Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

CHARACTERIZATION OF THE COUNT RATE PERFORMANCE AND EVALUATION OF THE EFFECTS OF HIGH COUNT RATES ON MODERN GAMMA CAMERAS

CHARACTERIZATION OF THE COUNT RATE PERFORMANCE AND EVALUATION OF THE EFFECTS OF HIGH COUNT RATES ON MODERN GAMMA CAMERAS Michael Stephen Silosky, B.S. Supervisory Professor: S. Cheenu Kappadath, Ph.D. Evaluation of count rate performance (CRP) is an integral component of gamma camera quality assurance and measurement of system dead time (τ) is important for quantitative SPECT. The CRP of three modern gamma cameras was characterized using established methods (Decay and Dual Source) under a variety of experimental conditions. For the Decay method, input count rate was plotted against observed count rate and fit to the paralyzable detector model (PDM) to estimate τ (Rates method). A novel expression for observed counts as a function of measurement time interval was derived and the observed counts were fit to this expression to estimate τ (Counts method). Correlation and Bland-Altman analysis were performed to assess agreement in estimates of τ between methods. The dependencies of τ on energy window definition and incident energy spectrum were characterized. The Dual Source method was also used to estimate τ and its agreement with the Decay method under identical conditions and the effects of total activity and the ratio of source activities were investigated. Additionally, the effects of count rate on several performance metrics were evaluated. The CRP curves for each system agreed with the PDM at low count rates but deviated substantially at high count rates. Estimates of τ for the paralyzable portion of the CRP curves using the Rates and Counts methods were highly correlated (r=0.999) but with a small (~6%) difference. No significant difference was observed between the highly correlated estimates of τ using the Decay or Dual Source methods under identical experimental conditions (r=0.996). Estimates of τ increased as a power-law function with decreasing ratio of counts in the photopeak to the total counts and linearly with decreasing spectral effective energy. Dual Source method estimates of τ varied as a quadratic with the ratio of the single source to combined source activities and linearly with total activity used across a large range. Image uniformity, spatial resolution, and energy resolution degraded linearly with count rate and image distorting effects were observed. Guidelines for CRP testing and a possible method for the correction of count rate losses for clinical images have been proposed.

MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.

ANTI-TUMOR EFFECTS OF THE NOTCH PATHWAY IN GASTROINTESTINAL STROMAL TUMORS

Gastrointestinal Stromal Tumors (GIST) are sarcomas driven by gain-of-function mutations of KIT or PDGFRA. Although, the introduction of tyrosine kinase inhibitors has dramatically changed the history of this disease, evidences emerge that inhibition of KIT or PDGFRA are not sufficient to cure patients. The developmental pathway Notch has a critical role in the cell fate, regulating cell proliferation and differentiation. Dysregulation of Notch pathway has been implicated in a wide variety of cancers functioning as a tumor promoter or a tumor suppressor in a cell context dependent manner. Given that Notch activation deregulates the morphogenesis of mesenchymal cells in the GI track, that Notch acts as a tumor suppressor in neuroendocrine tumors, and finally that the cell of origin of GIST are the Interstitial Cell of Cajal that arise from a mesenchymal origin with some neuroendocrine features, we hypothesized that Notch pathway signaling may play a role in growth, survival and differentiation of GIST cells. To test this hypothesis, we genetically and pharmacologically manipulated the Notch pathway in human GIST cells. In this study, we demonstrated that constitutively active intracellular domain of Notch1 (ICN-1) expression potently induced growth arrest and downregulated KIT expression. We have performed a retrospective analysis of 15 primary GIST patients and found that high mRNA level of Hes1, a major target gene of Notch pathway, correlated with a significantly longer relapse-free survival. Therefore, we have established that treatment with the FDA approved histone deacetylase inhibitor SAHA (Vorinostat) caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch with dominant negative Hes-1 as well as pharmacological inhibition of Notch pathway with a γ-secretase inhibitor partially rescued GIST cells from SAHA treatment. Taken together these results identify anti-tumor effect of Notch1 and a negative cross-talk between Notch1 and KIT pathways in GIST. Consequently, we propose that activation of this pathway with HDAC inhibitors may be a potential therapeutic strategy for GIST patients.

A STUDY ON THE FUNCTION OF 14-3-3SIGMA IN REGULATING CANCER ENERGY METABOLISM

Metabolic reprogramming has been shown to be a major cancer hallmark providing tumor cells with significant advantages for survival, proliferation, growth, metastasis and resistance against anti-cancer therapies. Glycolysis, glutaminolysis and mitochondrial biogenesis are among the most essential cancer metabolic alterations because these pathways provide cancer cells with not only energy but also crucial metabolites to support large-scale biosynthesis, rapid proliferation and tumorigenesis. In this study, we find that 14-3-3σ suppresses all these three metabolic processes by promoting the degradation of their main driver, c-Myc. In fact, 14-3-3s significantly enhances c-Myc poly-ubiquitination and subsequent degradation, reduces c-Myc transcriptional activity, and down-regulates c-Myc-induced metabolic target genes expression. Therefore, 14-3-3σ remarkably blocks glycolysis, decreases glutaminolysis and diminishes mitochondrial mass of cancer cells both in vitro and in vivo, thereby severely suppressing cancer bioenergetics and metabolism. As a result, a high level of 14-3-3σ in tumors is strongly associated with increased breast cancer patients’ overall and metastasis-free survival as well as better clinical outcomes. Thus, this study reveals a new role for 14-3-3s as a significant regulator of cancer bioenergetics and a promising target for the development of anti-cancer metabolism therapies.

Development and Characterization of Novel Ras Inhibitors

Ras genes are mutated in 15% of human cancers. Ras GTPases operate as molecular switches regulating cellular processes including proliferation, differentiation, and apoptosis. The three main isoforms of Ras – H-Ras, K-Ras, and N-Ras – inhabit distinct nanodomains of the plasma membrane and intracellular compartments including the Golgi. However, the role of single endogenous Ras isoforms on these compartments remains unclear as most studies have utilized ectopically expressed and mutant forms of Ras proteins. In an effort to develop novel tools that will allow us to abrogate individual endogenous Ras isoforms, we targeted the catalytic domain of p120RasGAP to the plasma membrane with the hypervariable region (HVR) of H-Ras (GAP-CTH) or K-Ras (GAP-CTK) and to the Golgi using the HVR of H-Ras with insertion of a point mutation (GAP-CTH181S). We performed GST-RBD pull-downs on cells expressing each GAP construct and stimulated with epidermal growth factor (EGF). We found that GAP-CTH and GAP-CTK specifically inhibited H-Ras or K-Ras, respectively. However, we did not detect any effect of GAP-CTH181S on Ras activation. Additionally, we used confocal microscopy to verify the ability of GAP constructs to abrogate Ras activation in distinct sub-cellular compartments. We found that GAP-CTH inhibits H-Ras activation on the plasma membrane, while GAP-CTK inhibits K-Ras activation on the plasma membrane. On the contrary, GAP-CTH181S inhibited H-Ras activation on the Golgi. We also analyzed the effects of these GAP constructs on the activation of ERK and Akt in response to EGF stimulation. We found that EGF stimulation of the MAPK pathway was inhibited by GAP-CTK but none of the other GAP constructs, while Akt activation was not inhibited by any GAP construct. Finally, we assayed cellular proliferation and differentiation. We found that GAP-CTK and GAP-CTH were equipotent inhibitors of cellular growth, whereas GAP-CTH181S was less potent. We also found that GAP-CTK and GAP-CTH inhibited differentiation with similar potency, while GAP-CTH181S was more potent. This approach may be adapted to investigate any Ras-dependent signaling pathway. Therefore, it has the potential to become a powerful tool for studying Ras isoform-specific signaling outputs.

FANCM AND FAAP24 MAINTAIN GENOMIC STABILITY THROUGH COOPERATIVE AND UNIQUE FUNCTIONS

Fanconi anemia (FA) is a rare recessive genetic disease with an array of clinical manifestations including multiple congenital abnormalities, progressive bone marrow failure and profound cancer susceptibility. A hallmark of cells derived from FA patients is hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C (MMC) and cisplatin, suggesting that FA- and FA-associated proteins play important roles in protecting cells from DNA interstrand crosslink (ICL) damage. Two genes involved in the FA pathway, FANCM and FAAP24, are of particular interest because they contain DNA interacting domains. However, there are no definitive patient mutations for these two genes, and the resulting lack of human genetic model system renders their functional studies difficult. In this study, I established isogenic human FANCM- and FAAP24-null mutants through homologous replacement-mediated gene targeting in HCT-116 cells, and systematically investigated the functions of FANCM and FAAP24 inchromosome stability, FA pathway activation, DNA damage checkpoint signaling, and ICL repair. I found that the FANCM-/-/FAAP24-/- double mutant was much more sensitive to DNA crosslinking agents than FANCM-/- and FAAP24-/- single mutants, suggesting that FANCM and FAAP24 possess epistatic as well as unique functions in response to ICL damage. I demonstrated that FANCM and FAAP24 coordinately support the activation of FA pathway by promoting chromatin localization of FA core complex and FANCD2 monoubiqutination. They also cooperatively function to suppress sister chromatid exchange and radial chromosome formation, likely by limiting crossovers in recombination repair. In addition, I defined novel non-overlapping functions of FANCM and FAAP24 in response to ICL damage. FAAP24 plays a major role in activating ICL-induced ATR-dependent checkpoint, which is independent of its interaction with FANCM. On the other hand, FANCM promotes recombination-independent ICL repair independently of FAAP24. Mechanistically, FANCM facilitates recruitment of nucleotide excision repair machinery and lesion bypass factors to ICL damage sites through its translocase activity. Collectively, my studies provide mechanistic insights into how genome integrity is both coordinately and independently protected by FANCM and FAAP24.

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

The Role of the Arched Helicases in Exosome-Mediated Function

RNA processing and degradation are two important functions that control gene expression and promote RNA fidelity in the cell. A major ribonuclease complex, called the exosome, is involved in both of these processes. The exosome is composed of ten essential proteins with only one catalytically active subunit, called Rrp44. While the same ten essential subunits make up both the nuclear and cytoplasmic exosome, there are nuclear and cytoplasmic exosome cofactors that promote specific exosome functions in each of the cell compartments. To date, it is unclear how the exosome distinguishes between RNA substrates. We hypothesize that compartment specific cofactors may promote the substrate specificity of the exosome. In this work, I characterize several cofactors of the exosome, both nuclear and cytoplasmic. First, I describe the arch domain, which is a unique domain in a nuclear and a cytoplasmic cofactor of the exosome. Specifically, I show that the arch domain of the nuclear exosome cofactor, Mtr4, is required for specific exosome-mediated activities and overlaps functionally with the exosome-associated exonuclease, Rrp6. Further, I show that the arch domain of Ski2 is required for the degradation of normal and aberrant mRNAs. Additionally, this work describes in detail the Mtr4 domains involved in the physical association with other RNA processing proteins. Further, I characterize the minimal Mtr4-binding region in a third exosome cofactor, Trf5. Understanding how exosome cofactors synergistically promote exosome function will provide us a better understanding of how the exosome complex precisely regulates its catalytic activities. As described here, cofactors play a major role in determining the substrate specificity of the nuclear and cytoplasmic exosome. Moreover, specific accessory domains, which are not involved in the catalytic function of the cofactor, are required for substrate targeting of the eukaryotic RNA exosome.

Design and Optimization of Four-dimensional Cone-beam Computed Tomography in Image-guided Radiation Therapy

The influence of respiratory motion on patient anatomy poses a challenge to accurate radiation therapy, especially in lung cancer treatment. Modern radiation therapy planning uses models of tumor respiratory motion to account for target motion in targeting. The tumor motion model can be verified on a per-treatment session basis with four-dimensional cone-beam computed tomography (4D-CBCT), which acquires an image set of the dynamic target throughout the respiratory cycle during the therapy session. 4D-CBCT is undersampled if the scan time is too short. However, short scan time is desirable in clinical practice to reduce patient setup time. This dissertation presents the design and optimization of 4D-CBCT to reduce the impact of undersampling artifacts with short scan times. This work measures the impact of undersampling artifacts on the accuracy of target motion measurement under different sampling conditions and for various object sizes and motions. The results provide a minimum scan time such that the target tracking error is less than a specified tolerance. This work also presents new image reconstruction algorithms for reducing undersampling artifacts in undersampled datasets by taking advantage of the assumption that the relevant motion of interest is contained within a volume-of-interest (VOI). It is shown that the VOI-based reconstruction provides more accurate image intensity than standard reconstruction. The VOI-based reconstruction produced 43% fewer least-squares error inside the VOI and 84% fewer error throughout the image in a study designed to simulate target motion. The VOI-based reconstruction approach can reduce acquisition time and improve image quality in 4D-CBCT.

TRIM24-REGULATED ESTROGEN RESPONSE IS DEPENDENT ON SPECIFIC HISTONE MODIFICATIONS IN BREAST CANCER CELLS

In this dissertation, I discovered that function of TRIM24 as a co-activator of ERα-mediated transcriptional activation is dependent on specific histone modifications in tumorigenic human breast cancer-derived MCF7 cells. In the first part, I proved that TRIM24-PHD finger domain, which recognizes unmethylated histone H3 lysine K4 (H3K4me0), is critical for ERα-regulated transcription. Therefore, when LSD1-mediated demethylation of H3K4 is inhibited, activation of TRIM24-regulated ERα target genes is greatly impaired. Importantly, I demonstrated that TRIM24 and LSD1 are cyclically recruited to estrogen responsive elements (EREs) in a time-dependent manner upon estrogen induction, and depletion of their expression exert corresponding time-dependent effect on target gene activation. I also identified that phosphorylation of histone H3 threonine T6 disrupts TRIM24 from binding to the chromatin and from activating ERα-regulated targets. In the second part, I revealed that TRIM24 depletion has additive effect to LSD1 inhibitor- and Tamoxifen-mediated reduction in survival and proliferation in breast cancer cells.

Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention

Despite having been identified over thirty years ago and definitively established as having a critical role in driving tumor growth and predicting for resistance to therapy, the KRAS oncogene remains a target in cancer for which there is no effective treatment. KRas is activated b y mutations at a few sites, primarily amino acid substitutions at codon 12 which promote a constitutively active state. I have found that different amino acid substitutions at codon 12 can activate different KRas downstream signaling pathways, determine clonogenic growth potential and determine patient response to molecularly targeted therapies. Computer modeling of the KRas structure shows that different amino acids substituted at the codon 12 position influences how KRas interacts with its effecters. In the absence of a direct inhibitor of mutant KRas several agents have recently entered clinical trials alone and in combination directly targeting two of the common downstream effecter pathways of KRas, namely the Mapk pathway and the Akt pathway. These inhibitors were evaluated for efficacy against different KRAS activating mutations. An isogenic panel of colorectal cells with wild type KRas replaced with KRas G12C, G12D, or G12V at the endogenous loci differed in sensitivity to Mek and Akt inhibition. In contrast, screening was performed in a broad panel of lung cell lines alone and no correlation was seen between types of activating KRAS mutation due to concurrent oncogenic lesions. To find a new method to inhibit KRAS driven tumors, siRNA screens were performed in isogenic lines with and without active KRas. The knockdown of CNKSR1 (CNK1) showed selective growth inhibition in cells with an oncogenic KRAS. The deletion of CNK1 reduces expression of mitotic cell cycle proteins and arrests cells with active KRas in the G1 phase of the cell cycle similar to the deletion of an activated KRas regardless of activating substitution. CNK1 has a PH domain responsible for localizing it to membrane lipids making KRas potentially amenable to inhibition with small molecules. The work has identified a series of small molecules capable of binding to this PH domain and inhibiting CNK1 facilitated KRas signaling.

INDUCTION OF STRONGER AND LONG LASTING VACCINE IMMUNITY TO TUBERCULOSIS

Tuberculosis is a major cause of death due to an infection in mankind. BCG vaccine protects against childhood tuberculosis although, it fails to protect against adult tuberculosis. BCG vaccine localizes to immature phagosomes of macrophages, and avoids lysosomal fusion, which decreases peptide antigen production. Peptides are essential for macrophage-mediated priming of CD4 and CD8 T cells respectively through MHC-II and MHC-I pathways. Furthermore, BCG reduces the expression of MHC-II in macrophages of mice after infection, through Toll-like receptor-1/2 (TLR-1/2) mediated signaling. In my first aim, I hypothesized that BCG-induced reduction of MHC-II levels in macrophages can decrease CD4 T cell function, while activation of other surface Toll-like receptors (TLR) can enhance CD4 T cell function. An in vitro antigen presentation model was used where, TLR activated macrophages presented an epitope of Ag85B, a major immunogen of BCG to CD4 T cells, and T cell derived IL-2 was quantitated as a measure of antigen presentation. Macrophages with BCG were poor presenters of Ag85B while, TLR-7/9/5/4 and 1/2 activation led to an enhanced antigen presentation. Furthermore, TLR-7/9 activation was found to down-regulate the degradation of MHC-II through ubiquitin ligase MARCH1, and also stimulate MHC-II expression through activation of AP-1 and CREB transcription elements via p38 and ERK1/2 MAP kinases. I conclude from Aim-I studies that TLR-7/9 ligands can be used as more effective ‘adjuvants’ for BCG vaccine. In Aim-II, I evaluated the poor CD8 T cell function in BCG vaccinated mice thought to be due to a decreased leak of antigens into cytosol from immature phagosomes, which reduces the MHC-I mediated activation of CD8 T cells. I hypothesized that rapamycin co-treatment could boost CD8 T cell function since it was known to sort BCG vaccine into lysosomes increasing peptide generation, and it also enhanced the longevity of CD8 T cells. Since CD8 T cell function is a dynamic event better measurable in vivo, mice were given BCG vaccine with or without rapamycin injections and challenged with virulent Mycobacterium tuberculosis. Organs were analysed for tetramer or surface marker stained CD8 T cells using flow cytometry, and bacterial counts of organisms for evaluation of BCG-induced protection. Co-administration of rapamycin with BCG significantly increased the numbers of CD8 T cells in mice which developed into both short living effector- SLEC type of CD8 T cells, and memory precursor effector-MPEC type of longer-living CD8 T cells. Increased levels of tetramer specific-CD8 T cells correlated with a better protection against tuberculosis in rapamycin-BCG group compared to BCG vaccinated mice. When rapamycin-BCG mice were rested and re-challenged with M.tuberculosis, MPECs underwent stronger recall expansion and protected better against re-infection than mice vaccinated with BCG alone. Since BCG induced immunity wanes with time in humans, we made two novel observations in this study that adjuvant activation of BCG vaccine and rapamycin co-treatment both lead to a stronger and longer vaccine-mediated immunity to tuberculosis.

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

Exploring the Impact of Decreased AMPK Pathway Activity on Brain Injury Outcome

Each year, 150 million people sustain a Traumatic Brain Injury (TBI). TBI results in life-long cognitive impairments for many survivors. One observed pathological alteration following TBI are changes in glucose metabolism. Altered glucose uptake occurs in the periphery as well as in the nervous system, with an acute increase in glucose uptake, followed by a prolonged metabolic suppression. Chronic, persistent suppression of brain glucose uptake occurs in TBI patients experiencing memory loss. Abberant post-injury activation of energy-sensing signaling cascades could result in perturbed cellular metabolism. AMP-activated kinase (AMPK) is a kinase that senses low ATP levels, and promotes efficient cell energy usage. AMPK promotes energy production through increasing glucose uptake via glucose transporter 4 (GLUT4). When AMPK is activated, it phosphorylates Akt Substrate of 160 kDa (AS160), a Rab GTPase activating protein that controls Glut4 translocation. Additionally, AMPK negatively regulates energy-consumption by inhibiting protein synthesis via the mechanistic Target of Rapamycin (mTOR) pathway. Given that metabolic suppression has been observed post-injury, we hypothesized that activity of the AMPK pathway is transiently decreased. As AMPK activation increases energy efficiency of the cell, we proposed that increasing AMPK activity to combat the post-injury energy crisis would improve cognitive outcome. Additionally, we expected that inhibiting AMPK targets would be detrimental. We first investigated the role of an existing state of hyperglycemia on TBI outcome, as hyperglycemia correlates with increased mortality and decreased cognitive outcome in clinical studies. Inducing hyperglycemia had no effect on outcome; however, we discovered that AMPK and AS160 phosphorylation were altered post-injury. We conducted vii work to characterize this period of AMPK suppression and found that AMPK phosphorylation was significantly decreased in the hippocampus and cortex between 24 hours and 3 days post-injury, and phosphorylation of its downstream targets was consistently altered. Based on this period of observed decreased AMPK activity, we administered an AMPK activator post-injury, and this improved cognitive outcome. Finally, to examine whether AMPK-regulated target Glut4 is involved in post-injury glucose metabolism, we applied an inhibitor and found this treatment impaired post-injury cognitive function. This work is significant, as AMPK activation may represent a new TBI therapeutic target.