The T cell-specific kinase, Protein Kinase C theta (PKCq) is essential to T cell activation and differentiation. PKCq integrates T cell receptor (TCR) and CD28 signaling, and ultimately activates transcription factors necessary for full T cell activation, proliferation, survival, and differentiation into T helper (Th) subsets. Th1, Th2, Th17 and Treg cells compose the four major lineages of T helper cells, differentiated from CD4 T cells, and each have different requirements for PKCq. PKCq, itself, is regulated through phosphorylation of specific resides, including tyrosine (Y)90 and threonine (T)538. Following T cell stimulation, PKCq is phosphorylated on Y90 by the kinase, LCK, and translocates to the cell membrane. There it remains associated with LCK in a structural complex known as the immunological synapse. Loss of PKCq in T cells produces a phenotype that is similar to loss of another important T cell protein, Notch1, suggesting these two proteins may function in the same signaling pathway. Our lab has shown that PKCq can interact with Notch1, but how this interaction regulates Notch1 function is not known. Due to the strong overlap between cellular functions regulated by PKCq and Notch1, understanding how these two proteins might function, cooperatively, can provide better insight into autoimmune diseases and may be useful in developing novel therapies We hypothesized that phosphorylation of a specific residue of PKCq (T538) is required for Notch1 cleavage and nuclear translocation. We also hypothesize that the phosphorylation status of PKCq (T538) will influence the ability of T cells to differentiate into specific T helper subsets. We used two means of inhibiting PKCq function to evaluate its regulation of Notch1 in differentiated T cells: 1) we blocked the association of PKCq and LCK, thereby preventing its movement to the immunological synapse and 2) we prevented PKCq phosphorylation on T538. We found that by preventing the phosphorylation of PKCq, we also decreased the level of cleaved Notch1 in Th1, Th2, and Th17 cells. We further determined that inhibiting PKCq decreased the amount of Notch1 that translocated to the nucleus in Th1 and iTreg cells. Understanding how PKCq interacts with and regulates Notch1 to influence T cell differentiation may lay the foundation for specifically modulating T cell responses.

The experimental results reported here test the hypothesis that incorporation of amino acid analogs into periodic proteins at multiple positions should yield new materials with unique physicochemical properties associated with the amino acid analogs. Two amino acid analogs with trifluoromethyl and thiophene side groups were featured in this study, i.e., trifluoroleucine and 3-thienylalanine. A coupled in vitro transcription-translation assay procedure was first established to assess the ability of the Escherichia coli protein synthesis machinery to use non-natural amino acids. Second, a general strategy for incorporating amino acid analogs in vivo was developed. This procedure uses a bacteriophage T7 expression system and high cell density fermentation. The method was used to make tens of milligrams of periodic proteins of the general sequence $\{$(GlyAla)$\sb3$GlyXxx$\}\sb{\rm n}$ where Xxx is either leucine, trifluoroleucine, phenylalanine or 3-thienylalanine. The proteins were designed to adopt a "lamellar" morphology in the solid state on crystallization. Incorporation of trifluoroleucine into the recombinant protein was established by elemental analysis, NMR analysis and mass spectroscopy. Spectroscopic analysis by $\sp{19}$F NMR showed that both 2S isomers of trifluoroleucine are used in protein synthesis. Periodic proteins containing leucine and the trifluoroleucine were crystallized. Structural analysis based on X-ray diffraction and infrared spectroscopy indicated that the chains form predominantly beta sheets with a lamellar morphology. Contact angle measurements indicated that the fluorinated protein had a lower surface energy compared with the non-fluorinated counterpart. These results demonstrate that protein materials with modified surface properties can be created by incorporating multiple trifluoromethyl groups at precise positions. A periodic protein containing 3-thienylalanine in place of phenylalanine was also prepared. Results from elemental and amino acid analyses, and NMR and UV spectroscopy indicated that the 3-thienylalanine was substituted at least 80% in the bulk sample and was not modified on incorporation. The 3-alkylthiophene side chain of 3-thienylalanine should be susceptible to electrochemical polymerization, opening a route to genetically engineered polymeric materials with useful electronic properties. Taken together, the results of this investigation establish the feasibility of preparing recombinant periodic proteins in vivo that contain useful amino acid analogs at multiple positions. The general strategy used should be useful in designing and fabricating additional protein-based materials with unique physicochemical properties.

The phytohormone indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. Biochemical evidence indicates that IAA can be synthesized both from tryptophan [Trp; Trp-dependent (D) pathways] and from an indolic precursor of Trp [Trp-independent (I) pathways]. At the genetic level, there is much support for Trp-D pathways, but there is little genetic evidence for Trp-I pathways. Mutants with altered Trp-I IAA synthesis were identified from genetic screens using the model plant Arabidopsis thaliana. From these screens, the allelic iss1-1 and iss1-2 mutants were identified that displayed an indole-dependent IAA overproduction phenotype consisting of fused leaves and increased lateral and adventitious root growth. iss1 mutants appear wild type (WT) when grown on medium supplemented with Trp. Using stable isotope labeling studies, iss1 and WT were both found to use primarily Trp-D synthesis when grown on unsupplemented medium. In contrast, iss1, but not WT, used primarily Trp-I synthesis, when grown on indole-supplemented medium. Surprisingly, iss1 seedlings also have a 170-fold increase in Trp when grown on indole, indicating that the increase in Trp-I IAA is not due to a loss of indole to Trp conversion but is suggestive of a role for ISS1 in Trp catabolism. Using map-based cloning, ISS1 was identified as an uncharacterized aminotransferase that is distantly related to aromatic aminotransferase (AroATs). ISS1 is highly conserved across the plant kingdom; however none of these ISS-related genes have been characterized. To demonstrate that ISS1 is an AroAT, heterologous expression of the ISS1 cDNA was found to fully rescue the yeast aro8 aro9 double mutant that is defective in redundant AroATs needed for the production of phenylalanine (Phe) and tyrosine (Tyr) in yeast. Based on the data presented in this thesis, the most likely role for ISS1 in planta is in Trp catabolism. However, the iss1 mutant also showed a decrease in the Phe-derived metabolite, coniferin, suggesting an additional role (either direct or indirect) for ISS1 in Phe metabolism. Although the precise role of ISS1 remains to be determined, these results presented provide genetic evidence for the existence of the Trp-I IAA pathway.

ABSTRACT Approximately 70% of patients with type I allergy in America, Europe, and Australia display lgE reactivity to grass pollen allergens. Timothy grass pollen is one of the most common in Europe. Bahia grass pollen is one of the more important grass aeroallergens in the Southeastern United States and is especially prevalent in the Gulf coast states. Cross­ reactivity between the proteins of these two grass pollens has not previously been demonstrated. A 32 kDa protein allergen ofbahia has, however, now been shown to share 63% homology with a Group I timothy grass pollen allergen. The purpose of the study reported here is to further evaluate bahia pollen proteins for their cross-reactivity with timothy using a series of monoclonal antibodies produced to Group I, V, and XIII timothy grass allergens. Protein extracts ofbahia grass pollen, timothy grass pollen, and a tree pollen control were evaluated for their ability to bind to IG 12, a monoclonal antibody directed against Group I timothy allergens, and a series of monoclonal antibodies directed against Group XIII timothy allergens, using Western blotting. The group I monoclonal reacted, not only with the timothy crude extract pollens, but also to a number of proteins in the crude extract and partially purified extracts ofbahia. The group XIII monoclonals reacted with a 55kDa protein of timothy and a protein ofbahia of similar molecular weight. The amino acid sequence similarity and the ability ofbahia proteins to react with monoclonal antibodies directed against timothy Group I and XIII allergens suggest that these two grass pollens share common epitopes and are cross reactive.

Prostate cancer is the second leading cause of cancer-related death, and the most commonly diagnosed type of cancer in men. By conventional paradigm, serine proteases have been shown to play a significant role in cancer cell invasion. Prostasin is a serine protease found in abundance in prostate epithelial cells, and also in other tissues and cell types in the body. Prostasin is absent in prostate cancer cell lines. When re-expressed in these prostate cancer cell lines, the invasiveness was decreased. The structural domain responsible for the anti-invasion activity exhibited by prostasin has not yet been characterized. Through site-directed mutagenesis of the serine active residue to an alanine residue, the role that the serine active site plays in anti- invasion can be explored. The cDNA clone of the mutated prostasin was transfected into the 293-EBNA cell line and cultured (293/ProM). The alanine-prostasin mutant, Ala-Pro, was partially purified and confirmed by Western blotting. Additionally, a binding assay was performed using mPBP (mouse prostasin-binding protein), a serpin class serine protease inhibitor. By way of immunoblotting, it was shown that normal wild-type prostasin forms an 82-kDa complex with the mPBP; however, the mutated prostasin does not. In future studies the invasiveness of prostate cancer cells transfected with the mutant prostasin cDNA can be investigated to determine whether prostasin's serine protease activity provides a mechanism for anti-invasion.

The embryonic cell cycle of Xenopus laevis consists of rapid oscillations between S and M phase occurring in the absence of gap phases and their associated regulatory checkpoints. The end of the 12th cell cycle marks the onset of the midblastula transition (MBT) when the cell cycle lengthens establishing gap phases, their associated checkpoint pathways, and the initiation of zygotic transcription. During cell cycles 2-12, cyclins A and B are translated and expressed once per cell cycle until the MBT when their protein levels decrease due to a newly transcribed zygotic factor that leads to the deadenylation and subsequent loss of their mRNAs. In contrast, cyclin E is expressed at a constitutively high level during cell cycles 2-12. Furthermore, cyclin E levels are terminally lost coincident with initiation of the MBT in the continued presence of its adenylated mRNA. Terminal disappearance of cyclin E appears to be maternally directed and is not affected by zygotic transcription, translation, replication, or the nuclear to cytoplasmic ratio. This has led to the hypothesis that cyclin E is part of an autonomous maternally directed timer that determines the timing of the MBT. To investigate this possibility we have used antisense oligonucleotides to knockdown cyclin E and assess the affects on the timing of the MBT. Premature knockdown of cyclin E did not affect the timing of the MBT indicating it is not part of the maternal timer. Furthermore, prior to the MBT cyclin E protein has an unusually long half life. However, despite an increased stability the constitutively high levels of cyclin E require a low level of translation. We have also determined that the stable pattern of adenylation observed for cyclin E1 is specified by three cis-acting elements in its' 3' UTR. Deletion of the NPS, eCPE/ARE3, and ARE2 abolished adenylation. Additionally, a putative stem loop in ARE2 is targeted by ElrA the Xenopus homolog of HuR and a member of the ELAV gene family. Loss of adenylation required disruption of ElrA binding. These findings demonstrate ElrA functions in the correct adenylation of cyclin E1 mRNA.

The innate immune system provides nonspecific defenses against pathogens. Many diseases occur because of malfunctions in the innate immune system. In the present thesis, I have investigated two independent mechanisms of innate immunity in the lung and liver. Both mechanisms involve responses to bacterial infection and/or components of the bacterial wall (lipopolysaccharide). In the first model, I studied the role of submucosal glands in lung innate immunity through the use of tracheal xenograft airways with and without glands. This work provides evidence that submucosal glands are a major source of antibacterials that are critical for maintaining sterile airways. In the second model, I studied host responses to a gram-negative bacterial cell wall component (lipopolysaccharide) and how the liver coordinates cytokine responses that lead to endotoxic shock. This work examined how hepatic induction of NFkappaB and TNFalpha influenced survival in this lethal murine model of endotoxemic shock. My findings suggest that during the course of lethal endotoxic shock, NFkappaB activation has a predominantly pro-inflammatory effect in the liver through the induction of TNFalpha, and that TNFalpha influences the role of NFkappaB as an anti-apoptotic factor in the liver. In conclusion, my thesis suggests that maintaining a homeostatic balance in response to pathogens is an important function of the complex innate immune system.

The KpnI, 1.2 and 1.5 kb families of interspersed repetitive DNAs from the African green monkey genome were isolated and characterized. Each family contains three populations of segments based on their sequence lengths and susceptibility to cleavage by the restriction enzymes KpnI and RsaI. The first population contains the smallest segments which are susceptible to both KpnI and RsaI cleavage and have fragment lengths of 1.2 kb (1.2 kb family) and 1.5 kb (1.5 kb family) respectively. The members in this population are referred to as KpnI-sensitive segments. The second population contains longer segments (> 2 kb) which represent fusions of members from different families. The fusion sequences are cleaved by KpnI at their termini but lack internal KpnI sites at the junctions that join the individual component members. The third population contains members from each family that are cleaved occasionally by KpnI (KpnI-resistant segments) and remained linked to the bulk of the high molecular weight DNA. KpnI 1.2 kb, 1.5 kb and KpnI-resistant populations were isolated and analyzed for the presence of internal RsaI sites. All members from both populations were cleaved by RsaI into a simple series of low molecular weight fragments. Some members from both the KpnI-sensitive and the KpnI-resistant populations were found to contain internal RsaI sites. Other members from both populations lacked internal RsaI sites. Genomic KpnI 1.2 kb segments were cloned and two recombinants pBK(1.2)14 and pBK(1.2)39 identified. The partial nucleotide sequence of clone Kpn(1.2)14 was determined. The sequence content of KpnI 1.2 and 1.5 kb families in DNA fragments that anchor DNA loops to the nuclear matrix (att-DNA) was also studied. The relative sequence content of both 1.2 and 1.5 kb families was found to be impoverished when compared to their content in total nuclear DNA. However, members in each family were found to be present in detectable amounts. The association of KpnI 1.2 and 1.5 kb family sequences with the nuclear matrix was also demonstrated by metrizamide gradient centrifugation of nuclear matrix complexes. The results suggest that some KpnI 1.2 and 1.5 kb segments are differentially associated with nuclear proteins.

Mutagenesis of the defective transducing bacteriophage lambda fus2 was used to isolate a collection of temperature-sensitive mutants of E. coli in the major ribosomal protein gene cluster. Four mutants were examined in detail. Two of the mutants were resistant to the ribosomal antibiotics neamine and spectinomycin. Another mutant was defective in 50S ribosomal subunit assembly at 42(DEGREES)C. The 30S subunit proteins S17 and S19 were changed in two different mutants. Each protein migrated as a more basic species in two-dimensional gels of ribosomal proteins. Ribosomes from each of the four mutants examined showed a temperature-dependent reduction in translational activity in cell-free assays. The kinetic assays showed declines in both the rate and extent of translation at three temperatures. Ribosomes from three of the four mutants were also found to have an increased rate of heat inactivation at 45(DEGREES)C compared to control particles. Mixed subunit assays idendtified a t.s. subunit in each mutant. A defect in reassociation at high temperature was found for the subunits from one mutant. Another mutant showed significantly high levels of misreading at 32(DEGREES)C and 42(DEGREES)C. Two mutants showed a decreased ability to bind 14C-phenylalanine tRNA at the two temperatures tested. The increased efficiency and utility of this mutagenesis method for the isolation of protein synthesis mutants is discussed.

<p>In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations.</p><p>Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species.</p><p>Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain.</p><p>In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing.</p><p>Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.</p>