Doctor of Philosophy

dissertationMicroRNA expression is dysregulated in many human cancers, including hematopoietic malignancies. Among hematopoietic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year in the US alone. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~30% of all AML cases, and confers a negative prognosis. MicroRNA expression has been shown to be highly dysregulated in FLT3-ITD+ AML; however, the functional relevance of many of these microRNAs on leukemic phenotypes remains unclear. We performed a genome-wide CRISPR-Cas9 screen to identify which microRNAs, and which of their putative mRNA targets, regulate FLT3-ITD+ AML cell growth. Our screen identified a number of microRNAs that function to suppress or promote FLT3-ITD+ AML cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. We also performed anticorrelation functional profiling to predict relevant microRNA-mRNA target pairs in this context, and identified miR-150 targeting of p53 as a critical relationship governing the growth of these cells. We validated one of our targets, miR-155, as a critical regulator of FLT3-ITD+ AML cell growth in vitro, where miR-155-deficient cells displayed a competitive growth disadvantage compared to cells with miR-155 intact. We extended these findings into an in vivo model of FLT3-ITD-driven myeloid malignancy, where mice containing a FLT3-ITD mutation but lacking miR-155 exhibited decreased myeloid expansion in the bone marrow, spleen, and blood compared to their FLT3-ITD miR-155+/+ counterparts. This phenotype was attributed to miR-155's role in promoting proliferation of the hematopoietic stem cell and myeloid progenitor cell compartments in the bone marrow. Further analysis revealed that miR-155 likely exerts these effects by regulating multiple pathways involved in cellular proliferation, including repressing the interferon response through targeting Cebpb, and activating AKT signaling through targeting of Ship1. These findings correlated with human AML data from The Cancer Genome Atlas dataset, where we found that FLT3-ITD+ AML samples had a decreased interferon signature and lower levels of Cebpb and Ship1 compared to FLT3-WT AML samples. Finally, we treated FLT3-ITD+ AML primary patient samples with a miR-155 inhibitor and observed decreased colony forming potential and increased apoptosis in these cells. These results suggest that miR-155 inhibition could be a novel therapeutic approach in FLT3-ITD+ AML

The Inflammatory Response to Different HIIT Modalities

Pro-inflammatory and anti-inflammatory cytokines have been documented to change in response to long-duration, aerobic exercise. Little work has been performed in high-intensity interval training (HIIT). Furthermore, it has yet to be elucidated whether the modality of HIIT affects this inflammatory response. PURPOSE: The purpose of this study was to investigate the changes in IL-8 and IL-10 cytokine concentrations pre-post and post-1hr following HIIT protocols using two different exercise modalities, treadmill running (TM) and stationary cycling (CE). METHODS: Eight young, healthy, recreationally active volunteers (1 female, 7 males; 25.50 ± 3.25 years) completed a 4x4 HIIT protocol (4-minute 85-95% HRmax active; 3-minute 60-70% HRmax recovery) on both a treadmill (TM) and a stationary cycle (CE). All participants completed both modalities in a randomized order 1 week apart. Blood samples were collected using Tasso+ devices attached to the upper arm. Plasma was isolated and frozen at -80C until protein analysis. IL-8 and IL-10 were measured using the Millipore HCYTOMAG60K assay. Data are presented as percent change, post- and 1h-post HIIT bouts. RESULTS: IL-8 was elevated by 8.11% immediately following TM HIIT and did not change following CE HIIT (0.3%). Following 1-hour of recover, IL-8 increased a further 28.96% after TM and increased by 2.08% after CE. IL-10 concentration was increased by 14.41% 1-hour following TM HIIT; however, it did not change 1 hour following CE HIIT. CONCLUSION: The immune system plays an important role in the maintenance of health. This study demonstrated that a higher-impact exercise (TM) elicits a proinflammatory response that persists for 1 hour after training. However, this pro-inflammatory response was not observed in the lower-impact CE modality. Accounting for differences in inflammatory response between modalities of HIIT is important when designing exercise programs for populations with impaired immune systems. Future work should be performed to identify differences in immune cell function following various HIIT modalities

Operational Use of the Air Quality Monitor on ISS and Potential for Air Quality Monitoring Onboard Submarines

The air quality monitor (AQM) began operations on the International Space Station (ISS) in March 2013 and was validated for operational use in January 2014. The AQM is a gas chromatograph-differential mobility spectrometer that currently monitors 22 target compounds in the ISS atmosphere. Data are collected twice per week, although data collection can be more frequent in contingency situations. In its second year, the AQM has provided data to decision-makers on several ISS contaminant related issues in both air and water. AQM has been used in strictly air incidents, such as a potential ammonia leak, and to investigate air contaminants affecting the water processing (excess ethanol). In the latter case data from water monitors and AQM were compared to understand the issue with the water processor. Additionally, the AQM has been moved to different ISS modules to determine whether air is sufficiently mixed between modules so that a central LAB module location is representative of the entire ISS atmosphere. Historic data on the ISS atmosphere in different modules from archival samples (ground lab analysis) suggest that the atmosphere is usually homogenous. This presentation will briefly describe the technical aspects of the AQM operations and summarize the validation results. The main focus of the presentation will be to discuss the results from the AQM survey of the ISS modules and to show how the AQM data has contributed to an understanding of environmental issues that have arisen on ISS. Presentation of a potential ammonia leak (indicated by an alarm) in 2015 will illustrate the use and value of the AQM in such situations

Capturing Heat from Spent Nuclear Fuel

ME450 Capstone Design and Manufacturing Experience: Winter 2021Spent Nuclear Fuel can be placed in dry cask storage, where it emits waste heat into the atmosphere. Our sponsor, Dr. Marianna Coulentianos, identified an opportunity to capture this heat for a beneficial application. Our team evaluated the feasibility of our sponsor’s previously proposed solutions and designed a system that could transfer heat from the dry cask. We focused specifically on quantifying the amount of heat that would be available for a therothecial application. In order to determine how much heat would theoretically be available, we constructed both mathematical and computational simulations of heat transfer through a duct system. The system we propose includes a square funnel feature at the cask interface, connected to a round, rigid duct system extending over the perimeter fence. It was observed that the outlet temperature of our proposed system is around 36-65℃, which we determined is most suitable for a greenhouse application. We calculated a return on investment of 5 years by growing tomatoes in a greenhouse of 1800 ft2. We are confident that our design is feasible and does not violate any regulations set forth by the Nuclear Regulatory Commission. However, more analysis is needed to further examine discrepancies between field data and our assumptions, as well as the scalability of our proposed solution. We also considered the social context of this solution. Eating fruits and vegetables grown on a nuclear site is likely to cause skepticism around our solution. While we believe that the radiation levels of this waste heat are too low to realistically affect horticultural applications, all food that is intended for human or animal consumption in the United States must register with the FDA before beginning these activities.Dr. Marianna Coulentianos: UM Mechanical Engineering Departmenthttp://deepblue.lib.umich.edu/bitstream/2027.42/167636/1/Team_17-Capturing_Heat_from_Spent_Nuclear_Fuel.pd

Seeing the Threat: Pilot Visual Detection of Small Unmanned Aircraft Systems in Visual Meteorological Conditions

One key challenge of integrating Unmanned Aircraft Systems (UAS) platforms into the National Airspace System (NAS) is the potential for midair collisions between manned aircraft and the unmanned system. The lack of an established UAS benchmark for Detect, Sense & Avoid Systems put the preponderance of avoidance efforts on manned aircraft pilots to visually see and avoid potential collision threats. The small size, unusual configurations, and diverse operational applications of unmanned systems make UAS platforms difficult to visually identify. This paper sought to determine the mean visibility distance of small UAS systems (sUAS) to an alerted pilot flying a general aviation aircraft in visual meteorological conditions (VMC). The study evaluated mean visibility distance to various sUAS platforms based on a scripted set of UAS convergence conditions. The study utilized a mixed method design in which a general aviation aircraft was flown into a UAS operations area. Study pilots were instructed to locate a flying UAS aircraft without bearing assistance. Both the UAS and manned aircraft were assigned vertically de-conflicted altitudes with the UAS aircraft executing a series of converging and crossing courses relative to the manned aircraft. The distance at which the pilot visually located the UAS platform was timestamped and electronically recorded via a GPS tracking device. The various conditions were analyzed to determine significant visibility differences among the various convergence conditions. Qualitative data was collected from participant comments and observations recorded by an in-flight safety observer

Algorithm-Based Meta-Analysis Reveals the Mechanistic Interaction of the Tumor Suppressor LIMD1 With Non-Small-Cell Lung Carcinoma

Non-small-cell lung carcinoma (NSCLC) is the major type of lung cancer, which is among the leading causes of cancer-related deaths worldwide. LIMD1 was previously identified as a tumor suppressor in lung cancer, but their detailed interaction in this setting remains unclear. In this study, we have carried out multiple genome-wide bioinformatic analyses for a comprehensive understanding of LIMD1 in NSCLC, using various online algorithm platforms that have been built for mega databases derived from both clinical and cell line samples. Our results indicate that LIMD1 expression level is significantly downregulated at both mRNA and protein levels in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), with a considerable contribution from its promoter methylation rather than its gene mutations. The Limd1 gene undergoes mutation only at a low rate in NSCLC (0.712%). We have further identified LIMD1-associated molecular signatures in NSCLC, including its natural antisense long non-coding RNA LIMD1-AS1 and a pool of membrane trafficking regulators. We have also identified a subgroup of tumor-infiltrating lymphocytes, especially neutrophils, whose tumor infiltration levels significantly correlate with LIMD1 level in both LUAD and LUSC. However, a significant correlation of LIMD1 with a subset of immune regulatory molecules, such as IL6R and TAP1, was only found in LUAD. Regarding the clinical outcomes, LIMD1 expression level only significantly correlates with the survival of LUAD (p0.1) patients. These findings indicate that LIMD1 plays a survival role in LUAD patients at least by acting as an immune regulatory protein. To further understand the mechanisms underlying the tumor-suppressing function of LIMD1 in NSCLC, we show that LIMD1 downregulation remarkably correlates with the deregulation of multiple pathways that play decisive roles in the oncogenesis of NSCLC, especially those mediated by EGFR, KRAS, PIK3CA, Keap1, and p63, in both LUAD and LUSC, and those mediated by p53 and CDKN2A only in LUAD. This study has disclosed that LIMD1 can serve as a survival prognostic marker for LUAD patients and provides mechanistic insights into the interaction of LIMD1 with NSCLC, which provide valuable information for clinical applications

Pilot Visual Detection of Small Unmanned Aircraft Systems (sUAS) Equipped with Strobe Lighting

When operating under Visual Flight Rules, pilots primarily rely on visual scanning to avoid other aircraft and airborne collision threats. Records from the Federal Aviation Administration indicate that near encounters with unmanned aircraft are on the rise, reaching 1,761 reported unmanned aircraft system (UAS) sightings or near-misses in 2016. This study sought to assess the effectiveness of pilot visual detection of UAS platforms that were equipped with strobe lighting. A sample of 10 pilots flew a general aviation aircraft on a scripted series of five intercepts with a small UAS (sUAS) that was equipped with strobe lighting. Participants were asked to indicate when they visually detected the unmanned aircraft. Geolocation information for both the aircraft and sUAS platform was compared to assess visibility distance. Findings were used to evaluate the efficacy of daytime strobe lighting as a method to enhance pilot sUAS detection, visibility, and collision avoidance. Participants detected the unmanned aircraft during 7.7% of the intercepts. Due to a lack of data points, the authors were unable to conclusively determine if strobe lighting improved UAS visual detection. The authors recommend further research to explore the effectiveness of using sUAS-mounted strobe lights for nighttime visual detection

Detecting and Assessing Collision Potential of Aircraft and Small Unmanned Aircraft Systems (sUAS) by Visual Observers

Visual observers are used to assist the Remote Pilot with maintaining sight of the unmanned aircraft as well as scanning the surrounding airspace for potential collision hazards. The purpose of this study was to examine the effectiveness of visual observers in detecting an intruding general aviation aircraft approaching the small unmanned aircraft system (sUAS) operations area. The study sought to determine the effectiveness of sUAS visual observers in detecting a general aviation aircraft collision hazard with a sUAS. Ten participants were asked to perform visual observer duties in support of a sUAS operation. Participants were asked to indicate when they were able to hear and see an aircraft that conducted a scripted series of close intercepts with a sUAS. Additionally, researchers assessed each visual observer’s ability to accurately judge the closure rate of the aircraft, by estimating the duration from initial sighting until the aircraft would intercept the airborne sUAS platform. Geolocation data from both the aircraft and sUAS were time correlated and compared to determine estimation accuracy. Findings were used to formulate operational recommendations to improve visual observer performance in detecting and assessing intruder aircraft collision potential

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.BackgroundThe proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3′, 5′ cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells.MethodsPrimary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot.Results8-Br-cAMP increased phosphorylated ERK (2.7- ± 0.6-fold, N = 7), and B-Raf kinase activity (3.6- ± 1.1-fold, N = 5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK.ConclusioncAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts