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Title: Development of validation guidelines for high pressure processing to inactivate pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in treated juices
Creator: Rolfe, Catherine
Date: 2020
Description: The fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the functional...
Show moreThe fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the functional juice division is cold pressure, which refers to the application of high pressure processing (HPP) at low temperatures for a mild treatment to inactivate foodborne pathogens instead of thermal pasteurization. HPP juice manufacturers are required to demonstrate a 5-log reduction of the pertinent microorganism to comply with FDA Juice HACCP. The effectiveness of HPP on pathogen inactivation is determinant on processing parameters, juice composition, packaging application, as well as the bacterial strains included for validation studies. Unlike thermal pasteurization, there is currently no consensus on validation study approaches for bacterial strain selection or preparation and no agreement on which HPP process parameters contribute to overall process efficacy.The purpose of this study was to develop validation guidelines for HPP inactivation and post-HPP recovery of pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in juice systems. Ten strains of each microorganism were prepared in three growth conditions (neutral, cold-adapted, or acid-adapted) and assessed for barotolerance or sensitivity. Pressure resistant and sensitive strains from each were used to evaluate HPP inactivation with increasing pressure levels (200 – 600 MPa) in two juice matrices (apple and orange). A 75-day shelf-life analysis was conducted on HPP-treated juices inoculated with acid-adapted resistant strains for each pathogen and examined for inactivation and recovery. Individual strains of E. coli O157:H7, Salmonella spp., and L. monocytogenes demonstrated significant (p <0.05) differences in reduction levels in response to pressure treatment in high acid environments. E. coli O157:H7 was the most barotolerant of the three microorganism in multiple matrices. Bacterial screening resulted in identification of pressure resistant strains E. coli O157:H7 TW14359, Salmonella Cubana, and L. monocytogenes MAD328, and pressure sensitive strains E. coli O15:H7 SEA13B88, S. Anatum, and L. monocytogenes CDC. HPP inactivation in juice matrices (apple and orange) confirmed acid adaptation as the most advantageous of the growth conditions. Shelf-life analyses reached the required 5-log reduction in HPP-treated juices immediately following pressure treatment, after 24 h in cold storage, and after 4 days of cold storage for L. monocytogenes MAD328, S. Cubana, and E. coli O157:H7 TW14359, respectively. Recovery of L. monocytogenes in orange juice was observed with prolonged cold storage time. These results suggest the preferred inoculum preparation for HPP validation studies is the use of acid-adapted, pressure resistant strains. At 586 – 600 MPa, critical inactivation (5-log reduction) was achieved during post-HPP cold storage, suggesting sufficient HPP lethality is reached at elevated pressure levels with a subsequent cold holding duration.
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Title: Sense of Community and Virtual Community Among People with Autism Spectrum Conditions
Creator: Rafajko, Sean I
Date: 2020
Description: Individuals with autism spectrum conditions (ASC) face poorer quality of life (QOL) and psychological well-being. Sense of community (SOC) has...
Show moreIndividuals with autism spectrum conditions (ASC) face poorer quality of life (QOL) and psychological well-being. Sense of community (SOC) has been studied in the general population as well as in other disability populations and found to be associated with increased QOL outcomes. However, SOC has never been examined quantitatively in the ASC population. Additionally, a number of communities exist online, and there has been recent research showing that people may feel sense of virtual community (SOVC), which may be particularly important to the ASC population, as internet use is higher in the population, and people with ASC report positive experiences with online communication and relationships. The purpose of this study was to examine SOC and SOVC in the ASC population. A sample of 60 participants with ASC completed an online survey about their communities, SOC, SOVC, QOL, and psychological distress, and their results were compared with a sample of 60 general population participants (N = 120). Results indicated that people with ASC reported participating in a greater number of smaller relational communities compared to the general population sample. There were no significant differences between the ASC and general population samples on levels of SOC or SOVC, suggesting that the differential relationship of the ASC group with their communities does not reduce the experience of SOC. SOC significantly contributed to QOL but not psychological distress. Results indicated that the magnitude of the relationship between SOC and SOVC on QOL was not different between those with ASC and those in the comparisons sample. Findings from this study help frame the different ways in which people with ASC interact with their communities and inform individual and community-level interventions.
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Title: LOW-DOSE CARDIAC SPECT USING POST-FILTERING, DEEP LEARNING, AND MOTION CORRECTION
Creator: Song, Chao
Date: 2019
Description: Single photon emission computed tomography (SPECT) is an important technique in use today for the detection and evaluation of coronary artery...
Show moreSingle photon emission computed tomography (SPECT) is an important technique in use today for the detection and evaluation of coronary artery diseases. The image quality in cardiac SPECT can be adversely affected by cardiac motion and respiratory motion, both of which can lead to motion blur and non-uniform heart wall. In this thesis, we mainly investigate imaging de-noising algorithms and motion correction methods for improving the image quality in cardiac SPECT on both standard dose and reduced dose.First, we investigate a spatiotemporal post-processing approach based on a non-local means (NLM) filter for suppressing the noise in cardiac-gated SPECT images. Since in recent years low-dose studies have gained increased attention in cardiac SPECT owing to its potential radiation risk, to further improve the image quality on reduced dose, we investigate a novel de-noising method for low-dose cardiac-gated SPECT by using a three dimensional residual convolutional neural network (CNN). Furthermore, to reduce the negative effect of respiratory-binned acquisitions and assess the benefit of this approach in both standard dose and reduced dose using simulated acquisitions. Inspired by the success in respiratory correction, we investigate the potential benefit of cardiac motion correction for improving the detectability of perfusion defects. Finally, to combine the benefit of above two types of motion correction, dual-gated data acquisitions are implemented, wherein the acquired list-mode data are further binned into a number of intervals within cardiac and respiratory cycle according to the electrocardiography (ECG) signal and amplitude of the respiratory motion.
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Title: Factor Analysis of the Neurobehavioral Symptom Inventory in Veterans with Posttraumatic Stress Disorder
Creator: Scimeca, Lauren
Date: 2020
Description: The Neurobehavioral Symptom Inventory (NSI) is a widely used measure of postconcussive symptoms in veteran populations. Previous psychometric...
Show moreThe Neurobehavioral Symptom Inventory (NSI) is a widely used measure of postconcussive symptoms in veteran populations. Previous psychometric studies used samples of veterans with mild Traumatic Brain Injury (mTBI) and high rates of comorbid Posttraumatic Stress Disorder (PTSD). The present study aims to determine the best-fitting factor structure of the NSI in veterans with PTSD and to evaluate the relationship between the best-fitting factor structure and the symptom clusters of PTSD. A confirmatory factor analysis (CFA) found that 4-factors had the best overall fit in veterans with PTSD. Correlational analyses found high rates of correspondence between the cognitive and affective factors of the NSI and the alterations in cognition and mood and hyperarousal symptom clusters of PTSD. The analyses reveal that symptoms of the NSI cluster in the same way in a sample of veterans with PTSD as they do in veterans with mTBI, suggesting that lingering postconcussive symptoms in veterans with PTSD are better characterized as non-specific generalized health symptoms on the NSI.
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Title: CONCEPTUAL COST ESTIMATION MODEL FOR BRIDGES WITH RESPECT TO ABC METHODS
Creator: Rajeei, Farshad
Date: 2020
Description: As the need for renovating and repairing structurally deficient and functionally obsolete bridges is increased, employing innovative methods...
Show moreAs the need for renovating and repairing structurally deficient and functionally obsolete bridges is increased, employing innovative methods which can lead to shorter construction time, better quality, longer durability, and less life-cycle costs become more popular in transportation agencies.Developing a model that has the capability of estimating the total construction cost of ABC projects and compare them with conventional methods costs [without using these methods] will help decision-makers at DOTs in understanding and assessing the benefits and costs of ABC methods at the planning phase of a project and in return, will lead to the elaboration in the use of ABC methods versus the conventional ones. But this decision making process is complicated since the number of executed ABC projects, especially those which done by SIBC and SPMT [two superstructure replacement method] is limited and as a result; there is a lack of historical knowledge to estimate the associated cost of these methods in future projects. Factors affecting this process include but are not limited to: construction costs, user costs, quality of work, impact on traffic, the safety of road users and construction workers, and the impact on surrounding communities and businesses. The main aim of this study is to make a model to estimate additional costs of using SIBC and SPMT methods and the saving in user costs.
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Title: PTSD Symptoms as a Potential Link Between Military Sexual Assault and Disordered Eating
Creator: Sandhu, Danielle
Date: 2020
Description: Despite increasing rates of sexual assault in the military and high rates of disordered eating and posttraumatic stress disorder (PTSD) among...
Show moreDespite increasing rates of sexual assault in the military and high rates of disordered eating and posttraumatic stress disorder (PTSD) among veterans, little is known about how these constructs are related. The present study examined whether PTSD symptoms mediate the relation between military sexual assault and disordered eating among female veterans. Prolific Academic was used to recruit 98 United States female veterans as participants for the study. Participants completed an online questionnaire of self-report measures assessing demographic characteristics, military sexual assault, PTSD symptoms, and disordered eating. Mediational analyses were conducted using the PROCESS v3 macro in IBM SPSS Statistics. Within the sample, 61% of female veterans reported being sexually assaulted while serving in the military. Military sexual assault was associated with higher levels of PTSD symptoms and disordered eating. Findings did not support the hypothesis that PTSD symptoms would mediate the relation between military sexual assault and disordered eating among women veterans. Given the heterogeneous nature of disordered eating, post-hoc mediational analyses were conducted to examine specific facets of eating pathology. Results indicated that PTSD symptoms fully mediated the relation between military sexual assault and bulimia and food preoccupation. Awareness of these psychopathological sequelae following military sexual assault may improve screening and intervention efforts at Veteran Affairs (VA) medical centers. The present study highlights the importance of future longitudinal studies that can establish temporal precedence in order to better understand the pathways leading to disordered eating in female veterans.
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Title: GROWTH KINETICS OF SALMONELLA ENTERICA DURING REHYDRATION OF DEHYDRATED PLANT FOODS AND SUBSEQUENT STORAGE
Creator: Ren, Yuying
Date: 2020
Description: Dehydrated plant foods have low water activities and do not support the growth of pathogenic bacteria like Salmonella enterica. Once...
Show moreDehydrated plant foods have low water activities and do not support the growth of pathogenic bacteria like Salmonella enterica. Once rehydration, the water activities will increase to > 0.92, and along with their neutral pHs, plant foods may be able to support the growth of S. enterica. Therefore, product assessments are required to determine the extent to which these products support growth of S. enterica. The purpose of this study was to determine the growth kinetics of S. enterica during rehydration with 5 or 25 °C water, and subsequent storage of dehydrated potatoes, carrots, and onions at 5, 10, and 25 °C. Fresh plant foods were dehydrated at 60°C (140°F) for 24 h. Dehydrated plant foods were inoculated with 4 log CFU/g of a 4-strain cocktail of S. enterica and dried for 24 h. Samples were rehydrated using 4-volumes of 5 or 25 °C water for 24 h. During rehydration, 30 g of sample was removed and drained for 10 min. Ninety mL of BPB was added to triplicate 10-g samples. Serial dilutions of the homogenate were plated onto TSA overlaid with XLD agar for enumeration of S. enterica. After 24 h rehydration, the remaining samples were drained and stored in containers at 5, 10, and 25°C for 7 d. S. enterica was enumerated at 1, 3, 5, and 7 d. Three independent trials were conducted. Growth kinetics were determined using DMFit and data were statistically analyzed using Student’s t-test (α=0.05). Overall, the growth rates of S. enterica when 5 °C water was used for rehydration were higher than when 25 °C water was used for potatoes and carrots. The highest growth rate of S. enterica was 3.74 log CFU/g per d on potatoes, leading to a 1 log CFU/g increase in S. enterica after only 0.27 d (16 h) which occurred during storage at 25 ℃ after 5℃ water rehydration. The highest growth rate on carrots was 1.98 log CFU/g per d (requiring only 0.51 d to increase 1 log CFU/g) when rehydrated with 5℃ water and stored at 25 ℃. The growth rates were the lowest during the storage of rehydrated onions. S. enterica required 12.5 d to increase 1 log CFU/g (the growth rate was 0.61 log CFU/g per d) when the onions were rehydrated with 25 ℃ water and stored at 25 ℃. The results of this study determined that S. enterica could survive and grow in dehydrated plant foods during rehydration and storage, highlighting the need for product assessments for these types of foods.
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Title: Analysis of High-Fidelity Experiments and Simulations of the Flow in Simplified Urban Environments
Creator: Stuck, Maxime
Date: 2020
Description: The mean ﬂow and turbulence statistics of the ﬂow through a simpliﬁed urban environment, which is an active research area in order to improve...
Show moreThe mean ﬂow and turbulence statistics of the ﬂow through a simpliﬁed urban environment, which is an active research area in order to improve the knowledge of turbulent ﬂow in cities, is investigated. This is useful for civil engineering, pedestrian comfort and for health concerns caused by pollutant spreading. In this work, we provide analysis of the turbulence statistics obtained both from highly-quality stereoscopic particle image-velocimetry (SPIV) measurements (from Monnier et al.) and well-resolved large eddy simulations (LES) by Torres et al. A detailed comparison of both databases reveals the impact of the geometry of the urban array on the ﬂow characteristics and provides for a good description of the turbulent features of the ﬂow around a simpliﬁed urban environment. The most prominent features of this complex ﬂow include coherent vortical structures such as the so-called arch vortex, the horseshoe vortex, or the roof vortex. These structures of the ﬂow have been identiﬁed by an analysis of the turbulence statistics. The inﬂuence of the geometry of the urban environment (and particularly the street width and the building height) on the overall ﬂow behavior has also been studied.
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Title: Development of Microfluidic Platform to Study Insulin Resistance
Creator: Tanataweethum, Nida
Date: 2020
Description: Insulin resistance, a precursor for the development of type 2 diabetes (T2D), propagates among heterologous tissues through dysregulated lipid...
Show moreInsulin resistance, a precursor for the development of type 2 diabetes (T2D), propagates among heterologous tissues through dysregulated lipid flux, as well as dysregulated glucose production, and secretion of cytokines, adipokines and hepatokines. Although T2D is characterized by systemic insulin resistance, disruption of insulin signaling in the liver and adipose tissue recapitulates many aspects of T2D, including enhance endogenous glucose production as well as defects of insulin action. Mechanistic studies often aim to provide fundamental understanding of the observations from human and animal studies. Due to the complexity of animal models and the multifactorial character of T2D, there is a strong need to develop advanced experimental systems such as in vitro models that can enable the recapitulation of the complex physiology of the in vivo system and enable investigation of the pathological pathways as well as identify novel treatment options. The overall goal of this study was to develop insulin resistant models of adipose tissue and liver to study the metabolic function of each organ as well as to the organ-organ crosstalk. To accomplish this goal, four specific aims were pursued: (1) Establish adipose tissue on-a-chip to study the metabolic function of the adipocytes in flow culture; (2) Develop towards an insulin resistant adipose on-a-chip to study the metabolic function of adipocytes in setting of insulin resistance; (3) Develop insulin resistant liver on-a-chip to investigate the metabolic function of hepatocytes in setting of insulin resistance; (4) Develop adipose-liver on-a-chip in setting of insulin resistance to identify the metabolic interaction between organs.
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Title: IMAGE-ANALYSIS WITH FIJI PROGRAM ON PERIPHERAL BLOOD MONOCULAR CELLS AFTER CONSUMPTION OF HIGH-FAT, HIGH CARBOHYDRATE MEAL WITH OR WITHOUT ADDITION OF SPICES – A SINGLE-CENTER RANDOMIZED, BLINDED, PLACEBO-CONTROLLED, 4-ARM, 24HR ACUTE CROSSOVER STUDY
Creator: Tsai, Meng Fu
Date: 2020
Description: Chronic low-grade inflammation plays a significant role in developing various chronic diseases, such as cardiovascular disease and type II...
Show moreChronic low-grade inflammation plays a significant role in developing various chronic diseases, such as cardiovascular disease and type II diabetes. Western-type diets characterized by high-fat (saturated fat) and high-carbohydrate (HFHC) calories induce oxidative stress leading to inflammation. Polyphenol rich foods, such as berries, tea, and herbs and spices, have antioxidant properties. Spices have been shown to have anti-inflammatory effects in cell and animal studies; however, data are limited in humans. In the present study, we hypothesized that bioactive polyphenolic compounds in herbs and species would reduce diet-induced inflammation in overweight and obese (OW/OB) individuals. In a randomized, single-blinded 4-arm, 24-h, crossover clinical trial, sixteen OW/OB adults consumed an HFHC meal with and without three herbs and spices combinations, including Italian herbs (rosemary, basil, thyme, oregano, and parsley), cinnamon and pumpkin pie spice (cinnamon, ginger, nutmeg, and allspice) on four separate occasions at least three days apart. Markers of inflammation were assessed before and at 2, 4, 5.5, and 7 hours after meal consumption by tracking nuclear translocation of nuclear factor kappa B (NF-κB), a transcription factor in inflammatory signaling, in human peripheral blood monocular cells (PBMCs) and by measuring plasma interleukin-6 (IL-6), a pro-inflammatory cytokine. Nuclear translocation of NF-κB and the proportion of PBMCs activated were estimated through a new method leveraging machine-learning immunofluorescence image analysis. Metabolic markers were also investigated by RX Daytona automated clinical chemistry analyzer. Statistical analysis was conducted using a statistical package for the social sciences (SPSS) (α<0.05, significance). Preliminary results suggested the pumpkin pie spice mixture may improve inflammatory status. Compared to the control meal, the meal with pumpkin spice reduced nuclear translocation of NF-κB and proportion of PBMCs activation, p=0.007, and p=0.005, respectively. The addition of herbs/spices in HFHC meal had no apparent effect on postprandial glucose, insulin, or IL-6 concentrations compared to the control meal. Increased triglyceride concentrations were suggested after consuming the meal with Italian herbs compared to control (p=0.004). Overall, the results of this research suggested the potential of pumpkin pie spice as having anti-inflammatory effects in the context of a typical western-style eating pattern. A major component of this research was to develop a new method for assessing real-time inflammation in the human body. While the method and data are encouraging, upgrading image resolution and programming will be the subject of future research.
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Title: WASTEWATER COLLECTION SYSTEM MODELING: TOWARDS AN INTEGRATED URBAN WATER AND ENERGY NETWORK
Creator: Wang, Xiaolong
Date: 2020
Description: Wastewater collection systems, among the oldest features of urban infrastructure, are typically dedicated to collect and transport wastewater...
Show moreWastewater collection systems, among the oldest features of urban infrastructure, are typically dedicated to collect and transport wastewater from users to water resource recovery facilities (WRRFs). Since the 1970s, wastewater engineers and scientists have come to understand that wastewater collection systems can bring benefits for urban water and energy networks, including thermal energy recovery and converting pipelines to bioreactors. However, there is little knowledge about the temporal and spatial changes of collection systems parameters that are important for these applications. Furthermore, the vast majority of existing studies of these applications have focused on laboratory or extremely small-scale systems; there have been few studies about beneficial applications associated with large-scale systems. The purpose of this study is to increase our understanding of how urban wastewater collection systems can bring potential benefits to urban water and energy systems. Models describing wastewater hydraulics, temperature, and water quality can provide valuable information to help evaluate thermal energy recovery and wastewater pretreatment feasibility. These kinds of models, and supporting data from a case study, were used in this study; sizes of the theoretical wastewater collection systems range from 2.6 L/s to 52 L/s, and the sample locations of the case study had flows ranging from 2.3 L/s to 24.5 L/s. A cost-benefit analysis of wastewater source heat pumps was used to evaluate the thermal energy recovery feasibility for different sizes of wastewater collection systems. Results show that the large collection system can support a large capacity heat pump system with a relatively low unit initial cost. Small collection systems have a slightly lower unit operating cost due to the relatively high wastewater temperature. When the heat pump system capacity design was based on the average available energy from the collection system, larger systems have lower payback times; the lowest payback time is about 3.5 years. The wastewater quality model was used to describe the dissolved oxygen (DO) and organic matter concentrations changes in the collection system. The model provides a framework for predicting pretreatment capability. Model results show that DO concentration is the limiting parameter for organic matter removal. Larger collection systems can provide more organic matter removal because they provide relatively longer retention times, and they offer the potential for greater DO reaeration. The model can also be used to identify environmental conditions in sewer pipelines, providing information for potential issues predication.
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Title: A MICROFLUIDIC INTESTINAL-MICROBIOTA PLATFORM TO STUDY DRUG METABOLISM
Creator: Wang, Chengyao
Date: 2020
Description: The intestine is the main site that orally administered drugs are primarily metabolized, absorbed, and distributed. The trillions of bacteria...
Show moreThe intestine is the main site that orally administered drugs are primarily metabolized, absorbed, and distributed. The trillions of bacteria that inhabit the intestine influence health and regulate important biochemical factors, such as the activity of enzymes pertinent to drug metabolism. However, this has not been systematically studied partly due to the challenges of recapitulating the unique and complex intestinal microenvironment that includes (1) the presence of mammalian and microbial cells and (2) a unique partitioned oxygenation proﬁle across the lumen to the subepithelial mucosa from anaerobic to the richly vascularized oxygenated. This thesis reports the development of a microfluidic device in which is integrated a membrane synthesized from a key element of mucosal basal lamina, collagen, and precisely controlled partitioned oxygen environment. The device enabled excellent cell viability and long-term function. More importantly, it enabled the coculture of intestinal epithelial cells and aerobic and anaerobic bacteria in the partitioned oxygen environment. These experiments on one hand allowed the measurement of cellular oxygen consumption rate under perfusion, which could be used to study microbial regulation of oxidative metabolism in epithelial cells. On the other hand, the device allowed a systematic examination of the role of different gut bacteria strains on the regulation of factors that are important in drug metabolism, namely, transporters and phase I enzymes. Our studies highlighted the importance of direct communication between the intestinal cells and the gut bacteria with major findings being that species-specific differences exist in the regulation of drug metabolism. This work will be useful for (1) the discovery of novel regulators of drug metabolizing enzymes, (2) developing new pharmacokinetic models, and (3) advancing precision medicine models for patients.
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Title: Exploiting contextual information for deep learning based object detection
Creator: Zhang, Chen
Date: 2020
Description: Object detection has long been an important research topic in computer vision area. It forms the basis of many applications. Despite the great...
Show moreObject detection has long been an important research topic in computer vision area. It forms the basis of many applications. Despite the great progress made in recent years, object detection is still a challenging task. One of the keys to improving the performance of object detection is to utilize the contextual information from the image itself or from a video sequence. Contextual information is defined as the interrelated condition in which something exists or occurs. In object detection, such interrelated condition can be related background/surroundings, support from image segmentation task, and the existence of the object in the temporal domain for video-based object detection. In this thesis, we propose multiple methods to exploit contextual information to improve the performance of object detection from images and videos.First, we focus on exploiting spatial contextual information in still-image based object detection, where each image is treated independently. Our research focuses on extracting contextual information using different approaches, which includes recurrent convolutional layer with feature concatenation (RCL-FC), 3-D recurrent neural networks (3-D RNN), and location-aware deformable convolution. Second, we focus on exploiting pixel-level contextual information from a related computer vision task, namely image segmentation. Our research focuses on applying a weakly-supervised auxiliary multi-label segmentation network to improve the performance of object detection without increasing the inference time. Finally, we focus on video object detection, where the temporal contextual information between video frames are exploited. Our first research involves modeling short-term temporal contextual information using optical flow and modeling long-term temporal contextual information using convLSTM. Another research focuses on creating a two-path convLSTM pyramid to handle multi-scale temporal contextual information for dealing with the change in object's scale. Our last work is the event-aware convLSTM that forces convLSTM to learn about the event that causes the performance to drop in a video sequence.
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Title: Population Dynamics of Listeria monocytogenes in Nut, Seed and Legume Butters
Creator: Zhang, Xinyuan
Date: 2020
Description: Nut, seed, and legume butters are low water activity foods and do not support the growth of foodborne pathogens. Research has determined that...
Show moreNut, seed, and legume butters are low water activity foods and do not support the growth of foodborne pathogens. Research has determined that some pathogens, such as Listeria monocytogenes, can survive for long periods of time in butters, such as almond butter. However, information on the persistence of L. monocytogenes in butters is lacking. The purpose of this study was to determine the population dynamics of L. monocytogenes in butters stored at 5 and 25°C. Nut (almond, hazelnut, pecan), seed (pumpkin, sesame, sunflower), legume (peanut and soy) and butters containing chocolate (hazelnut and peanut) were inoculated with a 4-strain cocktail of rifampicin-resistant L. monocytogenes at 4 (high inoculation) or 1 log CFU/g (low inoculation). High inoculation butters were mixed by hand for 15 min and 100-g portions were weighed into deli-style containers with lids and stored at 5 or 25°C for 12 mo (370 d). Low inoculation butters were stored in 25- g portions in stomacher bags at 25°C for 6 mo (180 d). During storage, 25 g from the 100- g high inoculation portion or 25 g from the low inoculation samples, in triplicate, were homogenized with 225 mL BPB (or BLEB for FDA BAM enrichments when necessary) and serial dilutions of the homogenate were plated onto BHIA with rifampicin for enumeration of L. monocytogenes. Data were statistically analyzed using Student’s t-test (α=0.05). The average initial population of L. monocytogenes in the butters was 3.58±0.25 log CFU/g for the high inoculation butters; L. monocytogenes was detected through enrichments for all low inoculation butters. After 12 mo storage at 5°C, the population of L. monocytogenes decreased by 1.34, 1.27, 1.72, 2.04 and 0.93 log CFU/g in almond, hazelnut, peanut with chocolate, hazelnut with chocolate and pecan butter, respectively, when inoculated at the higher level. Significantly less population reduction was observed in pumpkin, sesame, soy, peanut and sunflower butters (1.08, 0.61, 0.84, 0.05 and 0.40 log CFU/g, respectively). After 12 mo storage at 25°C, the L. monocytogenes population in all butters, with the exception of sunflower butter, decreased to below the limit of enumeration (1.67 log CFU/g), but the pathogen was still present via enrichment. For low inoculation butters, L. monocytogenes was present as determined by enrichment in all butters in at least one of two trials after 6 mo. The results of this study provide information on the survival of L. monocytogenes in different butter types when stored at different temperatures.
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Title: AMPLIFICATION AND PURIFICATION OF RECOMBINANT PRO-DEATH BAXΔ2 PROTEINS FOR STRUCTURE ANALYSIS
Creator: Zhou, Yi
Date: 2020
Description: BaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from...
Show moreBaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from Baxα to induce apoptosis. The current computationally predicted model of BaxΔ2 is based on known Baxα structure, which is considered biased. Therefore, the elucidation of the BaxΔ2 crystal structure is critical. The goal of this project was to obtain a sufficient amount of purified recombinant Bax∆2 protein for crystallization. We cloned full-length BaxΔ2 fused with a poly-histidine tag on either N-terminus (His-Bax∆2) or C-terminus (Bax∆2-His) into an inducible bacterial expression vector. We found that His-Bax∆2 proteins were expressed better than Bax∆2-His, which totally inhibit host growth. However, the protein concentration of His-Bax∆2 was still too low to be detected by Coomassie blue staining. To increase His-Bax∆2 expression and avoid cytotoxicity, we further tested different bacterial host cells and applied the chaperone system. However, all attempts could not overcome Bax∆2 cytotoxicity and the protein expression levels were not high enough to be feasible for further large-scale purification. The mechanism underlying how Bax∆2 inhibits bacterial growth is still a mystery because Bax∆2 eukaryotic targets (mitochondria and caspases) do not exist in bacteria. Further experiments are required to explore the mechanism of Bax∆2 cytotoxicity in bacteria, so as to finally optimize and elevate the BaxΔ2 protein yields.
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