Doctor of Philosophy / Department of Agronomy / P.V. Vara Prasad / Productivity of grain crops is highly sensitive to changing climates and crop management practices. Response of finger millet [Eleusine coracana (L.) Gaertn.] to high temperature stress, and intensive management practices such as increased seeding rates and fertilizer application are not clearly understood. The objectives of this research were to determine the effects of (a) season-long, and short episodes of high temperature stress on growth and yield traits of finger millet, (b) seeding rates and nitrogen fertilizer application rates on grain and biomass yield, and (c) to evaluate the finger millet minicore collection for high grain and biomass yield. Controlled environment studies were conducted to determine the effects of high temperature stress on physiological, growth and yield traits. Field studies were conducted in Manhattan and Hays (Kansas) and Alupe (Kenya) to determine the effects of seeding and nitrogen fertilizer rates on growth and yield traits. Finger millet minicore collection was evaluated under field conditions in India, for phenology, growth and yield traits. Season long high temperature stress of 36/26 or 38/28°C compared to 32/22°C decreased panicle emergence, number of seeds per panicle, grain yield and harvest index. Finger millet was most sensitive to short episodes (10 d) of high temperature (40/30°C) during booting, panicle emergence and flowering stages, resulting in lower number of seeds, and grain yield. Finger millet responded to the interaction between environmental (locations) and temporal (years) factors. In general, locations with higher rainfall had greater grain and biomass yield than those with low rainfall. There was no influence of seeding rates (3.2 or 6.0 kg ha[superscript]-1) at Hays and Alupe. However, in one of the two years in Manhattan, higher seeding rate of 6.0 kg ha[superscript]-1 increased grain yield compared to 3.2 kg ha[superscript]-1. There was no influence of nitrogen rates (0, 30, 60 or 90 kg ha[superscript]-1) on grain or biomass yield at all three locations. However, higher fertilizer rates had greater percentage lodging. The finger millet minicore collection displayed large ranges for most quantitative traits including days to flowering, plant height, number of fingers panicle[superscript]-1, grain yield, biomass yield, and lodging; and had >60% heritability. Some of the genotypes from the minicore collection have the potential to increase grain and biomass yield and abiotic stress tolerance of finger millet.

As reações luminosas da fotossíntese em plantas envolvem quatro complexos proteicos multi-unidades na membrana dos tilacóides incluindo o fotossistema II (PSII), o complexo citocromo b6f, o fotossistema I (PSI) e o complexo ATP sintase. Uma atividade apropriada desse processo exige um mecanismo de controle de qualidade mediado por chaperonas, DnaJs e proteases, como o complexo FtsH. Esse conjunto de proteínas garantem um dobramento correto de proteínas, as montagens devidas dos complexos e a degradação de algumas subunidades danificadas quando necessário. Neste trabalho nós mostramos o envolvimento de FIP, uma proteína com um domínio dedo-de-zinco localizada nos tilacóides de cloroplastos de A. thaliana, no mecanismo de resposta à estresses abióticos. Plantas mutantes fip foram, fenotipicamente, mais tolerantes à estresses abióticos de alta luminosidade, elevado potencial osmótico e excesso de sal. Também mostramos que a expressão de FIP é diminuída em resposta às diferentes condições de estresse, assim como o acúmulo de transcritos de genes relacionados à estresse foi menor nas plantas mutantes fip. Análises por immunoblot mostraram que os mutantes fip acumulam menos proteínas PsaA e PsaB do fotossistema I e plastocianina (PC) do que as plantas selvagens, no entanto não são afetados quanto ao acúmulo de proteínas do fotossistema II e do Complexo do Citocromo b6f sob condições controle. Esses mutantes também acumulam menos FtsH5 nos tilacóides, sem afetar a eficiência dos fotossistemas I e II. Foi testado também o potencial redutase do domínio dedo-de-zinco da proteína recombinante FIP (6xHis-FIP) em ensaios in vitro de redução de insulina. Vimos que FIP apresenta atividade redutase, significantemente, maior que o controle negativo nas condições testadas. Considerando todos os resultados obtidos até o momento, acreditamos que FIP possa estar agindo como uma redutase na membrana dos tilacóides, tendo como alvos não somente FtsH5, mas também outras proteínas com resíduos de cisteína nas suas estruturas, e que sua atividade tem influência no acúmulo de proteínas dependentes de redução para a maturação como PsaA, PsaB e PC. Uma investigação mais aprofundada da atividade de FIP nos cloroplastos ainda é necessária para o completo entendimento da sua função. / The light-driven photosynthetic reactions in plants take place within four multi- subunit protein complexes in the thylakoid membranes, including photosystem II (PSII), the cytochrome b6f complex, photosystem I (PSI) and the ATP synthase complex. Regulation of all these molecular machineries requires a fine-tuning control mechanism mediated by specific proteins, including chaperones, DnaJs, and proteases, such as the FtsH complex. These set of proteins guarantee the proper folding, assembly and degradation of the photosynthetic complexes\' subunits. In this work we showed the involvement of FIP, a zinc-finger protein localized in the thylakoid membranes in A. thaliana, in the abiotic stress response mechanism. Mutants fip knockdown plants were phenotypically more tolerant to abiotic stresses like high light, increased osmotic potential and salt excess. We also showed that FIP is down-regulated by different abiotic stresses, with lower levels of stress-related gene transcripts accumulation in mutant fip plants. Analysis of accumulation of photosynthetic proteins by immunoblot under control conditions showed that mutants fip displayed lower levels of PsaA, PsaB (PSI) and Plastocyanin (PC) proteins than wild-type plants, however are not affected for PSII and Cyt b6f proteins accumulation under the same growth conditions. In addition, the mutants accumulated slightly less FtsH5 proteins in thylakoid membranes, without affecting PSII and PSI efficiency. We tested the putative reductase activity probably mediated by FIP zinc-finger domain, using the recombinant form of the protein 6xHis-FIP in in vitro insulin reduction assays. FIP presented a reductase activity higher than the negative control under the same assay conditions. Taking all together, these results suggest that FIP may be acting as a reductase in the thylakoid membranes, having as targets not only FtsH5 but other targets with available cysteine residues, depending on the reduction step for proper accumulation such as PsaA, PsaB and PC. Further investigations regarding the role of FIP in chloroplasts are still necessary to completely understand its function.

Transcriptional regulation of genes is vital to cell success making it an important aspect of research. Transcriptional regulation can occur in many ways; transcription factors bind to the promoter region and block transcription, disrupt an activator protein, or interact with histones to lead to higher order chromatin. Plant HomeoDomain can recognize and bind to different methylation states of histone tails. PHD proteins use other functional regions to carry out functions. Two associated domains having DNA-binding capacity were characterized in this study; the ARID domains of JARID1A and JARID1C and the DDT domains of BAZ1A, BAZ1B and BAZ2A. These genes are important because of their roles in various diseases such as cancer. The consensus sequences for BAZ1A-DDT is GGACGGRnnGG, GnGAGRGCRnnGGnG, RAGGGGGRnG and CRYCGGT. Consensus sequences for BAZ1B-DDT were CGnCCAnCTTnTGGG and YGCCCCTCCCCnR. Consensus sequences for BAZ2A-DDT were TACnnAGCnY and CnnCCRGCnRTGnYY. Consensus sequence for JARID1A-ARID was GnYnGCGYRCYnCnG. Consensus sequences for JARID1C-ARID was RGGRGCCRGGY. / by Emmanuel MacDonald. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Transcriptional regulation by the family of SNAG (Snail/Gfi-1) zinc fingers has been shown to play a role in various developmental states and diseases. These transcriptional repressors have function in both DNA- and protein-binding, allowing for multiple interactions by a single family member. This work aims to characterize the SNAG members Slug, Smuc, Snail, Scratch, Gfi-1, Gfi-1B, and IA-1 in terms of both DNA-protein and protein-protein interactions. The specific DNA sequences to which the zinc finger regions bind were determined for each member, and a general consensus of TGCACCTGTCCGA, was developed for four of the members. Via these studies, we also reveal thebinding affinities of E-box (CANNTG) sequences to the members, since this core is found for multiple members' binding sites. Additionally, protein-protein interactions of SNAG members to other biological molecules were investigated. The Slug domain and Scratch domain have unknown function, yet through yeast two-hybrid screening, we were able to determine protein interaction partners for them as well as for other full length SNAG members. These protein-interacting partners have suggested function as corepressors during transcriptional repression. The comprehensive information determined from these studies allow for a better understanding of the functional relationship between SNAG-ZFPs and other genes. The collected data not only creates a new profile for each member investigated, but it also allows for further studies to be initiated from the results. / by Cindy Chiang. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Transcriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional repressors shown to play a role in various diseases such as cancer. The SNAG transcription factors contain a conserved SNAG repression domain and DNA binding domain zinc fingers. The specific DNA sequences to which each SNAG-ZFP binds, as well as a general consensus -TGCACCTGTCCGA, have been determined. Also, putative protein-protein interactions in which the Slug domain participates has been identified via binding assays. All these results contribute to better understanding of SNAG-ZFP functions. / by Cindy Chung-Yue Chiang. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Zhang, Zhiyi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves ). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

There are approximately 20,000 genes in the human genome. Around 2% of these genes code for transcriptional repressors known as KRAB-ZFPs. It is already known that Zinc-Finger Proteins contain two main functional domains at either end of the polypeptide. In today's database, you will find a KRAB (Kruppell-associated Box) domain at one end and a tandem array of Zinc-finger repeats at the other end. The carboxyl terminal tandem Zinc-finger repeats function as sequence-specific DNA-binding domains. The amino terminal KRAB domain serves as a repressor domain, which will recruit a co-repressor termed KAP-1 (KRAB Associated Protein-1). Located in between these two domains is a region of uncharacterized DNA referred to as the "Linker Region". This thesis will explore the DNA-binding domains of 6 known KRAB-ZFPs, as well as utilize the linker regions to derive an evolutionary history for this superfamily. / by Alain Chamoun. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

The two-finger banjo style languishes as a small footnote in the lexicon of old time banjo music—very important to a passionate (and lucky!) few, but not known by too many others. This research is a starting point to understanding the meaning of two-finger banjo; through a review of primary literature, interviews, witnessing performances, and individual investigation of playing two-finger banjo, I have been able to document some understandings about the style. These understandings informed further appreciation of old time music, the old time music (and banjo) communities, and the art of making music in this way—while describing the journey, I gained insights from scholars, folklorists, musicians, recordings, and made many discoveries that I documented in this thesis. I have concluded that one of the best ways to find meaning in any pursuit is by engaging with your community and connecting yourself to your art.

Hepatitis B virus chronically infects 350-400 million people worldwide. It often leads to hepatocellular carcinoma, which causes >1 million deaths yearly. Current therapies prevent new viral genome formation but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted hepatitis B virus DNA for cleavage using zinc finger nucleases, which cleave as dimers. Co-transfection of our zinc finger nuclease pair with a target plasmid containing the hepatitis B virus genome resulted in specific cleavage. After three days in culture, 26% of the target remained linear, while ~10% was cleaved and mis-joined tail-to-tail. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the zinc finger nuclease sites. Targeted cleavage and misrepair destroys the XbaI site. After three days in culture, ~6% of plasmids were XbaI resistant. 13 of 16 clones sequenced contained frameshift mutations that would lead to dramatic truncations of the viral core protein. These results demonstrate for the first time the feasibility of targeting episomal viral DNA genomes in cells using zinc finger nucleases. This strategy is broadly applicable toward inactivating other DNA viruses within cells. A major concern for the therapeutic use of zinc finger nucleases is off-target cleavage. To measure specificity, we employed in vitro assays and developed a bioinformatics method to find off-target cleavage sites in cultured cells. These sites can then be PCR amplified and tested using a mutation detection assay that we developed.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.