BACKGROUND:Translational medicine is attracting much attention worldwide and many translational research organizations (TROs) have been established. In China, translational medicine has developed rapidly, but faces many challenges. This study was aimed at exploring these challenges faced by emerging TROs in China.METHOD:A qualitative, multiple case study approach was used to assess the challenges faced by TROs in China. Data were collected between May and August 2012.RESULTS:Eight cases were identified. Overall, four themes that characterized TROs in China emerged from analyses: 1. objectives, organizer, and funding resources, 2. participating partners and research teams, 3. management, and 4. achievements. All TROs had objectives related to translating basic discovery to clinic treatment and cultivating translational researchers. In terms of organizer and funding resources, 7 out of 8 TROs were launched only by universities and/or hospitals, and funded mostly through research grants. As for participating partners and multidisciplinary research teams, all but one of the TROs only involved biomedical research institutions who were interested in translational research, and characterized as clinical research centers / 7 out of 8 TROs involved only researchers from biomedicine and clinical disciplines and none involved disciplines related to education, ethnicity, and sociology, or engaged the community. Current management of the TROs were generally nested within the traditional research management paradigms, and failed to adapt to the tenets of translational research. Half of the TROs were at developmental stages defined as infrastructure construction and recruitment of translational researchers.CONCLUSIONS:TROs in China face the challenge of attracting sustainable funding sources, widening multidisciplinary cooperation, cultivating multi-disciplinary translational researchers and adapting current research management to translational research. Greater emphasis should be placed on increasing multidisciplinary cooperation, and innovating in education programs to cultivate of translational researchers. Efforts should be made to reform research management in TROs, and establish sustainable funding resources.

Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. / October 2016

Introduction: Sepsis is a leading cause of death in critically ill patients. Conventional methods of diagnostics in patients with suspected sepsis have limited sensitivity and long turnaround times. These factors contribute to indiscriminate use of broad-spectrum antibiotics and antimicrobial resistance. Our goal was to investigate where direct molecular analysis of circulating cell-free DNA in plasma could identify the microbial etiology of sepsis and improve assessment of prognosis. Methods: We conducted a prospective study of 30 consecutive patients suspected of sepsis in the Surgical Trauma ICU and compared with 22 healthy volunteer controls. Longitudinal plasma samples were collected at the time of workup for sepsis (day 0) and on days 7 and 14. Blood samples were collected in Streck Cell-Free DNA tubes and processed within 24 hours. DNA was extracted using QIAamp Circulating Nucleic Acid Kit. We measured mtDNA levels in plasma using real-time quantitative PCR targeting the mitochondrial NADH1 gene. Absolute mtDNA copies were calculated by comparing with known standards, pre-quantified using droplet digital PCR. Whole genome sequencing of cell-free DNA from plasma samples of three patients with positive blood cultures and 1 healthy volunteer control were performed to detect pathogen DNA. Results: We analyzed 72 serial plasma samples from 30 patients with suspected sepsis. Median mtDNA levels in controls were 602 ± 636 copies/µL of plasma (median ± IQR). In comparison, median mtDNA levels at day 0 in patients with SIRS were 3318 ± 1960 copies/µL (p<10⁻⁸, area under the ROC curve: 0.939). mtDNA levels were correlated with peripheral WBC count and respiratory rate (p=0.026 and 0.013 respectively) but not with temperature, heart rate or systolic BP. 3/30 patients died within the same hospital stay and last recorded mtDNA levels were higher as compared to survivors (p=0.028). Three out of thirty patients with a diagnosis of sepsis had positive blood cultures. Concordant results were found between conventional microbiology and next-generation sequencing of cell-free plasma DNA in 1 patient. Conclusions: Circulating mtDNA levels in patients with suspected sepsis are five-fold higher than healthy controls. Longitudinal changes in mtDNA are correlated with conventional markers of systemic inflammatory response and can be a biomarker for outcomes. Sequencing of cell-free DNA in plasma of sepsis patients can enable identification of bacteria and viral pathogens, although additional optimization of laboratory and informatics protocols is needed.

Introduction Following polytrauma and major surgery patients experience a period of immunosuppression, which can predispose them to the development of nosocomial infections. This thesis examines how polytrauma and surgery influences T-helper (Th) cell differentiation and antigen presentation, whilst exploring how the detrimental immunomodulatory effects of these insults may be reversed. Methods Serial blood samples were drawn from two cohorts of patients at The Royal London Hospital; following severe polytrauma (n=112) or major abdominal surgery (n=119). mRNA levels of candidate cytokines and transcription factors were assayed, along with protein levels of key cytokines IL-10 and IL-6. Associations between these data, acquisition of nosocomial infection and outcome were described. As a validated surrogate of immune competence CD14+HLA-DR levels were quantified. In vitro models explored the reversibility of tissue damage induced immunosuppression and determined the role of individual circulating mediators in altering host immune function. Results A consistent up-regulation in gene expression of prototypical anti-inflammatory pathways in conjunction with features of depressed pro-inflammatory Th cell pathways was detected across both cohorts. This was accompanied by early down-regulation of CD14+HLA-DR. Gene expression changes were quantitatively associated with the subsequent acquisition of nosocomial infections. Allogeneic blood transfusion exacerbated these findingsand was independently associated with an increased risk of nosocomial infection. Culture experiments determined that postoperative decreases in antigen presentation were IL-10 dependent and reversible in the presence of Interferon-Gamma and Granulocyte Monocyte- Colony Stimulating Factor. Conclusions This thesis describes a significant host immune response immediately following significant tissue damage which is dominated by features of immune suppression. Blood transfusion appears to have a distinct, additive effect. These data identify a potential role for targeted treatment with currently licenced immune stimulants (IFN-γ and GM-CSF). In addition exploitation of the IL-10 signalling pathway may be of importance as a strategy to reduce the incidence of nosocomial infections.

Bcl11b (B-cell lymphoma/leukemia 11b), also known as Ctip2 (Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2), is a C2H2 zinc finger transcriptional regulatory protein, which is an essential protein for post-natal life in the mouse and plays crucial roles in the development, and presumably function, of several organ systems, including the central nervous, immune, craniofacial formation and cutaneous/skin systems. Moreover, inactivation of Bcl11b has been implicated in the etiology of lymphoid malignancies, suggesting that Bcl11b may function as a tumor suppressor. Bcl11b was originally identified as a protein that interacted directly with the orphan nuclear receptor COUP-TF2. Later studies revealed that this C2H2 zinc finger protein can bind DNA directly in a COUP-independent manner, and it has been studied mostly as a transcription repressor. In T cells, gene repression mediated by Bcl11b involves the recruitment of class I HDACs, HDAC1 and HDAC2, within the context of the Nu It has become evident that post-translational modifications (PTMs) play essential roles in modulating activity of transcription regulators. By sensing extracellular signals, cells initiate a series of signaling cascades, which eventually transduce to transcription factors by PTMs, leading to changes of gene expression profile. cleosome Remodeling and Deacetylation (NuRD) complex. The hypothesis that Bcl11b functions as a transcriptional repressor has been supported by transcriptome analyses in mouse T cells and human neuroblastoma cells. However, approximately one-third of the genes that were dysregulated in the double positive (DP) cells of Bcl11b-null mice were down-regulated relative to control T cells, suggesting that Bcl11b may act as a transcriptional activator in some promoter and/or cell contexts. We have also found that Bcl11b functions as a transcriptional activator in a promoter context-dependent manner. However, how Bcl11b and its transcription regulatory activity is regulated still remain largely unknown. Here, we study the reversible, covalent modification of Bcl11b by phosphorylation and small ubiquitin-like modifier (SUMO). We have identifiedK679 and K877 as the two major Bcl11b SUMOylation sites by mutagenesis study. We have shown that phosphorylation and SUMOylation of Bcl11b are likely mutually exclusive processes, and phosphorylation of Bcl11b inhibits its SUMOylation by promoting the recruitment of SUMO specific protease SENP1. To study the function of Bcl11b SUMOylation, we fused SUMO1 to the amino terminus of Bcl11b. This generated a form of Bcl11b that was constitutively sumoylated without the complications of indirect effects associated with overexpression of SUMO1. Our data presented using the constitutive SUMO-Bcl11b demonstrated that SUMOylation compromises the transcription repression mediated by Bcl11b. Interestingly, when Bcl11b is fused to a cleavable form of SUMO, Bcl11b is targeted to ubiquitination pathway and it is degraded through proteasome machinery, suggesting that SUMOylation targets Bcl11b to the ubiquitination-proteasome machinery and deSUMOylation of SUMO conjugated Bcl11b is required for this process. These results described herein provide a framework for understanding the mechanisms underlying the transcription regulatory activities of Bcl11b, and how Bcl11b is regulated by post-translational modifications, including phosphorylation and SUMOylation. These studies may contribute to a better understanding of the molecular and cellular basis for Bcl11b function in vivo. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from June 9, 2011 - June 9, 2012

Melanoma is rapidly increasing in incidence throughout the world. Early stages are curable with surgical approaches with excellent prognosis. However, a substantial proportion of patients progress to metastatic disease with survival rates of less than 5% making melanoma the culprit for over 65% of all skin-cancer related deaths. Novel agents targeting the immune system and the signaling pathways of melanoma are generating new promise, but chemotherapy remains an important therapeutic alternative, despite low response rates. The resistance of melanoma to chemotherapy is in part due to DNA repair mechanisms that allow cells to survive alkylation damage. Several novel agents targeting the abrogation of DNA repair pathways alone and in combination with cytotoxic agents have been developed with varying measures of success. In this dissertation, we first identified the epigenetic silencing of the DNA mismatch repair (MMR) gene MLH1 as a determinant of response and survival for melanoma patients treated with alkylator-based chemotherapy (dacarbazine/ temozolomide). We then determined the safe dosage of the epigenetic agent decitabine that can be administered in combination with temozolomide. The safety, tolerability and efficacy of the combination of decitabine and temozolomide were evaluated in a Phase II population. We finally determined the pharmacokinetic and pharmacodynamic effects of treatment with the combination of decitabine and temozolomide in the blood and tumor tissues of metastatic melanoma patients.

Immunotherapy utilizing cytokines or immune regulatory check point blockade has consistently demonstrated superior clinical efficacy in melanoma when compared to tumor peptide immunization strategies reported to date. In this project, I conducted 2 model studies representing alternative immunotherapeutic approaches (non-antigen specific combination of interferon-á2b and an anti-CTLA4 monoclonal antibody, IFN-Treme compared to a tumor antigen specific multi-epitope vaccine given in adjuvant with the potent combination of a TLR-9 agonist and GM-CSF) designed to overcome tumor immune evasion and conducted separately in a similar patient population. In addition to evaluating safety and clinical efficacy, I tested the following hypotheses: (1) Clinical benefits are likely to be associated with markers of reversal of immune tolerance (autoimmunity). (2) Clinical benefits may be predicted by baseline peripheral biomarkers of immune tolerance/suppression (C-reactive protein, CRP and absolute lymphocyte count, ALC). (3) Superior antitumor efficacy is likely to be associated with more effective downregulation of the host suppressor immune response (circulating T regulatory cells, T-reg and myeloid derived suppressor cells, MDSC). My findings supported superior clinical efficacy that was associated with more significant modulation of immune tolerance by the combination of IFN-Treme. Autoimmunity correlated with improved clinical outcome among the recipients of IFN-Treme (but not the vaccine) and suggested more significant reversal of immune tolerance. Baseline CRP and ALC were significantly predictive of therapeutic benefit with the IFN-Treme combination and may serve as variables for stratification of future trials, as these are validated in larger studies. Finally, my findings supported more significant downregulation of the host suppressor immune response by the nonspecific IFN-á/Treme regimen as compared to the vaccine-TLR agonist/GM-CSF combination. There was apparent increase in CD4+CD25hi+ CD39+ Treg but this was associated with an increase in the overall CD4+ T cell population suggesting that direct inhibition of CTLA4 suppressive effects on T effector cells leading to their expansion and prolonged activation is likely more important than the regimens effect on T-reg. In addition, I saw parallel downregulation in several populations of MDSC following treatment with IFN-Treme which may have had a role in the reduction of immune suppression and superior clinical outcome observed.

The Neurospora crassa arg-2 gene encodes the small subunit of carbamoyl phosphate synthetase, the first enzyme in fungal arginine (Arg) biosynthesis. The arginine attenuator peptide (AAP), specified by an upstream open reading frame (uORF), stalls ribosomes at its termination codon in response Arg to control the translation of arg-2. In project 1, the effect of AAP and Arg on ribosome peptidyl transferase center (PTC) activity was analyzed in N. crassa and wheat germ cell-free translation extracts using the transfer of nascent AAP to puromycin as an assay. The results show that inhibition of PTC activity by the AAP and Arg is the basis for the AAP’s function. The mode of PTC inhibition appears unusual because neither a specific amino acid nor a specific nascent peptide chain length was required for AAP to function. In eukaryotic translation initiation, the stringency of start codon selection impacts initiation efficiencies at AUG codons in different contexts and at near-cognate codons (NCCs) that differ from AUG by a single nucleotide. In project 2, a codon-optimized firefly luciferase reporter was used to examine the stringency of start codon selection in N. crassa. In vivo and in vitro results indicated that the hierarchy of initiation in N. crassa is similar to that in human cells. The preferred context was more important for efficient initiation from NCCs than from AUG. In project 3, the use of NCCs was also specifically examined for the N. crassa cpc-1 gene. cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying a transcription activator, which drives the primary transcriptional response to amino acid starvation. In vitro studies showed that uORF1 and uORF2 in cpc-1 are functionally analogous to uORF1 and uORF4 in GCN4. uORF1 promotes reinitiation at downstream start codons. uORF2 inhibits translation from the main cpc-1 start codon. Four NCCs in the CPC1 reading frame and upstream of uORF2 can also be used for translation initiation. In summary, we explored uORF-mediated translational regulation and the use of NCCs as initiation codons. Taken together, these studies establish N. crassa as a model system to examine mechanisms contributing to translational control including initiation and termination.

My thesis project explores the ability of architecture to represent a culture in a meaningful way. The embassy is technically in Brazil but surrounded by the District of Columbia, USA. This poses questions about culture, ecology, and geography. The challenge is how to design a cultural-specific building without it becoming a World's Fair pavilion or a cultural cliché. World's Fair pavilions often represent just a moment within a nation's ongoing architectural discourse, as is fitting for their temporary status. Some embassies are cultural clichés, which look like how uneducated residents of the host county perceive a foreign land and are invitations to form cultural stereotypes. My approach to answering this thesis question is in the process of translating Brazilian culture through the lens of my own culture using the medium of design and construction. This process can be described as translational architecture. The act of translation does not consist merely of mimicry but a transformation into something similar yet substantively different. This act of translation disrupts the implicit superiority of the original because the translation can have its own life, character, and depth. / Master of Architecture

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