para o substrato ácido poligalacturônico não metilado, a enzima revelou Km = 3,88 mg.mL-1, Vmax = 21.96 μM.s-1 e Kcat = 3.137 s-1, para o substrato pectina de citrus parcialmente metilada, a enzima apresentou Km = 4,98 mg.mL-1, Vmax = 17,19 μM.s-1 e Kcat = 2.456 s-1. Os resultados da análise de expressão sugerem que as pectinases de S. levis são enzimas digestivas atuantes no intestino médio. Este trabalho representa as primeiras pectinases de inseto produzidas em sistema heterólogo de Pichia pastoris e caracterizadas quanto a condições ótimas de atividade, termoestabilidade e parâmetros cinéticos.

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "July 1998." Typescript. Vita. Includes bibliographical references (leaves 215-225). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-130). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-130). Also available on the Internet.

Oocyte activation deficiency (OAD) is an infertile condition observed in patients who have experienced recurrent total fertilisation failure (TFF) following intracytoplasmic sperm injection treatment. This condition was considered to be an idiopathic factor for a long time but strong clinical evidence now suggests that dysfunctional forms of phospholipase C zeta (PLCζ) may be predominant causative factors for OAD. Genetic contribution has played a role in patients suspected of having OAD, as four PLCζ exonic mutations have been discovered and characterised as being the cause of infertility. In this study, a novel nonsense mutation, PLCζK322Stop, was identified in the PLCζ XY-linker region of Patient LR. This variant results in the truncation of approximately half of PLCζ, therefore was non-functional when activity was tested. Patient LR, which also exhibited a previously reported mutation, PLCζH233L, may suggest that the patient is sub-fertile, as opposed to being infertile, as initially expected. Although research has purely focused upon the coding regions of PLCζ, it was obvious that our knowledge of PLCζ regulatory elements remain very limited. Next generation sequencing (NGS) was therefore employed to detect variants in the non-coding regions of PLCζ, promoter and introns, which may have resulted in the observed phenotypic diversity of PLCζ expression in fertile and infertile patients. As a result of mapping failure, an alternative approach was considered to identify variants within human PLCζ, and this involved using the single nucleotide polymorphism (SNP) database. Over 2500 SNPs were localised in the intronic regions of PLCζ and thus, it could be speculated that these variants may help elucidate the wide variation of PLCζ expression reported. Additionally, two particular patients with TFF (79 and 107) were investigated in this study to identify an association with PLCζ and their infertile state. For Patient 79, multiple PLCζ immunofluorescence analysis was performed and a significant improvement in PLCζ expression was observed one year after his first investigation. This may have been the result of an external factor, which influenced protein expression. As for Patient 107, a novel substitution mutation, PLCζV193E, was identified and was predicted to affect PLCζ stability and folding. There is global interest to create a safer and alternative OAD therapy, namely a human recombinant PLCζ protein (hrPLCζ). The first method, using a bacterial cell line resulted in successful purification and identification but the product proved to be inactive following mouse oocyte microinjection. The second method involved production of a mammalian-expressed hrPLCζ, which was successfully purified and identified but due to time restrictions, could not be tested for functionality. Concurrently, the findings in this thesis have reinforced the association between PLCζ and OAD, and provided improved options for the diagnosis and treatment of OAD.

xii, 83 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / My dissertation research has centered on understanding how nuclear encoded proteins affect chloroplast gene expression in higher plants. I investigated the functions of three proteins that belong to families whose members function solely or primarily in mitochondrial and chloroplast gene expression; the Whirly family (ZmWHY1) and the pentatricopeptide repeat (PPR) family (ZmPPR5 and ZmPPR10). The Whirly family is a plant specific protein family whose members have been described as nuclear DNA-binding proteins involved in transcription and telomere maintenance. I have shown that ZmWHY1 is localized to the chloroplast where it binds nonspecifically to DNA and also binds specifically to the atpF group II intron RNA. Why1 mutants show reduced atpF intron splicing suggesting that WHY1 is directly involved in atpF RNA maturation. Why1 mutants also have aberrant 23S rRNA metabolism resulting in a lack of plastid ribosomes. The PPR protein family is found in all eukaryotes but is greatly expanded in land plants. Most PPR proteins are predicted to localize to the mitochondria or chloroplasts where they are involved in many RNA-related processes including splicing, cleavage, editing, stabilization and translational control. Our results with PPR5 and PPR10 suggest that most of these activities may result directly from the unusually long RNA binding surface predicted for PPR proteins, which we have shown imparts two biochemical properties: site-specific protection of RNA from other proteins and site-specific RNA unfolding activity. I narrowed down the binding site for PPR5 and PPR10 to ∼45 nt and 19 nt, respectively. I showed that PPR5 contributes to the splicing of its group II intron ligand by restructuring sequences that are important for splicing. I used in vitro assays with purified PPR10 to confirm that PPR10 can block exonucleolytic RNA decay from both the 5' and 3' directions, as predicted by prior in vivo data. I also present evidence that PPR10 promotes translation by restructuring its RNA ligand to allow access to the ribosome. These findings illustrate how the unusually long RNA interaction surface predicted for PPR proteins can have diverse effects on RNA metabolism. This dissertation includes both previously published and unpublished co-authored material. / Committee in charge: Eric Selker, Chairperson, Biology; Alice Barkan, Advisor, Biology; Victoria Herman, Member, Biology; Karen Guillemin, Member, Biology; J. Andrew Berglund, Outside Member, Chemistry

Two of the most pervasive threats to species biodiversity are invasive species and habitat loss and degradation. Invasive species are often relatively insensitive to disturbance and many expand their range into disturbed and fragmented habitats. This dissertation uses an interdisciplinary approach to investigate how anthropogenic habitat disturbance is precipitating a range expansion in an invasive toad species, Bufo nebulifer, which is driving a decline in its native congener, B. fowleri. I employed a remote sensing and GIS study using historical data to compare changes in the two species distributions and habitat changes, a molecular genetic study to identify interspecific hybrids and their potential effects on the parental species, and an experimental ecology study to look at the effects of competition and predation on the two species. The results of the landscape level analyses of species' distributional changes in different disturbance levels showed that both species' distributions have changed significantly. The distributions of the two species are inversely affected by habitat disturbance; the distribution of B. fowleri in highly degraded habitat has contracted while the expansion of B. nebulifer increased substantially. The molecular genetic study successfully demonstrated the use of nuclear and mitochondrial markers to identify cryptic hybrids and their maternal lineage. Three hybrids were detected using nuclear introns and a morphologically cryptic hybrid was identified using mitochondrial DNA as the progeny of a cross that was previously thought to be inviable. Although relatively few hybrids were currently found, the identification of a cryptic hybrid implies that the rate of historical hybridization may have been drastically underestimated. Ecological studies showed that competition with B. nebulifer tadpoles had a negative effect on both body size measures and survival to metamorphosis for B. fowleri tadpoles. The addition of predators to experiment did not favor the survival of B. fowleri over B. nebulifer. Bufo fowleri's inability to compete with its invasive congener could be a driving mechanism for the decline of B. fowleri and the expansion of B. nebulifer. The methods discussed in this dissertation offer promising and practical new approaches for evaluating and managing changes in the distribution of species of conservation concern.