Cellular stress invokes a protective response in which heat shock factor 1 (HSF1) is activated to increase heat shock protein (Hsp) expression. HSF1 exists as a latent monomer in unstressed cells. Upon stress HSF1 forms homotrimers, increasing its affinity for the heat shock DNA element upstream of all Hsp genes. A second conformational change is required for HSF1 to gain transcriptional competence. During prolonged heat shock or following the resumption of normal conditions HSF1 DNA-binding and transcriptional activities are reduced and HSF1 returns to the monomeric state in a process called attenuation. During the activation/deactivation cycle HSF1 is modified by small ubiquitin-related modifier (SUMO-1) conjugation and undergoes several phosphorylation and dephosphorylation events that modulate HSF1 activity. Hyperphosphorylation of HSF1 is hypothesized to trigger HSF1 transcriptional activity. HSF1 also interacts with a dynamic series of Hsp90/Hsp70-based chaperone heterocomplexes that negatively regulate DNA-binding, and transcriptional activity, and promote attenuation. This thesis was aimed at characterizing the mechanisms regulating HSF1 DNA-binding, and transcriptional activity. Expression of human HSF1 in Xenopus oocytes altered the set-point of DNA-binding in response to heat indicating that both the cellular environment and innate properties of the molecule allow HSF1 to set its activation/deactivation set-point in response to stress in vivo. HSF1 DNA-binding but not transcription was activated in oocytes treated with a high temperature heat shock. Further characterization of this observation determined that HSF1 activated by a brief high temperature heat shock inhibited transcriptionally competent HSF1 from activating transcription. It was hypothesized that this phenomenon exists to ensure the eventual death of the cell due to the accumulation of excessive damage and potential mutation caused by severe stress. The most significant observation made in this thesis is that Hsp expression was detected in oocytes injected with reporter plasmid only during recovery from a high temperature heat shock. These results led to the proposal of a model in which HSF1 trimers are either assembled in a transcriptionally incompetent form or one that has the potential to become transcriptionally competent during stress, prior to DNA-binding. The identity of HSF1-binding proteins that interact with HSF1 at different stages of activation/deactivation was characterized in an effort to assign regulatory roles to these proteins. HSF1 was detected in a high molecular weight complex (350-600 kDa) during all phases of the activation/deactivation cycle. HSF1 at different stages of activation was tested for interaction with specific molecular chaperones by electrophoretic mobility supershift analysis. Hsp90, p23, FKBP52, Hip and Hop are all associated with transcriptionally active and inactive HSF1 suggesting that interaction of HSF1 with any of these molecules does not activate HSF1 transcriptional activity. These results do not exclude the possibility that the function of these molecular chaperones may change during activation of HSF1 transcription or that post-translational modifications may be the primary mechanism that drives HSF1 from a transcriptionally inactive to active form.

Cells are continuously exposed to a variety of physiological and environmental stresses that can lead to protein aggregation and/or denaturation, and eventually cell death. In order to ensure survival, cells have evolved a stress response that monitors, detects, and responds to changes within the cellular environment. The stress response is characterized by the up-regulation of heat shock protein (hsp) genes whose products can mediate the assembly and/or degradation of misfolded or aggregated proteins within the cell. This stress-induced upregulation of heat shock protein encoding genes is under the regulation of heat shock transcription factor 1 (HSF1) and its associated proteins that together form what is known as the HSF1 heterocomplex. In eukaryotic cells, HSF1 exists as a non-DNA binding monomer in the absence of stress. Upon exposure to stress, HSF1 undergoes trimerization and acquires the ability to bind heat shock elements (HSEs) located upstream of all hsp genes and after further modification, can become converted into a transcriptionally active form. Following prolonged stress or after removal of stress, HSF1 loses its ability to bind DNA and transcription ceases in a process termed attenuation. <p>Several studies have suggested that the DNA-binding and transcriptional activities of HSF1 are regulated by phosphorylation and dephosphorylation events and by chaperone-based folding mechanisms similar to those involved in the regulation of glucocorticoid receptors. Protein phosphatase 5 (PP5) has been identified as a member of the glucocorticoid receptor chaperone complex and its phosphatase activity has been shown to regulate the maturation and activation of the receptor. It has been suggested that PP5 may regulate HSF1 in a manner similar to that of glucocorticoid receptors however it has not yet been determined how PP5 interacts with the HSF1 heterocomplex or if PP5 functions to regulate HSF1-DNA binding and/or HSF1 transactivation.<p>Utilizing the Xenopus model system, I tested the hypothesis that PP5 regulates the DNA binding and transcriptional activities of HSF1 through interactions with the HSF1 heterocomplex. Increasing the activity of PP5, either through the elevation of PP5 protein levels or by activating endogenous PP5, resulted in decreased HSF1-DNA binding as well as accelerated attenuation after the removal of stress. Conversely, inhibiting the phosphatase activity of PP5 using okadaic acid or by immunotargetting, where an antibody recognizing PP5 was microinjected into the nuclei of oocytes, resulted in delayed HSF1 attenuation. Transcription assays performed using activated PP5 also demonstrated that PP5 acts to decrease HSF1-mediated transcription. Immunoprecipitation and gel mobility supershift assays were also used to show that PP5 interacts with the HSF1 heterocomplex and PP5-HSP90 binding mutants illustrated that PP5 may exert its repressive effects independently of binding directly to HSP90.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Have you thought of why you get tired or why you get hungry? Something in your body keeps track of time. It is almost like you have a clock that tells you all those things. And indeed, in the suparachiasmatic region of our hypothalamus reside cells which each act like an oscillator, and together form a coherent circadian rhythm to help our body keep track of time. In fact, such circadian clocks are not limited to mammals but can be found in many organisms including single-cell, reptiles and birds. The study of such rhythms constitutes a field of biology, chronobiology, and forms the background for my research and this thesis. Pioneers of chronobiology, Pittendrigh and Aschoff, studied biological clocks from an input-output view, across a range of organisms by observing and analyzing their overt activity in response to stimulus such as light. Their study was made without recourse to knowledge of the biological underpinnings of the circadian pacemaker. The advent of the new biology has now made it possible to "break open the box" and identify biological feedback systems comprised of gene transcription and protein translation as the core mechanism of a biological clock. My research has focused on a simple transcription-translation clock model which nevertheless possesses many of the features of a circadian pacemaker including its entrainability by light. This model consists of two nonlinear coupled and delayed differential equations. Light pulses can reset the phase of this clock, whereas constant light of different intensity can speed it up or slow it down. This latter property is a signature property of circadian clocks and is referred to in chronobiology as "Aschoff's rule". The discussion in this thesis focus on develop a connection and also a understanding of how constant light effect this clock model.

The L1 region of the porcine adenovirus (PAdV)-3 genome encodes a protein of 622 amino acids named IIIa. Although it binds a neighboring group of nine (GON) hexons at the capsid level and cement the icosahedral shell that contains the viral DNA, little is known regarding its function with respect to viral life cycle. Moreover, the known location of IIIa protein in the capsid may help to express targeting ligands for altering the tropism of PAdV-3. The objective of this study was to characterize the IIIa protein of porcine adenovirus Type 3 (PAdV-3). <p> In order to characterize the IIIa protein, polyclonal antisera were raised in rabbits against different regions of IIIa. Anti-IIIa sera detected a specific protein of 70 kDa in PAdV-3 infected cells using Western blot assay. Immunofluorescence studies indicated that IIIa is predominantly localized in the nucleus of PAdV-3 infected cells. Analysis of PAdV-3 IIIa using antibodies specific for N- and C- terminal domains of the protein suggested that although the N-terminus and C-terminal domains of IIIa are immunogenic, they are not exposed on the surface of PAdV-3 virions. These results were further confirmed by our inability to isolate a chimeric PAdV-3 virion containing a heterologous protein fused to the N-terminus or C-terminus of IIIa. <p>Functional analysis suggested that IIIa may transactivate the major late promoter and down regulate the early region (E) 1A promoter. In order to locate the domains of IIIa responsible for different functions, in-frame deleted/truncated forms of IIIa were constructed. Analysis of the deleted/truncated forms of IIIa suggested that a) the sequences located between amino acids 273-410 and between amino acids 410-622b) affect the nuclear localization and transactivation function respectively.<p>Since protein- protein interactions are important for the biological functions of the protein, we determined the interaction of PAdV-3 IIIa with other viral proteins. IIIa was found to interact with DNA binding protein (DBP), E3 13.7 kDa protein, hexon, fiber, and pIX. These results suggest that PAdV3 IIIa may do more in the viral life cycle than merely act as cement between the hexons to maintain capsid stability and may actually be involved in regulating early to late gene transcription at appropriate stages during viral infection.

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

We previously described BT2, a BTB/POZ domain containing protein, as an activator of telomerase in Arabidopsis thaliana. In the current study, I present evidence of its interesting roles in mediating multiple hormone, stress and metabolic responses in plants. Steady-state expression of BT2 mRNA was regulated diurnally and was under the control of circadian clock, with a maximum expression in the dark. BT2 mRNA was responsive to nutrient status and to multiple biotic and abiotic stress signals. Using bt2 loss-of-function and BT2 over-expressing lines, I show that BT2 suppresses sugar and ABA-mediated responses during germination. BT2 is also essential for transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. Loss of BT2 in several well-characterized 35S enhancer activation-tagged lines such as yucca1d, pap1d, jaw1d etc., resulted in suppression of the activation phenotypes. The suppression of the phenotypes was due to decreased transcription of the activation-tagged genes. I further demonstrate that BT2 genetically interacts with CULLIN3. I propose that BT2 and CULLIN3 are components of a ubiquitin ligase complex. Together with associated proteins BET9 and BET10, the BT2 complex is required for CaMV 35S enhancer-mediated activation of gene expression and may regulate expression of target genes involved in multiple responses to fluctuating biotic and abiotic conditions. I also found that BT2 protein levels are tightly regulated in plants. BT2 protein was primarily localized in the nucleus and was developmentally regulated. BT2 turn-over was regulated in part by the 26S-proteosome, and rare codons present in its open reading frame affected BT2 protein accumulation. In addition to BT2, its orthologs, BT1, BT3, BT4 and BT5, also responded to light, clock and nutrients, with some differences. Moreover, BT1, BT3 and BT4 were also required for 35S enhancer-mediated activation of gene expression. I propose that BT family proteins assemble into multi-protein complexes to mediate multiple responses to changing environmental and nutritional conditions.

Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females &#8722; both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.

Advances in optical imaging techniques have allowed quantitative studies of many biological systems. This dissertation elaborates on our efforts in both developing novel imaging modalities based on detection of optical absorption and applying high-sensitivity fluorescence microscopy to the study of biology. / Chemistry and Chemical Biology