Dissecting the Role of a lncRNA and Involvement of \u3cem\u3ePlasmodium\u3c/em\u3e Infections in the Innate Immune Response: A Dissertation

The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections

Genetic Models Reveal cis and trans Immune-Regulatory Activities for lincRNA-Cox2

An inducible gene expression program is a hallmark of the host inflammatory response. Recently, long intergenic non-coding RNAs (lincRNAs) have been shown to regulate the magnitude, duration, and resolution of these responses. Among these is lincRNA-Cox2, a dynamically regulated gene that broadly controls immune gene expression. To evaluate the in vivo functions of this lincRNA, we characterized multiple models of lincRNA-Cox2-deficient mice. LincRNA-Cox2-deficient macrophages and murine tissues had altered expression of inflammatory genes. Transcriptomic studies from various tissues revealed that deletion of the lincRNA-Cox2 locus also strongly impaired the basal and inducible expression of the neighboring gene prostaglandin-endoperoxide synthase (Ptgs2), encoding cyclooxygenase-2, a key enzyme in the prostaglandin biosynthesis pathway. By utilizing different genetic manipulations in vitro and in vivo, we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate Ptgs2. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo

The long non-coding RNA LUCAT1 is a negative feedback regulator of interferon responses in humans

Long non-coding RNAs are important regulators of biological processes including immune responses. The immunoregulatory functions of lncRNAs have been revealed primarily in murine models with limited understanding of lncRNAs in human immune responses. Here, we identify lncRNA LUCAT1 which is upregulated in human myeloid cells stimulated with lipopolysaccharide and other innate immune stimuli. Targeted deletion of LUCAT1 in myeloid cells increases expression of type I interferon stimulated genes in response to LPS. By contrast, increased LUCAT1 expression results in a reduction of the inducible ISG response. In activated cells, LUCAT1 is enriched in the nucleus where it associates with chromatin. Further, LUCAT1 limits transcription of interferon stimulated genes by interacting with STAT1 in the nucleus. Together, our study highlights the role of the lncRNA LUCAT1 as a post-induction feedback regulator which functions to restrain the immune response in human cells

Suppression of systemic autoimmunity by the innate immune adaptor STING

Cytosolic DNA-sensing pathways that signal via Stimulator of interferon genes (STING) mediate immunity to pathogens and also promote autoimmune pathology in DNaseII- and DNaseIII-deficient mice. In contrast, we report here that STING potently suppresses inflammation in a model of systemic lupus erythematosus (SLE). Lymphoid hypertrophy, autoantibody production, serum cytokine levels, and other indicators of immune activation were markedly increased in STING-deficient autoimmune-prone mice compared with STING-sufficient littermates. As a result, STING-deficient autoimmune-prone mice had significantly shorter lifespans than controls. Importantly, Toll-like receptor (TLR)-dependent systemic inflammation during 2,6,10,14-tetramethylpentadecane (TMPD)-mediated peritonitis was similarly aggravated in STING-deficient mice. Mechanistically, STING-deficient macrophages failed to express negative regulators of immune activation and thus were hyperresponsive to TLR ligands, producing abnormally high levels of proinflammatory cytokines. This hyperreactivity corresponds to dramatically elevated numbers of inflammatory macrophages and granulocytes in vivo. Collectively these findings reveal an unexpected negative regulatory role for STING, having important implications for STING-directed therapies

Innovation and access to technologies for sustainable development: diagnosing weaknesses and identifying interventions in the Transnational Arena

Sustainable development – improving human well-being across present generations without compromising the ability of future generations to meet their own needs – is a central challenge for the 21st century. Technological innovation can play an important role in moving society toward sustainable development. However, poor, marginalized, and future populations often do not fully benefit from innovation due to their lack of market or political power to influence innovation processes. As a result, current innovation systems fail to contribute as much as they might to meeting sustainable development goals. This paper focuses on how actors and institutions operating in the transnational arena can mitigate such shortfalls. To identify the most important transnational functions required to meet sustainable development needs our analysis undertook three main steps. First, we developed a framework to diagnose blockages in the global innovation system for particular technologies. This framework was built on existing theory and new empirical analysis. On the theory side, we drew from the literatures of systems dynamics; technology and sectoral innovation systems, science and technology studies, the economics of innovation, and global governance. On the empirical front, we conducted eighteen detailed case studies of technology innovation in multiple sectors relevant to sustainable development: water, energy, health, food, and manufactured goods. We use the framework to analyze our case studies in the common language of (1) technology stocks, (2) non-linear flows between stocks substantiated by specific mechanisms, and (3) characteristics of actors and socio-technical conditions (STCs) which mediate the flows between stocks . We identify blockages in the innovation system for each of the cases, diagnosing where in the innovation system flows were hindered and which specific sets of STCs and actor characteristics were associated with these blockages. Figure E.1 displays the components of our framework and how they relate

Does green tea affect postprandial glucose, insulin and satiety in healthy subjects: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Results of epidemiological studies have suggested that consumption of green tea could lower the risk of type 2 diabetes. Intervention studies show that green tea may decrease blood glucose levels, and also increase satiety. This study was conducted to examine the postprandial effects of green tea on glucose levels, glycemic index, insulin levels and satiety in healthy individuals after the consumption of a meal including green tea.</p> <p>Methods</p> <p>The study was conducted on 14 healthy volunteers, with a crossover design. Participants were randomized to either 300 ml of green tea or water. This was consumed together with a breakfast consisting of white bread and sliced turkey. Blood samples were drawn at 0, 15, 30, 45, 60, 90, and 120 minutes. Participants completed several different satiety score scales at the same times.</p> <p>Results</p> <p>Plasma glucose levels were higher 120 min after ingestion of the meal with green tea than after the ingestion of the meal with water. No significant differences were found in serum insulin levels, or the area under the curve for glucose or insulin. Subjects reported significantly higher satiety, having a less strong desire to eat their favorite food and finding it less pleasant to eat another mouthful of the same food after drinking green tea compared to water.</p> <p>Conclusions</p> <p>Green tea showed no glucose or insulin-lowering effect. However, increased satiety and fullness were reported by the participants after the consumption of green tea.</p> <p>Trial registration number</p> <p>NCT01086189</p

Untangling the clinicopathological significance of MRE11-RAD50-NBS1 complex in sporadic breast cancers

The MRE11-RAD50-NBS1 (MRN) complex is critical for genomic stability. Although germline mutations in MRN may increase breast cancer susceptibility such mutations are extremely rare. Here we have conducted a comprehensive clinicopathological study of MRN in sporadic breast cancers. We have protein expression profiled for MRN and a panel of DNA repair factors involved in double strand break (DSB) repair (BRCA1, BRCA2, ATM, CHK2, ATR, Chk1, pChk1, RAD51, γH2AX, RPA1, RPA2, DNA-PKcs), RECQ DNA helicases (BLM, WRN, RECQ1, RECQL4, RECQ5), nucleotide excision repair (ERCC1) and base excision repair (SMUG1, APE1, FEN1, PARP1, XRCC1, Pol β) in 1650 clinical breast cancers. The prognostic significance of MRE11, RAD50 & NBS1 transcripts and their miRNA regulators (hsa-miR-494 and hsa-miR-99b) were evaluated in large clinical datasets. Expression of MRN components were analyzed in the cancer genome atlas breast cancer cohort (TCGA-BRCA). We show that low nuclear MRN is linked to aggressive histopathological phenotypes such as high tumor grade, high mitotic index, ER- and high-risk Nottingham Prognostic Index (NPI). In univariate analysis low nuclear MRE11 and low nuclear RAD50 was associated with poor survival. In multivariate analysis, low nuclear RAD50 remained independently linked with adverse clinical outcome. Low RAD50 transcripts was also linked with reduced survival. In contrast, overexpression of hsa-miR-494 and hsa-miR-99b microRNAs was associated with poor survival. We observed large scale genome wide alterations in MRN deficient tumours contributing to aggressive behaviour. We conclude that MRN status may be a useful tool to stratify tumours for precision medicine strategies

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist