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.

Environmental exposures during a critical window of developmental plasticity can alter disease susceptibility later in life. The nuclear receptor CAR (constitutive androstane receptor) is a known xenobiotic sensor that is activated by environmental chemicals to regulate the transcription of genes involved in hepatic drug metabolism, energy homeostasis, and hepatic carcinogenesis. This study investigated long-term gene expression changes in adult mouse liver followed by neonatal activation of CAR by its agonists at environmentally and physiologically relevant doses. The results demonstrated that previously reported long-term expression changes of Cyp2 genes, which encode monooxygenases, caused by neonatal exposure to the CAR-specific agonist, TCPOBOP (1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene), were likely due to the persistence of residual TCPOBOP in the liver. Additionally, I showed that the neonatal exposure to another CAR agonist, phenobarbital, could induce altered Cyp2 gene regulation at adulthood, which may be due to epigenetic reprogramming. These findings provide new insights into the role of CAR in liver physiology and the impact of early life environmental exposures on disease susceptibility. They also highlight the importance of careful experimental design in studying the long-term effects of environmental chemicals and could have implications for the development of therapeutics targeting CAR in liver- related disorders.

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.

Acute Myeloid Leukemia (AML) cells - like many other cancer cells - exploit lipid metabolic pathways to support their high demands for energy and biomass, while maintaining an immature phenotype. Though lipid synthesis and catabolism may be equally important to sustain AML cell survival, much of the focus in the field has been on the oxidation of fatty acids (FAs).To help close this gap, we queried publicly available data to identify FA biosynthetic enzymes that may be essential for AML cells. We found that the expression of Fatty Acid Desaturase 1 (FADS1) – a key regulator of polyunsaturated fatty acid (PUFA) biosynthesis – significantly correlates with poor outcomes in AML and is enriched in subtypes associated with chemotherapy resistance and intermediate-to-poor prognoses. Through an shRNA-mediated loss-of-function approach, we have demonstrated that FADS1 downregulation slows leukemia cell proliferation in vitro and impedes disease propagation in vivo. Interestingly, pharmacological inhibition of FADS1 selectively impairs AML cell colony formation while sparing normal hematopoietic stem and progenitor cells. FADS1-deficient cells display increased expression of mature myeloid markers, distinct morphology, and superior phagocytosis capabilities compared to control cells. These results suggest that disruption of PUFA biosynthesis pushes AML cells towards differentiation, overcoming a block that is a hallmark of malignant hematopoiesis. Next, we employed mass spectrometry to examine complex lipids and found that FADS1 knockdown (FADS1 KD) cells accumulate lipids containing very long-chain fatty acids (VLCFAs – i.e., ≥ 22 carbons). These FA species are mostly saturated and monounsaturated and such accumulation is found across several lipid classes such as diglycerides, triglycerides, and phospholipids. To determine the source of VLCFAs upon FADS1 KD, we measured the expression of several genes involved in lipid synthesis and catabolism but found no change that could explain our observations. Strikingly, the mRNA and protein expression of CD36, a scavenger receptor that mediates FA import, is increased 4-fold in FADS1-deficient cells. We speculate that AML cells become heavily reliant on FA import after FADS1 KD due to an inability to assemble building blocks used for proliferation. The greater influx of FA then directly boosts the activity of elongase enzymes by increasing substrate availability. Because membrane lipid composition can influence cellular function, we performed an RNA-Seq analysis to identify molecular pathways that may be responsible for the anti-leukemia effects of FADS1 downmodulation. This analysis revealed that FADS1 KD dramatically upregulates pathways related to plasma membrane function such as GTPase and integrin binding, and membrane microdomain. FADS1-deficient cells also activate STING-dependent type I interferon responses and toll-like receptor signaling. Remarkably, genetic deletion of STING mitigated the anti-leukemia effects of FADS1 inhibition indicating that AML cells rely on PUFA homeostasis to preserve a malignant state. Highlighting the therapeutic potential of these findings, pharmacological interrogation of PUFA biosynthesis cooperated with a STING agonist to potentiate AML cell death. Collectively, our data supports the hypothesis that FADS1 reinforces the differentiation blockade and molecular pathogenesis of AML, unveiling a previously unrecognized vulnerability in this aggressive type of blood cancer. / Biomedical Sciences

MicroRNAs (miRNAs) are short non-coding sequences that function in maintaining tissue and cell specificity. These characteristics have made them ideal candidiates for body fluid identification. This study aims to determine if miRNA expression profiles can be used to differentiate vaginal fluid samples collected at different time points within a single menstrual cycle, as well as miRNA markers that can be used to differentiate menstrual blood from peripheral blood. The HTG EdgeSeq® workflow was used to prepare and analyze the samples. The HTG Reveal software was used to analyze the expression profiles of these samples by conducting pairwise comparisons. The top differentially expressed probes based on “rawP” values were assessed for observable trends in their fold changes. Potential miRNA markers for vaginal fluid, peripheral blood, and menstrual blood in this study and those from existing literature were identified. Week 3 appears to be the most different among the vaginal fluid only samples within the menstrual cycle. Minimal differences were observed between the samples of Weeks 1 and 2, thus the identified differentially expressed probes could not be reliably used to differentiate all timepoints in the menstrual cycle. MiR-200b-3p is the only probe out of four possible menstrual blood markers with supporting evidence from the data collected in this study and in literature. Several other miRNAs (miR-106a, miR-144-3p, miR-16-5p, miR-25-3p, miR-451a, miR-486-5p, and miR-93-5p) show potential as blood markers without distinguishing between peripheral and menstrual blood. Possible miRNA markers for vaginal fluid in general were also determined: miR-124-3p, miR-128-1-5, mir-147b, miR-193b-5p, miR-5585-3p, and miR-612.

Biofluid identification plays a pivotal role in forensic case investigation, as it assists in crime scene reconstruction and provides guidance for subsequent analysis. Current forensic biofluid detection techniques, such as alternate light sources, catalytic color tests, and lateral flow immunochromatographic assays, exhibit limitations in terms of sensitivity and specificity, and most can only detect one biofluid at a time. Developing a multiplex and confirmatory assay will significantly benefit forensic biofluid identification, with nucleic acid-based assays emerging as the most promising candidates. Among various nucleic acids, microRNAs boast biofluid specificity and exceptional stability, rendering them an optimal choice for biofluid identification assay and the focal point of this study. In this study, we investigated a semi-automated, extraction-free microRNA gene expression profiling method developed by HTG Molecular Diagnostics, Inc., namely HTG EdgeSeq microRNA Whole Transcriptome Assay, utilizing seven biofluid types frequently encountered in sexual assault cases, including peripheral blood, menstrual blood, saliva, vaginal fluid, and semen with varying sperm counts. The research focused on three aspects: the assay’s compatibility with whole blood samples and dried biofluid swab samples, its ability to identify and classify different biofluid types based on their microRNA expression patterns, and its potential to differentiate biofluids within mixtures. Our results demonstrate that HTG EdgeSeq microRNA Whole Transcriptome Assay effectively identified several differentially expressed microRNAs in each biofluid. Examples that align with the existing literature include miR-451a and 486-5p for all kinds of blood, miR-185-5p for peripheral blood, and miR-888-5p and 891a-5p for semen. Previously unreported microRNAs with biofluid-specific expressions were also discovered, such as miR-4306 for peripheral blood and miR-184 for saliva. The scatter plots and heat maps generated from experimental data exhibit distinctive microRNA expression profiles for different biofluid types, potentially enabling their identification and classification. Furthermore, the analysis of mixtures reveals that differentially expressed microRNAs in mixtures corresponded to those detected in the single-source biofluids, signifying the assay’s capacity to resolve biofluid mixtures. The assay also yields optimal results for whole blood and dried biofluid swab samples, displaying its compatibility with sample types frequently found in forensic casework. The outcomes from this study suggest the potential of HTG EdgeSeq microRNA Whole Transcriptome Assay to be employed in forensic biofluid identification, as it demonstrates high reproducibility, multiplex capacities, and compatibility with common forensic casework samples. Future studies encompassing a broader spectrum of biofluid types, and a greater number of individuals are required to validate the findings in this study and optimize the workflow for applying the assay in forensic laboratories.

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.

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.