<i>Hevea brasiliensis </i>latex contains a large quantity of high molecular weight rubber and is thus the primary commercial source of natural rubber. Rubber and other non-rubber isoprenoids in <i>Hevea </i>latex are synthesised from isopentenyl diphosphate (IPP) generated from the cytoplasmic mevalonate (MVA) pathway and the plastidic methyl erythritol phosphate pathway (MEP). This study utilised two rubber tree clones (RRIM600 and PB235) that show visibly contrasting levels of yellow carotenoids for the measurement of latex isoprenoids (carotenoids, rubber and isoprenoid intermediates) and transcript levels of the genes involved in isoprenoid biosynthesis. Metabolite extraction and analysis showed that four major carotenoids namely lutein, zeaxanthin, α-carotene and β-carotene were consistently present in both RRIM600 and PB235 latex. β-carotene was found to be the major carotenoid, at 1.2 μg/g in PB235 and 0.8 μg/g in RRIM600 fresh latex samples. However, the analytical method developed to measure isoprenoid intermediates needed to be further optimised to increase extraction efficiency. To enable accurate measurement of transcript levels of key genes involved in the isoprenoid biosynthetic pathway, a set of reference transcripts was constructed by merging short-reads (RNA-seq) and long-reads (Iso-seq and full-length cDNA sequences) data from <i>Hevea brasiliensis</i>. This produced a comprehensive set of 193,997 transcript sequences with good level of coverage of predicted transcripts and highly conserved core plant genes. Not only did the reference transcriptome update the annotation of rubber gene models, additional transcript variants were also discovered. Manual curation of gene models for key steps associated with rubber and carotenoids resulted in a repertoire of 115 genes, with 151 corresponding transcript variants. Subsequently, differential expression analysis on the basis of mapping RRIM600 and PB235 RNA-seq reads to the reference transcriptome revealed isoform-specific expression of genes for biosynthesis of carotenoids (PSY isoform 2), IPP (AACT2 and HMGR1) and rubber (REFSRPP gene members). In addition, the levels of these genes correlated positively with the carotenoid and rubber content measurements from the same latex of PB235 and RRIM600 used for metabolite extraction. Finally, the utility of the reference transcript catalogue was demonstrated by the characterisation of the REFSRPP gene family, which is involved in rubber elongation steps. REFSRPP gene family showed a local expansion which appear to be unique to <i>Hevea</i>. A pilot study has demonstrated there is considerable diversity of the genomic region containing the duplicated REFSRPP genes.

Friedreich ataxia (FRDA) is a lethal autosomal recessive neurodegenerative disorder caused by expanded GAA repeats in the FXN gene, resulting in local epigenetic changes and reduced expression of the mitochondrial protein frataxin. The disease is characterised by neurodegeneration of large sensory neurons of the dorsal root ganglia and spinocerebellar tracts. It has been recently reported that a novel frataxin antisense transcript, FAST-1, is overexpressed in FRDA patient derived fibroblasts. However, the lack of fundamental information about FAST-1 gene such as size, sequence, length and its origin has hindered the understanding of its interactions with FXN gene. Therefore, I proposed to investigate these characteristics of FAST-1 in a panel of FRDA cells and mouse models. Firstly, using Northern blot hybridisation with small and large riboprobes, I identified two bands with different sizes (~500 bp and 9 kb size), representing potential FAST-1 transcripts. Then to confirm the exact size and the location of the FAST-1 gene, I performed 5’- and 3’ RACE experiments, followed by cloning and sequencing. This analysis resulted in identification of the 5’- and 3’-ends of FAST-1, which mapped to nucleotide positions ‘-359’ and ‘164’ of the FXN gene, giving the total length of FAST-1 as 523 bp size. Strikingly, the full-length 523 bp FAST-1 transcript also corresponds to one of the Northern blotting results where I identified a band at approximately 500 bp size, indicating that the Northern blotting may have correctly identified the same full-length FAST-1 transcript. Subsequently, by optimising number of experimental parameters within our lab, I developed a robust qRT-PCR method to quantify FAST-1 expression levels. Using this technique, I analysed the expression pattern of this antisense transcript in various FRDA cell lines and mouse models. I confirmed the original finding of increased FAST-1 levels in human FRDA fibroblasts, and further quantified FAST-1 levels in FRDA mouse model cell lines and tissues. However, no consistently altered patterns of FAST-1 expression were identified in relation to FXN expression. Therefore, either they are not directly connected, as originally reported by De Biase et al., or their relationship varies between cell and tissue types. Lastly, improved understanding of epigenetic changes in FRDA and growing evidence on long-gene regulation led me to study the ‘neighbouring genes’ rather than just focusing on the FXN gene. Therefore, I studied a region of approximately 750 kb on both sides of the FXN and quantified the expression levels of two genes (PGM5 and PIP5K1β) on 5’- end and four genes (TJP2, FAM189A2, APBA1 and PTAR1) on 3’- end of FXN gene in human primary fibroblasts. I found that PGM5 and PIP5K1β genes, located at 5’- end of the FXN genes, were downregulated in FRDA fibroblasts and these findings coincide with the recent epigenetic changes identified in FRDA, where significant enrichment of gene repressive histone marks and increased DNA methylation were shown in upstream region of GAA repeats in intron 1 of the FXN gene. Out of four genes that were studied in the 3’- end of the FXN gene, only one gene (APBA1) was downregulated, which suggests that there are fewer repressive epigenetic marks downstream of the GAA repeat.

Pervasive transcription is widespread amongst eukaryotic genomes, and produces long noncoding RNAs (lncRNAs) in addition to classically annotated transcripts such as messenger RNAs (mRNAs). LncRNAs are heterogeneous in length and map to intergenic regions or overlap with annotated genes. Analogous to mRNAs, lncRNAs are transcribed by RNA polymerase II, regulated by common transcription factors, and possess 5’ caps and perhaps 3’ poly(A) tails. However, lncRNAs perform distinct functions, acting as scaffolds for ribonucleoprotein complexes or directing proteins to nucleic acid targets. The act of transcribing a lncRNA can also affect the local chromatin environment. Furthermore, whereas mRNAs are predominantly turned over in the cytoplasm, both nuclear and cytoplasmic pathways reportedly participate in lncRNA degradation. In this study, I address the question of when and how lncRNAs and mRNAs are distinguished in the cell. Messenger RNAs interact with a defined series of protein factors governing their production, processing and decay, and I hypothesised that lncRNAs might be similarly regulated. I therefore sought to determine which mRNA-binding proteins, if any, also bind lncRNAs. I reasoned that this would reveal the point at which lncRNAs and mRNAs diverge, and how differences in their biogenesis and turnover equip them for different roles. I selected factors from key stages of mRNA metabolism in Saccharomyces cerevisiae, and identified their transcriptome-wide targets using CRAC (crosslinking and analysis of cDNAs). CRAC can detect interactions with low abundance transcripts under physiological conditions, and reveal where within each transcript a protein is bound. Analyses of binding sites in mature mRNAs and intron-containing pre-mRNAs revealed the order in which the tested factors interact with mRNAs, and which region they bind. The poly(A)-binding protein Nab2 bound throughout mRNAs, consistent with an architectural role, whereas the cytoplasmic decay factors Xrn1 and Ski2 bound to poly(A) tails, which might act as hubs to coordinate turnover. The RNA packaging factors Tho2 and Gbp2, and nuclear surveillance factors Mtr4 and Trf4 bound abundantly to intron-containing premRNAs, indicating that they act during or shortly after transcription. The tested factors bound lncRNAs to various extents. LncRNA binding was most abundant for Mtr4 and Trf4, moderate for Tho2, Gbp2, the cap binding complex component Sto1, and the 3’ end processing factors Nab2, Hrp1 and Pab1, and lowest for Xrn1, Ski2 and the export receptor Mex67. This suggests that early events in lncRNA and mRNA biogenesis are similar, but unlike mRNAs, most lncRNAs are retained and degraded in the nucleus. Analyses of two documented classes of lncRNA, cryptic unstable transcripts (CUTs) and stable unannotated transcripts (SUTs), revealed some differences. SUTs were most similar to mRNAs, with canonical cleavage and polyadenylation signals flanking their 3’ ends, and poly(A) tails bound by the poly(A)-binding protein Pab1. CUTs lacked these characteristics, and in comparison to SUTs bound more abundantly to Mtr4 and Trf4 and less so to Ski2, Xrn1 and Mex67. Furthermore, CUTs accumulated upon Hrp1 depletion, suggesting that Hrp1 functions non-canonically to promote CUT turnover. Mtr4, Trf4 and Nab2 also bound abundantly to promoter-proximal RNA fragments generated from ~1000 protein coding genes. These fragments possessed short oligo(A) tails (hallmarks of nuclear surveillance substrates), were not bound to cytoplasmic factors, and apparently correspond to a population of ~150-200 nt promoter-proximal lncRNAs. Notably, CRAC analyses of Mtr4 and Sto1 targets in yeast subjected to a media shift revealed widespread changes in the abundance and surveillance of mRNAs, promoter-proximal transcripts and CUTs, which at many loci were arranged in a complex transcriptional architecture. Overall, the transcriptome-wide binding analyses presented here reveal that lncRNAs diverge from mRNAs prior to export, and are predominantly retained in the nucleus. Transcript fate is apparently determined during 3’ end processing, with CUTs diverging from mRNAs early in transcription via a distinct termination pathway coupled to rapid turnover, and SUTs diverging during or shortly after cleavage and polyadenylation, making them more stable and perhaps prone to escape to the cytoplasm. Promoter-proximal transcripts might arise from termination associated with an early checkpoint in Pol II transcription. The diverse behaviours of lncRNAs arise from their association with distinct subsets of RNA binding proteins, some of which perform different roles when bound to different types of transcript. In conclusion, my results provide the foundation for a mechanistic understanding of how distinct classes of non-coding Pol II transcripts are produced, and how they can perform diverse functions throughout the nucleus.

Temperate maize is one of few model species that relies mainly on endogenous indicators of the plant’s developmental stage to cue the onset of reproductive development. The INDETERMINATE1 (ID1) transcription factor is a key regulator of the floral transition and id1 mutants are very late-flowering. ID1 is expressed and remains localized in developing leaves, while florigenic signals originate in mature, photosynthetically active leaves. Since very little is known about the molecular components of the floral transition in maize, and in autonomously flowering species at large, this work utilized id1 mutants to analyze the transcriptional and physiological alterations associated with the floral transition in maize. Analyses of functional categories of transcriptional change between developing leaves of id1 non-flowering mutants and normal flowering maize suggest a role for ID1 in energy metabolism and epigenetic regulation of leaf development. In addition, a novel family of -glucosidase genes were found to be expressed exclusively in immature leaves of normal flowering maize in a pattern similar to the ID1 gene suggesting that these genes may act in concert downstream of ID1. Further, profiling of transcript and metabolite alterations in mature leaves, which are likely the source of floral cues, suggest that coordination of resource storage in the form of transitory starch is an important signal for floral promotion in maize. Finally, analysis of the floral transition in Balsas teosinte, the progenitor of modern maize and an obligate short-day plant, suggests that ID1 may define a regulatory module unique to the autonomous floral regulation pathway in maize and related grass species.

Several mechanisms have been proposed for the pol I transcript termination in Schizosaccharomyces pombe. Two well known models are “Pause and Release” and “Torpedo”. Each mechanism explains the role of some of the cis- and trans-factors in transcript termination and the eventual maturation of the ribosomal RNA, but neither mechanism can explain all the experimental observations. A recent study has suggested that each of the two mechanisms can terminate the pol I transcription independently but with significantly less efficiency than the presence of both mechanisms. To help clarify the reasons for the discrepancies in these data, in this study the suggested mechanisms were examined further in three areas by using alternative techniques. First, the effect of uracil concentration or selection times on the transformation frequency of alternative 3’external transcribed spacer (3’ETS) constructs were assessed. Consistent with the previous results a construct containing the full 3’ETS showed the higher transformation frequencies compared with a construct containing only the hairpin or only the termination sites. However, results showed neither the uracil concentration nor selection times have a significant effect on the transformation frequency. Second, to further confirm the “pause and release” mechanism, the termination sites identified by S1 nuclease studies were analyzed using ligation-mediated RT-PCR. The 3’ terminus of the mature 25S rRNA was demonstrated readily but, unexpectedly, the 3’termini of the 3’ETS precursor molecules were not detected, possibly because of their specific structure. Finally, the 3’ extended rRNA precursors were studied by semi-quantitative RT-PCR. These appeared not to correspond with past nuclease protection analyses nor did they demonstrate downstream exonuclease function, observations which question our current understanding of Pol I transcript termination.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-12-16T11:19:46Z No. of bitstreams: 1 michelemunkpereira.pdf: 1917144 bytes, checksum: 6ea13cf025292a61936d375b8b3710e2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-12-16T11:29:32Z (GMT) No. of bitstreams: 1 michelemunkpereira.pdf: 1917144 bytes, checksum: 6ea13cf025292a61936d375b8b3710e2 (MD5) / Made available in DSpace on 2016-12-16T11:29:33Z (GMT). No. of bitstreams: 1 michelemunkpereira.pdf: 1917144 bytes, checksum: 6ea13cf025292a61936d375b8b3710e2 (MD5) Previous issue date: 2010-03-12 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A maturação in vitro de oócitos é um processo importante para a produção in vitro de embriões bovinos. O objetivo do presente estudo foi investigar o efeito da interação do soro e tensão de oxigênio na maturação in vitro, sobre a maturação nuclear, viabilidade das células do cumulus, abundância de transcritos oocitários, desenvolvimento pré-implantacional e apoptose embrionária. Foram utilizados quatro grupos experimentais: G1 (10% soro de vaca no cio [SVC] em 20% de O2); G2 (0,1% álcool polivinílico [PVA] em 20% de O2), G3 (10% SVC em 5% de O2) e G4 (0.1% PVA em 5% O2). A proporção de oócitos em metáfase II, viabilidade das células do cumulus, taxa de blastocistos e número total de células não foi afetado (P>0,05) quando o soro foi substituído por PVA em 5% de O2, enquanto a taxa de blastocistos e número total de células foi maior (P<0.05) no grupo com soro comparado com PVA, ambos em 20% O2. O índice apoptótico foi menor em blastocistos produzidos a partir de oócitos maturados com PVA em 5% de O2 (G4) em relação aos de outros grupos (G1, G2 e G3), mas não foi encontrada diferença (P>0,05) na taxa de maturação, viabilidade das células do cumulus e de blastocistos. Foram encontradas diferenças (P<0,05) na quantidade de transcritos específicos em oócitos maturados sobre as diferentes condições. Conclui-se que a presença de soro durante a maturação é importante para o desenvolvimento embrionário em tensão de 20% de O2, e a suplementação com PVA em 5% de O2 fornece o melhor ambiente de maturação para os oócitos, resultando em blastocistos com baixo índice apoptótico. / In vitro maturation is an important step for in vitro embryo production. The objective of the present study was to investigate the effect of the interaction of serum and oxygen tension during in vitro maturation on nuclear maturation, viability of cumulus cells, oocyte transcript amount, preimplantation development and blastocyst apoptosis. Four experimental groups were designed: G1 (10% estrus cow serum [ECS] with 20% O2); G2 (0.1% polyvinyl alcohol [PVA] with 20% O2), G3 (10% ECS with 5% O2) e G4 (0.1% PVA with 5% O2). Proportion of metaphase II oocytes, viability of cumulus cells, blastocyst rates and the total cell number were not affected (P>0.05) when the ECS was replaced by PVA under 5% O2, whereas higher (P<0.05) blastocyst rate and total cell number were found with ECS than PVA, both under 20% O2. Apoptosis index were lower in blastocysts from oocytes matured with PVA under 5% O2 (G4) than blastocysts from other groups (G1, G2 and G3), but no differences (P>0.05) were found in nuclear maturation, viability of cumulus cells and blastocyst rate. Differences (P<0.05) in amount of specific transcripts were found in oocytes matured under different conditions. In conclusion, presence of serum during maturation is important for embryo development only when under 20% O2, and maturation with PVA and 5% O2 provides better environment for oocyte, resulting in blastocysts with low apoptosis index.

Abstract In eukaryotic cells, fatty acids are mainly synthesized in the cytoplasm, but recently, in yeast and in humans, the ability to synthesize fatty acids has been characterized in mitochondria. This mitochondrial pathway is similar to bacterial type II fatty acid synthesis (FAS). The main feature of mitochondrial FAS in yeast is the respiratory deficient phenotype and loss of cytochromes when any of genes encoding enzymes for mitochondrial FAS is deleted. Mitochondrial FAS has been demonstrated to have an important role in lipoic acid production, namely it synthesizes octanoyl-ACP, which is used as a precursor for lipoic acid. However, the role and function of mitochondrial FAS is not yet fully understood. Many components of the mitochondrial FAS pathway in yeast have been identified according to their similarity to bacterial counterparts, but 3-hydroxyacyl-ACP dehydratase does not show any easily recognizable similarity to bacterial dehydratases and thus remained unidentified. In this study 3-hydroxyacyl-ACP dehydratases of mitochondrial FAS were characterized from the yeast Saccharomyces cerevisiae, humans, and the human pathogen Trypanosoma brucei. The yeast 3-hydroxyacyl-ACP dehydratase (Htd2p) was identified by using a genetic screen, and this protein was shown to be encoded by open reading frame (ORF) YHR067w. The product of this gene shows mitochondrial localization and exhibits hydratase 2 activity. The deletion of HTD2 leads to a respiratory deficient phenotype, loss of cytochromes, reduced lipoic acids levels and changes in mitochondrial morphology. The ORF encoding human 3-hydroxyacyl-ACP hydratase (HsHTD2) was identified by functional complementation of the respiratory deficient phenotype of the yeast htd2 mutant with a human cDNA library. The complementing cDNA was previously identified as the RPP14 transcript encoding the 14 kDa subunit of the human RNase P complex. It was found that this transcript contains another 3' ORF, which encodes a protein that displays hydratase 2 activity and has mitochondrial localization. The bicistronic nature of the transcript is conserved in vertebrates and indicates a genetic link between mitochondrial FAS and RNA processing. The mitochondrial 3-hydroxyacyl-ACP hydratase in T. brucei is homologous to human HTD2, can complement the yeast respiratory deficient phenotype, exhibits hydratase 2 activity and localizes to the T. brucei mitochondrion.

Content of this thesis is dedicated to problematics of design and implementation of a web base video player with slides as a JavaScript plugin. As the outcome of this thesis is supposed to replace an already existing player , the first chapter is dedicated to it's throughout analysis . Following chapters are the dedicated to desing , implementation and testing of the player , using information acquired in the phase of analysis .

The purpose of this study was to assess the impact of a school division policy on early algebra on students' course taking patterns in high school. Over the past two decades, there has been significant growth in the number of students taking Algebra 1 in middle school. Research about the advantages and drawbacks to completing Algebra 1 prior to high school have mixed conclusions, with some suggesting that students benefit from the opportunity to take more advanced mathematics and science courses in high school and others concluding that students are more likely to fail and need to repeat courses if they take Algebra 1 early (Stein et al., 2011). Most of the research has focused on students taking Algebra 1 in eighth grade. At the same time, there is an ever-growing group of students seeking to take Algebra 1 even earlier, as evidenced by expansive growth in the number of students accessing Advanced Placement Calculus prior to twelfth grade (College Board, 1997; College Board, 2017). To assess the impact of early Algebra 1, the researcher considered transcript data for two cohorts of students in a large, suburban school district who took Algebra 1 in seventh or eighth grade. Statistical analysis was performed to assess whether students were likely to access the highest level mathematics courses available to them, whether they were staying in mathematics courses throughout all years of high school, and what patterns might emerge in mathematics and science course taking for students based on when they took Algebra 1. The findings indicated that students in this cohort who took Algebra 1 in eighth grade were more likely to complete the highest level mathematics courses available to them than those who took Algebra 1 in seventh grade, but they also took, on average, fewer total mathematics and science courses. For all students taking middle school Algebra 1, there were sharp declines in students accessing honors-level mathematics coursework as they advanced through the mathematics sequence. / Doctor of Education / The purpose of this study is to assess the impact of a school division policy on early algebra on students' course taking patterns in high school. Over the past two decades, there has been significant growth in the number of students taking Algebra 1 in middle school. Research about the advantages and drawbacks to completing Algebra 1 prior to high school have mixed conclusions, with some suggesting that students benefit from the opportunity to take more advanced mathematics and science courses in high school and others concluding that students are more likely to fail and need to repeat courses if they take Algebra 1 early (Stein et al., 2011). Most of the research has focused on students taking Algebra 1 in eighth grade. At the same time, there is an ever-growing group of students seeking to take Algebra 1 even earlier, as evidenced by expansive growth in the number of students accessing Advanced Placement Calculus prior to twelfth grade (College Board, 1997; College Board, 2017). To assess the impact of early Algebra 1, the researcher considered transcript data for two cohorts of students in a large, suburban school district who took Algebra 1 in seventh or eighth grade. Statistical analysis was performed to assess whether students were likely to access the highest level mathematics courses available to them, whether they were staying in mathematics courses throughout all years of high school, and what patterns might emerge in mathematics and science course taking for students based on when they took Algebra 1. The findings indicate that students in this cohort who took Algebra 1 in eighth grade were more likely to complete the highest-level mathematics courses available to them than those who took Algebra 1 in seventh grade, but they also took on average fewer total mathematics and science courses. For all students taking middle school Algebra 1, there were sharp declines in students accessing honors-level mathematics coursework as they advanced through the mathematics sequence.

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