<p> COPD is characterized by an abnormal regulatory T cell (Treg) response with a shift towards a Th1 and Th17 cell responses. However, it is unclear if the function of Treg cells is impaired by smoking and in COPD. In addition, the miRNA profile of Treg cells in COPD is unknown and whether miRNA deregulation contributes to COPD immunopathogenesis. We set the objective to study Treg cell function isolated from peripheral blood of patients with COPD versus controls and to compare their miRNA profiles. We also were interested in exploring the function of some of the differentially expressed Treg cell miRNAs. We assessed the Treg cell function by observing their suppressive activity on autologous effector T cells and analyzed their miRNA expression initially by microarray analysis then conducted real time RT-PCR validation for selected miRNAs. In Silico target gene analysis for the validated miRNAs suggested that miR-199-5p is particularly relevant to Treg cell physiology so its function was investigated further using CCD-986Sk and MOLT-4 cells. We found no difference in Treg cell function between COPD and controls but we were able to identify 6 and 96 miRNAs that were differentially expressed in COPD versus control Treg cells. We confirmed that miR-199a-5p was repressed by approximately 4 fold in Treg cells of COPD patients compared to cells in healthy smokers. Importantly, miR-199a-5p had significant overrepresentation of its target genes in the Treg cell transcriptome, with many targets associated with the TGF-&beta; activation pathway. We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan&reg; arrays of the Human TGF-&beta; Pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favor of a Th1 and Th17 response.</p>

<p> The yeast vacuole is a dynamic organelle that is functionally analogous to the mammalian lysosome and serves as a model for the study of membrane fusion and fission. Mechanisms of membrane fission and fusion dynamics have been well conserved from yeast to humans. However, the regulatory mechanisms that govern cellular fission and fusion dynamics remain poorly understood. Our lab has previously established that Env7 is a conserved yeast palmitoylated protein kinase that localizes to the yeast vacuole and negatively regulates vacuole membrane fusion during budding and hyperosmotic stress. Phosphorylation of Env7 is dependent on another vacuolar membrane kinase, Yck3, and is essential to Env7 stability and negative regulation of vacuolar membrane fusion. In this study, we aim to further our understanding of the role Env7 plays at the vacuole by 1) characterizing the phosphorylation of Env7 as a function of cell cycle using cell cycle arrest and synchronization techniques, and 2) generating functional biochemically tagged Yck3 to be used in interaction and phosphorylation assays with Env7. Cell cycle arrest and synchronization techniques have not previously been established in our lab. Here, we report reliable protocols of inducing cell cycle arrest using &alpha;-factor mating pheromone and Hydroxyurea. Results show that Env7 is hyperphosphorylated when cell cycle is arrested at G₁ phase using &alpha;-factor mating pheromone. In both cell cycle arrest approaches, vacuoles show significant increase in fragmentation, and Env7 remains localized to the membrane of fragmented vacuoles. In cell culture synchronized with &alpha;-factor, Env7 shows an increase in phosphorylation between S-phase and G₂, with decreased phosphorylation in M and G₁. We were successful in engineering biochemically tagged Yck3 and established that the expressed 6XHis-Yck3 is functional and able to restore phosphorylation of Env7 <i>in vivo</i>. We also established that overexpressed 6XHis-Yck3 localized correctly to the vacuolar membrane. These tools will be used in future studies on interactions and regulation of membrane fusion.</p><p>

<p> My research using the model plant <i>Arabidopsis thaliana </i> is focused on unraveling the signal transduction pathways involved in elicitor-mediated plant defense, particularly defense pathways involved in resistance to fungal pathogens. I have isolated a group of related genes, the <i>ATL</i> family, which appear to play a direct role in defense against fungal pathogens. Previous research has shown that ATL proteins can be induced by chitin and they are involved in basal resistance to the fungal pathogens. </p><p> ATL9, an <i>Arabidopsis</i> RING zinc finger protein, is an E3 ubiquitin ligase that can be induced by chitin and is involved in basal resistance to the fungal pathogen, <i>Golovinomyces cichoracearum</i> (<i>G. cichoracearum</i>). In order to understand the expression and regulation of ATL9, I studied the expression pattern of <i>ATL9</i> and the functions of its different protein domains. Using a p^ATL9:<i> GUS</i> transgenic <i>Arabidopsis</i> line I found that ATL9 is expressed in different tissues in <i>Arabidopsis</i> at various developmental stages and that GUS activity was induced rapidly upon wounding. Previous research in our lab also showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. Protein prediction software indicated that ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Data from confocal microscopy and western analysis indicate that both the PEST domain and the RING domain have effects on ATL9 degradation. To study the importance of these domains in ATL9's function, I constructed a series of deletion mutants and generated transgenic <i>Arabidopsis</i> plants. As expected, transgenic <i> Arabidopsis</i> containing the deletion constructs showed that both the RING domain and the TM domains are important to its resistance phenotype against <i> G. cichoracearum</i>. Interestingly, the PEST domain was also shown to be significant for the resistance to fungal pathogens. Additionally, I discovered that ATL9 can bind the defense related proteins FBS1, PCC1, and PDF1.2 directly and degrade them via the proteasome. Finally, I propose a hypothesized mechanism is proposed describing the function of ATL9 and its possible interaction with other proteins in enhancing the plant defense response.</p><p>

<p> Over half of all pregnancies worldwide occur in malaria endemic regions. Placental malaria, a serious condition caused by the malaria parasite <i> Plasmodium falciparum</i>, occurs when malaria-infected red blood cells adhere to the tissue of the placenta, with potentially devastating consequences for both mother and infant. Placental malaria infections are responsible for approximately 30% of preventable low birth weight newborns, 20% of stillbirths, and 200,000 infant deaths per year in Africa alone. Placental malaria infection is mediated by VAR2CSA, a <i>P. falciparum</i> protein that is expressed by the parasite only when in a pregnant woman, and translationally repressed outside of pregnancy. However, the mechanisms by which this repression and expression occur or, indeed, how the parasite senses when its host is pregnant are unknown. Elucidation of the genetic determinants of this specific translational regulation could provide insight for therapeutic development for placental infection. Additionally, further study of overall translation and its pharmacologic inhibition under &ldquo;normal&rdquo; circumstances may help identify novel therapies for malaria in general.</p><p> Utilizing a novel <i>in vitro</i> translation system derived from <i>P. falciparum</i> cultures, I have shown that synthesis of VAR2CSA is repressed under normal conditions, and that multiple elements in the 5&rsquo; untranslated region of the <i>var2csa</i> gene contribute to this repression. Further, this repression occurs only in <i>P. falciparum </i>, and not mammalian <i>in vitro</i> translation systems, indicating a <i>P. falciparum</i>-specific mechanism of inhibition. Importantly, I have found that circulating factors present in maternal serum during the first and second trimesters of pregnancy relieve repression of VAR2CSA translation, identifying two placental enzymes as candidate factors. Both enzymes serve to alter the pH of the microenvironment and, in fact, increasing pH in the <i>P. falciparum</i> <i>in vitro </i> translation system mimics the increase in VAR2CSA production induced by pregnant serum. Separately, I have utilized this <i>in vitro</i> translation system to identify inhibitors of translation among clinically approved antimalarial drugs and found that none utilize this mechanism of action. Importantly, this disproved the recent assertion that mefloquine inhibits translation, while also underscoring the therapeutic potential for targeting the translational apparatus as a novel and orthogonal mechanism of action. </p><p>

<p> Although castration-resistant prostate cancers no longer respond to anti-androgen therapies, the androgen receptor (AR) is still required to promote tumor survival. However, the signaling pathways downstream of AR that promote this survival are not well known. We recently identified an AR-dependent survival pathway whereby AR induction of integrin &alpha;6&beta;1 and adhesion to laminin activates NF-&kappa;B/RelA signaling and Bcl-xL. This pathway acts in parallel with the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin &alpha;6&beta;1 is required to effectively kill tumor cells adherent to laminin. However, PTEN-null castration-resistant tumors were not effectively killed by this combination. I discovered that BNIP3, a hypoxia-induced BH3-only, pro-mitophagic Bcl-2 family member, is induced by androgen in castration-resistant cells through integrin &alpha;6&beta;1 and HIF1&alpha;. Furthermore, castration-resistant cells adherent to laminin were much more efficient at inducing autophagy in response to androgen. Androgen blocked the ability of the PI3K inhibitor PX866 to kill castration-resistant tumors, but this was reversed by loss of BNIP3. Although BNIP3 was dispensable for androgen-induced autophagy, its mitophagy function was required for BNIP3 to promote resistance to PX866. Thus, enhanced hypoxia signaling in cooperation with AR/&alpha;6&beta;1/HIF1&alpha; signaling on laminin in castration-resistant cells drives the expression of BNIP3 and enhances autophagy, both of which contribute to PX866 resistance through induction of mitophagy.</p><p>

<p> Metastasis is the most lethal step in the progression of cancer, and the five-year survival rate for metastatic lung cancer patients is less than 5%. An essential step for metastasis is resistance to anoikis, a cell death program physiologically induced by detachment of cells from the extracellular matrix. Heat shock transcription factor 1 (HSF1) is the master regulator of heat shock proteins (HSP), and HSF1 and HSP promote cell survival and protein homeostasis during stress. In cancer, HSF1 dynamically controls a network of genes beyond HSP, is a mediator of malignant transformation, and promotes metastasis. HSF1 has been linked to anchorage-independent growth, but whether it exerts its effect by supporting anoikis resistance is largely unknown. Using NSCLC cells, we identified HSF1 as a novel supporter of anoikis resistance. Knockdown of HSF1 sensitizes NSCLC cells to anoikis, yet HSF1 expression or activation does not confer anoikis resistance to normal bronchial epithelial cells, suggesting parallel oncogenic pathways may be required to inhibit anoikis. Consistent with the ability of HSF1 to regulate HSP, HSF1 knockdown partially inhibited HSP72, HSP40, and HSP27. However, targeted inhibition of each HSP did not induce anoikis, suggesting the mechanism of HSF1 is unrelated to these HSP. Intriguingly, HSF1 activation markers were increased in response to cell detachment in H460 cells. Except for HSP60 in A549 cells, cell detachment did not induce HSP, further suggesting an alternative mechanism for HSF1. Interestingly, knockdown of HSP60 sensitized A549 cells to anoikis, despite HSF1 knockdown having no effect on HSP60. This work provides novel evidence that HSF1 and HSP60 can promote anchorage independence by supporting anoikis resistance, and may be valuable targets for future efforts to therapeutically suppress metastasis.</p><p>

<p> The majority of breast cancers express the estrogen receptor (ER+) and are treated with anti-estrogen therapies, particularly the inhibitor tamoxifen. However, many women treated with tamoxifen develop resistance, leading to metastatic disease, which is responsible for the majority of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen sensitive and resistant breast cancer cells, a patient derived tamoxifen-resistant xenograft model, and genome-wide transcription and translation studies, we show that tamoxifen resistance is mediated by selective mRNA translational reprogramming. Tamoxifen resistant translation is mediated by increased expression of translation factor eIF4E, increased mTOR activity to promote eIF4E availability, and increased MNK activity to promote eIF4E Ser209 phosphorylation. Tamoxifen re-sensitization is restored only by reducing eIF4E expression or mTOR activity and blocking MNK1-directed eIF4E phosphorylation. Of the translationally upregulated mRNAs specific to tamoxifen resistant cells, we show that Runx2, which encodes a regulator of ER signaling that antagonizes estrogen responses and promotes breast cancer metastasis, significantly increases tamoxifen resistance and restores sensitivity when silenced. Moreover, tamoxifen resistant but not sensitive patient ER+ breast cancer specimens demonstrate strongly increased levels of mTOR and MNK activity and eIF4E protein. eIF4E levels, availability and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through translatome reprogramming.</p><p>

<p> Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo <i>de novo</i> vessel formation.&nbsp;Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess <i> in vitro</i> endothelial colony forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over-expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.</p><p>

<p> Enhancers are regulatory elements that orchestrate cell type specific gene expression patterns. They can be separated from their target genes by large distances and activate transcription by coming into physical proximity with promoters in three-dimensional nuclear space. Complex regulatory networks with multiple enhancers often cooperate to control the same target gene. Antigen receptor loci have proved to be a rich ground for understanding enhancer-mediated gene regulation. The loci undergo somatic recombination of their V, (D), J segments to create a diverse repertoire of antigen receptors that can counteract a wide range of foreign antigens. V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor loci in a lineage and cell stage specific manner. Unexpectedly we find that both active and inactive antigen receptor loci enhancers cooperate to disseminate their effects in a localized and long-range mode. We demonstrate the importance of short-range contacts between active enhancers that constitute an <i>Igk</i> super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each enhancer. We further establish that in T cells, the <i> Igk</i> enhancer MiE&kappa; exerts a long-range effect on another antigen receptor locus <i>Tcrb</i> located 29MB away on chromosome 6. MiE&kappa; deletion leads to inefficient <i>Tcrb</i> recombination resulting in a block in T cell development, an effect that is associated with a long-range contact and cooperation between the MiE&kappa; and the <i> Tcrb</i> enhancer, E&beta;. MiE&kappa; deletion alters enrichment of the transcription factor CBF&beta; on E&beta; in a manner that impacts <i> Tcrb</i> recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer sharing between active and inactive elements in lineage and stage specific control. Finally, we expand our assessment of enhancer cooperation to the entire chromosome 6. We detect nearly nine hundred putative regulatory elements that are active in either DP or pre-B cells with less than 20% common to the two cell types. We also demonstrate how long-range contacts between enhancers and promoters coincide with target gene expression status, and provide a resource for identifying the regulatory elements that control T and B cell specific gene expression patterns.</p><p>

<p> Autophagy is a cellular recycling process in which cytoplasmic proteins and organelles are sequestered in a double membrane vesicle, delivered to the lysosome, and degraded following fusion of the two vesicles. A key part of the initiation signaling for autophagy is the generation of phosphoinositol 3-phosphate (P13P) by class III phosphoinositol 3-kinase also knows as Vps 34. In humans there are eight P13K isoforms divided into three classes, four class I enzymes, three class II enzymes, and a single class III enzyme. Of these eight enzymes, only the class III isoform is thought to participate directly in autophagic signaling. A quantitative microscopy based, loss-of-function survey of all eight P13K isoforms was used to determine their relative contribution to autophagic signaling, as measured by LC3 positive autophagic vesicles. As predicted, knockdown of P13K-class III reduced the number of autophagic vesicles in cells. Interestingly, knockdown of the P13K-class II&alpha; isoform had an even more potent effect on reducing the number of autophagic vesicles than knockdown of P13K-class III. In follow up studies, knockdown of P13K-class II&alpha; reduced endogenous LC3 conversion, caused the accumulation of p62 and lipid droplets, and colocalized with endosomal markers. These results suggest P13K-class II&alpha; may act to promote autophagy through the shuttling of endosomal vesicles into the autophagic pathway and approaches to test this hypothesis will be discussed. The requirement of P13K-class II&alpha; for autophagy is an important finding as it indicates a role for class II P13Ks in autophagy.</p>