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Title: ANTIBODY AND PEPTIDE CONJUGATES OF BIFUNCTIONAL CHELATORS FOR TARGETED CANCER THERAPY AND IMAGING
Creator: Kang, Chi Soo
Date: 2014, 2014-07
Description: A bifunctional ligand that can rapidly form a stable complex with a metal while conjugated to a tumor-targeting moiety is essential for...
Show moreA bifunctional ligand that can rapidly form a stable complex with a metal while conjugated to a tumor-targeting moiety is essential for targeted therapy and imaging of cancer. New bifunctional chelators in the NETA and DEPA series were developed for targeted radiation cancer therapy. The chelators showed rapid complexation kinetics with 90Y and 177Lu, and the corresponding radiolabeled complexes were stable in vitro and in vivo. Trastuzumab, Panitumumab, and c(RGDyK) conjugates (targeting HER-2, HER-1, and integrin αvβ3, respectively) of the novel bifunctional ligands were prepared. The conjugates displayed fast complexation kinetics with 90Y, 177Lu, and 205/6Bi, and the antibody and peptide conjugates labeled with 90Y, 177Lu, or 205/6Bi exhibited excellent stability and tumor targeting in mice bearing human colorectal cancer (LS-174T). Transferrin (Tf) and bile acid (BA) conjugates of novel chelators in the NE3TA series were prepared for targeted iron chelation therapy (ICT) and positron emission tomography (PET) imaging applications of cancer. Tf and BA conjugates of NE3TA had high cytotoxicity on HeLa, HT29, and PC3 cancer cells. The Tf-N-NE3TA and BA-N-NE3TA also displayed rapid complexation kinetics with 64Cu, and the Tf and BA conjugates of N-NE3TA labeled with 64Cu were stable and had high cellular uptake in HeLa and PC3 cancer cells. Tf-N-NE3TA was further conjugated with a fluorescence dye (Cy5.5) to develop targeted theranostic agent of cancer. Tf-N-NE3TA-Cy5.5 showed great radiolabeling efficiency with 64Cu, and the Tf-N-NE3TA-Cy5.5 labeled with 64Cu exhibited high cellular uptake in HeLa, HT29, and PC3 cancer cells. In summary, we have developed new bifunctional ligands having excellent chelation chemistry of 90Y, 177Lu, 205/6Bi, and 64Cu, and the tumor-specific antibody and peptide conjugates of the novel bifunctional chelators have a great potential for targeted cancer therapy and imaging as well as theranostic applications of cancer.
Ph.D. in Biology, July 2014
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Title: EFFECTS OF THERMAL TREATMENTS ON ELISA DETECTION OF MILK PROTEINS
Creator: Lu, Yingshuang
Date: 2011-07-27, 2011-07
Description: Cow’s milk allergy is one of the most prevalent food allergies in the United States. Commercial ELISA test kits, based on antigen-antibody...
Show moreCow’s milk allergy is one of the most prevalent food allergies in the United States. Commercial ELISA test kits, based on antigen-antibody reactions, are increasingly used by food manufacturers to detect the presence of allergen residues. Milk is a common ingredient used in a variety of foods that are subjected to different degrees of cooking. How thermal processing may affect the quantitative analysis of milk allergens by ELISA test kits needs to be determined. This study first examined the performance of four total protein assays (Pierce 660nm, Modified Lowry, Coomassie, BCA) for quantification of protein residues in thermally processed milk. BCA and Lowry assays were affected by lactose and its Maillard reaction byproducts resulting in the overestimation of protein concentrations in the samples. Pierce 660 nm assay was not affected and therefore was picked as the method for total protein analysis when evaluating commercial ELISA kits. Performances of four ELISA test kits (Veratox for Total Milk Allergen, BioKits BLG Assay Kits, ELISA SYSTEMS Casein and Beta-Lactoglobulin Residue assays) in comparison with the Pierce 660 nm assay for detection of thermally treated milk samples was also evaluated. NIST non-fat milk powder standard reference material 1549 and Backpacker’s Pantry Powdered Whole Milk were either heated in water at 60◦C, 63◦C, boiled, or autoclaved for 10 or 30 min, dry-heated in a mini-oven at 100 - 232◦C for 10 min, or fried in corn oil at 150◦C or 180◦C for 3 min in a deep fryer. Milk proteins in the oil samples were analyzed either directly or after extraction with phosphate-buffered saline with 0.05% Tween (PBST) followed by partitioning in hexane to remove residual oil. The results show that all four ELISA test kits were able to accurately quantify the amount of milk proteins in uncooked oils. Inclusion of the PBST/hexane extraction step prior to test kit analyses did not improve the detection. Elevated heat resulted in a lower level of proteins extracted. While the amount of protein residues determined by Veratox and ELISA SYSTEMS Casein kits were similar to those obtained by the Pierce assay, the BioKits BLG kits registered the highest amount of proteins in samples. On the other hand, ELISA SYSTEMS Beta-Lactoglobulin assay registered the lowest level of proteins in these samples. These results suggest that heat treatment could affect the solubility and possibly the antigenic properties of proteins. Depending on the test kits used, these changes could result in over- or underestimation of protein residues in thermally treated foods.
M.S. in Food Safety and Technology, July 2011
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Title: MULTIPLE SIGNAL TRANSDUCTION EVENTS AND MODULATION OF OLFACTORY RESPONSES IN MOUSE OLFACTORY SENSORY NEURONS
Creator: Yu, Yiqun
Date: 2012-04-30, 2012-07
Description: In the mammalian olfactory system, one olfactory sensory neuron (OSN) expresses a single olfactory receptor (OR) gene. I studied two subsets...
Show moreIn the mammalian olfactory system, one olfactory sensory neuron (OSN) expresses a single olfactory receptor (OR) gene. I studied two subsets of mouse OSNs, hereafter refered to as Ho-OSNs and AB-OSNs, in intact mouse olfactory turbinates using calcium imaging. Both of Ho-OSNs and AB-OSNs were located in the most ventral olfactory receptor zone. Ho-OSNs were specifically responsive to 2-heptanone (Ho), heptaldehyde (H) and cis-4-heptenal (cH). Dose-dependent analysis indicated that their responses to individual odorant was saturated within one logarithm unit of concentrations, a typical characteristic of isolated OSNs. Binary mixture and crossadpatation studies showed that these three odorants bound to the same binding sites in Ho-OSNs. However, these three structurally similar odorants activated distinct signaling pathways in Ho-OSNs. In detail, 2-heptanone-evoked intracellular calcium elevation was mediated by AC-cAMP signaling, while heptaldehyde triggered the PLC-DAG pathway. The cis-4-heptenal-evoked [Ca2+]i increases resulted from a combination of cAMP mediated activation and suppression involving PLC signaling. Furthermore, the PLCmediated intracellular calcium alteration was independent of IP3 signaling. A further complexity is that these olfactory receptors were able to interact with other types of Gprotein coupled receptors (GPCRs), such as purinergic receptors. I determined that both the P2X3 and P2Y2 receptor subtypes were expressed in Ho-OSNs. Application of purinergic agonists elevated [Ca2+]i increases in Ho-OSNs. It was discovered that the ATP-induced calcium response required either intracellular or extracellular calcium, while depletion of intracellular calcium stores blocked the UTP-evoked [Ca2+]i increases. Purinergic agonists were able to modulate the odor response in Ho-OSNs, and purinergic antagonist experiments showed that modulation of heptaldehyde-induced calcium responses was due to activation of P2X3 receptor subtypes while heptanone-induced calcium responses was not. Although AB-OSNs were found adjacent to Ho-OSNs, they had complete separate response profiles. AB-OSNs were sensitive to acetophenone (Ace) and benzaldehyde (Ben). In AB-OSNs, both acetophenone and benzaldehyde activated the PLC signaling pathway. Pharmacological characterization indicated that in AB-OSNs, P2X1 and P2Y2 receptors were present, which is different from that in Ho-OSNs. P2X and P2Y agonists modulated odorant responses in AB-OSNs. Purinergic signaling differentially regulated the various odorant responses in AB-OSNs. The acetophenoneevoked [Ca2+]i increases were negativly modulated through activation of the P2Y2 receptor, while the calcium response induced by benzaldehyde was modulated by P2X1 receptor activation. Collectively, these studies suggest that a complex signaling mechanism exists in OSNs, which has important implications for understanding the mechanism of information process in the olfactory system.
Ph.D. in Biology, July 2012
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Title: COMPARISON BETWEEN TWO CHITIN-PURIFIED PRO-DEATH PROTEINS IN MITOCHONDRIA TARGETING ANALYSIS
Creator: Yeap, Xin Yi
Date: 2011-04-19, 2011-05
Description: Bax protein belongs to the Bcl-2 family. It is pro-apoptotic and the most common form is Bax α. When a cell receives death stimuli, Bax...
Show moreBax protein belongs to the Bcl-2 family. It is pro-apoptotic and the most common form is Bax α. When a cell receives death stimuli, Bax protein will oligomerize and target to mitochondria. Another isoform of Bax called Bax 2, which has lost exon 2 and has a frame shift mutation, is verified to be more potent in inducing apoptosis than Bax α. Here, we would like to understand more about Bax 2 mitochondria targeting ability compared to the more common Bax α form. We first set up a cell-free system which contained purified Bax protein and purified mitochondria. The mitochondrial binding protein was identified using fractionation and Western blot with Bax isoformspecific antibodies. Integration of Bax protein into the mitochondrial membrane was determined using the alkaline stripping method. The results suggest that the majority of Bax α targeted and integrated into the mitochondria membrane, while the majority of Bax 2 did not target to mitochondria in the cell-free system. This may suggest that Bax 2 might need additional help from certain cytosol components such as co-factors to target to mitochondria or that Bax 2 induces cell death through a mechanism other than mitochondrial targeting.
M.S. in Biology, May 2011
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Title: THE MOLECULAR MECHANISM OF BAXΔ2-MEDIATED CELL DEATH AND ITS TISSUE DISTRIBUTION IN COLON CANCER
Creator: Zhang, Honghong
Date: 2014, 2014-07
Description: Bax is a pro-death tumor suppressor in the Bcl-2 family, and is frequently mutated in microsatellite instable tumors, especially Hereditary...
Show moreBax is a pro-death tumor suppressor in the Bcl-2 family, and is frequently mutated in microsatellite instable tumors, especially Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The loss of apoptotic Bax contributes to tumor development and chemoresistance. We recently uncovered that the combination of a Bax microsatellite mutation with a specific alternative splicing generated a unique Bax isoform (BaxΔ2) in Bax-negative cells. Similar to the prototype Baxα, BaxΔ2 is a potent pro-apoptotic molecule. However, the pro-apoptotic mechanism, therapeutic implication, and tumor tissue distribution of BaxΔ2 protein remain elusive. In this thesis research, we isolated and analyzed isogenic sub-cell lines that represent different Bax microsatellite statuses from colorectal cancer cells. We found that the colon cancer cells harboring Bax microsatellite G7/G7 alleles produced low levels of endogenous BaxΔ2 transcripts and proteins. BaxΔ2-positive cells were selectively sensitive to a subgroup of chemotherapeutics in comparison with BaxΔ2-negative cells. Different from other Bax isoforms, which mostly act through targeting mitochondria, BaxΔ2 recruited caspase-8 into the aggregates for activation, and consequently induced cell death independent of the mitochondrial pathway. Furthermore, the distribution of BaxΔ2 protein was mostly found in well-differentiated epithelial cells in primary colon tumor tissues or in primary squamous buccal cells, which contain Bax G7 mutation. However, not all cells harboring the Bax G7 mutation had a detectable level of BaxΔ2 proteins. These data suggest that, similar to Baxα, BaxΔ2 protein is pro-apoptotic, but not toxic to normal cells; expression of BaxΔ2 protein restores apoptotic program in Bax negative cells via a non-classical signaling pathway. Importantly, BaxΔ2 may provide a selective chemotherapeutic advantage for certain Bax-negative colon tumors.
Ph.D. in Biology, July 2014
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Title: The Effect of Salmonella Enteritidis on the Thermal Properties of Egg Albumen and Yolk
Creator: Chadha, Isha
Date: 2011-11-27, 2011-12
Description: Salmonella-inoculated and uninoculated fresh eggs were microwave processed to achieve pasteurization. However, quality degradation was...
Show moreSalmonella-inoculated and uninoculated fresh eggs were microwave processed to achieve pasteurization. However, quality degradation was observed in the albumen of inoculated eggs but not in the uninoculated eggs although the processing was the same. Previous work showed that dielectric properties of egg albumen and yolk do not change in the presence of the bacteria. A new hypothesis was therefore proposed that heat capacity is affected by its presence. If an egg has a lowered heat capacity it will become hotter for a given amount of microwave energy input than eggs having normal heat capacities. The goal of the experiments was to determine whether Salmonella at varying concentrations and different incubation times affects the temperature-dependent thermal properties of egg albumen and yolk during in-shell growth. Although the main concern was Salmonella’s effect on the heat capacity of albumen, the equipment used could measure thermal conductivity as well and measurements on yolk were included for completeness. The thermal properties of both egg albumen and yolk were measured by the KD2 Pro thermal properties analyzer (Decagon Instruments, Pullman, WA) via the supplied 30 mm dual-needle sensor. Variables were temperature (7oC, 30oC, 40oC, 50oC, 55oC and 60oC), bacterial inoculum (0, 103, 106, 108 cfu/ml) and incubation time (0 or 30 hours). The measurements showed no significant difference in thermal properties between inoculated and uninoculated samples of egg albumen and yolk regardless of change in temperature, bacterial concentration and incubation period. The absence of an effect on either the dielectric (previous study) or thermal (present study) parameters by the presence of Salmonella leaves unexplained the quality differences between similarly processed inoculated and uninoculated eggs. A biochemical explanation based on the interaction between metabolic byproducts and egg proteins, affecting how they denature, is a topic for future research.
M.S. in Food Safety and Technology, December 2011
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Title: AN EFFORT TO DISCOVER NOVEL BACTERIAL DSZ GENES TO HELP IMPROVE THE BIODESULFURIZATION OF CRUDE OIL
Creator: Salazar, Joelle Krieger
Date: 2011-04-25, 2011-05
Description: In this thesis study, soil samples were procured from various locations thought to contain high petroleum content, such as gas stations. Those...
Show moreIn this thesis study, soil samples were procured from various locations thought to contain high petroleum content, such as gas stations. Those samples were enriched for those that contained bacteria that were able to metabolize sulfur-containing compounds, such as DBT. The bacteria were then subjected to various experiments to determine if they possess any or all of the genes (dsz) encoding the three enzymes of the dibenzothiophene desulfurization pathway, as compared with R. erythropolis IGTS8. This feat was accomplished by using both growth experiments on medium containing dibenzothiophene as the sole sulfur source and genomic DNA extraction followed by PCR with dszABC universal primers. The resultant amplified DNA was then ligated to pGEM®-T Easy vector and transformed into electrocompetent E. coli cells. The transformants were subsequently screened and sent for sequencing. Using Blast, the sequences were analyzed. No obvious dsz homologies were identified although homologues in some cases to enzymes involved in sulfur metabolism were found. Several isolates were found to have dsz-like activity via the growth experiments as well as the Gibbs assay, which measures accumulation of the end product of the dsz-encoded pathway, 2-HBP Soil sample #32 showed promise in growth experiments in that the culture grew well in M9 with DBT as the sole sulfur source at temperatures of 30 °C, 37 °C, 45 °C, and 55 °C. Streaking the culture discovered two colonies with distinct morphology that were designated 32-W and 32-Y, for white and yellow colonies. 32-W and 32-Y also grew independently in M9 with DBT. Positive Gibbs Assays were obtained from the cultures 32-mixed, 32-W, and 32-Y.
M.S. in Biology, May 2011
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Title: EFFECTS OF CHROMIUM COMPOUNDS ON INSULIN-MEDIATED GLUCOSE UPTAKE
Creator: Yang, Liu
Date: 2012-04-13, 2012-05
Description: Chromium supplementation has been highlighted by its biological function in improvement of glucose metabolism. As essential trace nutrition,...
Show moreChromium supplementation has been highlighted by its biological function in improvement of glucose metabolism. As essential trace nutrition, chromium complex has great therapeutic potential in the alleviation of metabolic disorders, especially type II diabetes. This study is aimed at investigating the effect of two newly synthesized chromium (III) complexes Cr2(μ-OH)2(C4O4)2(H2O)4·(H2O)2, Cr2(μ-OH)2(μ1,2- C4O4)2(C2H6SO)4·(H2O)2 in comparison with a commercial compound CrCl3·6H2O on glucose uptake, as well as toxicity. We found that with 100 nM insulin stimulus, Cr2(μ- OH)2(C4O4)2(H2O)4·(H2O)2 significantly increased cellular glucose uptake, while Cr2(μ- OH)2(μ1,2-C4O4)2(C2H6SO)4·(H2O)2 inhibited the insulin-stimulated glucose uptake. Morphology study indicated that a relatively low concentration of these three complexes had little toxicity to cells within 24 h, but a higher concentration would lead to cell death. Cell growth curve supported the notion that the chromium (III) compounds in this study had no obvious cellular toxicity. Therefore, these results suggest that the newly synthesized less toxic chromium has a great potential in improvement of glucose metabolism in response to insulin. This study may provide valuable information in the treatment or management of diabetes. Keywords: Chromium, Insulin, Glucose uptake, Glucose metabolism, diabetes, type II diabetes
M.S. in Biology, May 2012
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Title: INTERACTION OF DAIRY BASED PROTEIN WITH PHENOLIC ANTIOXIDANT OF STRAWBERRY POWDER
Creator: Feng, Haoshi
Date: 2012-05-04, 2012-05
Description: Fruits contain an abundant amount of polyphenolic compounds that have antioxidant properties. These compounds have shown the ability of...
Show moreFruits contain an abundant amount of polyphenolic compounds that have antioxidant properties. These compounds have shown the ability of reducing the risk of major degenerative diseases, relief from allergies/asthma, weight loss and improved circulation. Dairy proteins have numerous benefits as enhancing muscle formation, weight and blood pressure control, beneficial effect in bone and dental health and protection against toxins, bacteria, and viruses. The combination of both fruit and dairy in commercial or homemade mixtures, such as smoothies, is readily available and popular. But in fact, the interactions which happen between proteins and fruit antioxidants could affect the availability and potential activity of antioxidants. This thesis will focus on the interaction of dairy proteins with antioxidant from freeze dried strawberry and measure the availability of the antioxidant capacity. Freeze dried strawberry powder at different concentrations (1.64%, 3.28% and 6.56%) was mixed with four types of proteins at different concentration to demonstrate the interaction over a range. The proteins used were skim milk powder (6.89%), whey from bovine milk (0.51%), casein from bovine milk (2.04%), and albumin from bovine serum (2.55%). The antioxidant content was measured by several analytical techniques using LCMS/MS, Oxygen Radical Absorbance Capacity (ORAC) assay and Folin-Ciocalteu assay. SDS-PAGE was used to examine the change of protein molecular weight before and after the reactions. The binding interaction resulted in significantly reduced ORAC and Folin- Ciocalteu assay values compared to the respective non-protein strawberry mixtures (P<0.001 and P<0.05). The SDS-PAGE images between protein strawberry mixtures and non-protein strawberry mixtures showed similar results. The (+)-catechin, cyanidin-3-O glucoside, pelargonidin-3-O-glucoside and kaempferol-3-coumaroylglucoside content were significantly reduced when comparing skim milk based strawberry powder mixtures and non-protein strawberry mixtures (P<0.05). This research indicates the binding interaction between dairy protein and freeze dried strawberry powder reduces the antioxidant activity of the freeze dried strawberry powder.
M.S. in Food Safety and Technology, May 2012
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Title: PANCREATIC ADENOCARCINOMA: SECRETORY MARKERS AND CORRELATION WITH PARKINSON’S DISEASE
Creator: Gayatri, Sitaram
Date: 2011-04-27, 2011-05
Description: Pancreatic adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation...
Show morePancreatic adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers. Proteins of importance in neurodegenerative diseases like Parkinson’s disease (PD) are believed to be of etiological significance and correlated to or be the cause/effect of pancreatic disease. Expression of proteins like PD autosomal recessive, early onset 7 (PARK-7/DJ-1), α-Synuclein (SNCA) both of which are known to be pathognomonic to Parkinson’s disease was found altered in various stages of the disease. Purine Nucleoside Phosphorylase (NP) has been exploited to be a therapeutic target in cancers. The expression of all these three proteins was found to be variable with the stage and existence of metastatic lesions in PDAC tissues. The protein levels of NP were higher in PDAC sera compared to the benign samples. The serum NP levels of other cancers viz. lung, kidney, colon and breast were also not elevated. While the serum levels of NP and those of its downstream metabolites have the potential to differentiating PDAC from benign, the expression levels of SNCA and PARK-7/DJ-1 allude to a possible nuance in the etiology of PD and PDAC.
M.S. in Biology, May 2011
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Title: THE CHARACTERIZATION OF TWO THERMOPHILIC STRAINS CONCERNING BIO-DESULFURIZATION OF DIBENZOTHIOPHENE BY THE 4S PATHWAY
Creator: Li, Enze
Date: 2016, 2016-05
Description: A moderately thermophilic Mycobacterium strain (denoted Myco-U) was isolated in our lab as a contaminant during the cultivation of another...
Show moreA moderately thermophilic Mycobacterium strain (denoted Myco-U) was isolated in our lab as a contaminant during the cultivation of another thermophilic Paenibacillus strain, and has been proved to harbor the genes that encode the enzymes for dibenzothiophene (DBT) biodegradation via the 4S pathway; this makes it possible to efficiently remove organosulfur compounds (e.g. DBT and DBTO2) from the crude petroleum. Through directed evolution (continuous passage and selection with increasing temperature), the Myco-U strain has shown moderate capacity of metabolizing DBT to 2-hydroxybiphenyl (2-HBP) by the 4S pathway at a thermophilic temperature (~53 ℃). In the middle-to-late stages of the selections, a stubborn contaminant (denoted Bacillus X) arose and has remained difficult to get rid of from our medium and cultures ever since, leading to a persistent contamination and repetition of our experiments. The reason why Bacillus X is regarded as a contaminant is that it does not obtain sulfur by the expected means (e.g., DBT desulfurization), but utilizes the tiny amount of sulfate from the vitamin mix and 50 μg/ml yeast extract in our medium to grow. This is confirmed by the facts that Bacillus X does not yield any detectable 2-HBP, and a significant difference of growth has been detected between cultures with DBT as the sole sulfur source and the positive controls, which employs Na2SO4 as sulfur source. Through 16S rDNA amplification, cloning, and sequencing, Bacillus X was identified as a Bacillus species, and shows the highest homology (99 % identical) to a specific uncultured bacterium clone (NCBI #Accession: JN882111.1). The chromogenic mechanism and impacts of pH and carbonate upon the Gibbs assay, by which 2-HBP is quantified, were investigated. When DBT is absent or at undetectably low levels, cultures measured by the assay display a yellowish-to-brownish background after adding the Gibbs reagent (2,6-dichloroquinone-4-chloroimide). According to our results, the Gibbs reaction prefers alkaline conditions and pH 8.0 ranks the optimal (compared with pH 7.0, 7.5, 8.5 and 9.0) at which the maximum A610nm is detected.
M.S. in Biology, May 2016
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Title: CHANGES OF BACTERIAL SPECIES AND HEME PROTEIN LEVEL IN ACTIVATED SLUDGE COMMUNITIES ACCLIMATED TO LOW AERATION
Creator: Li, Hainan
Date: 2016, 2016-05
Description: Increasing oxygen utilization of activated sludge under low dissolved oxygen (DO) conditions can lead to energy savings in wastewater...
Show moreIncreasing oxygen utilization of activated sludge under low dissolved oxygen (DO) conditions can lead to energy savings in wastewater treatment systems. Adaptation of sludge communities to low oxygen conditions may be facilitated by increased expression of heme proteins. One way to assess heme protein expression in sludge is to analyze the species present in the sludge community during the adaptation process. In the work reported here, growth of a sludge culture adapted by Kunkel to low aeration for 48 weekly passages was continued for an additional 26 passages (182 days). The activated sludge was cultured in synthetic wastewater under low DO conditions. Temperature, dissolved oxygen, and specific oxygen uptake rates (SOUR) were measured at the end of each weekly passage. The community structures of passages 50, 53 and 65 were determined by 16S rDNA cloning techniques. The type of heme present was identified using the pyridine hemochromogen method. The SOUR values measured increased slightly in the first part of the experiment, when the culture DO was very low, but then decreased in the latter part of the experiment, when the culture DO increased. The community structure diversity in passage 50 in Kunkel’s study and passage 50 from this work are totally different in terms of species present, even though the two cultures were both derived from Kunkel’s passage 48 and were grown under conditions which were matched as closely as possible for two additional passages. This indicates that the community structure is highly sensitive to small changes in growth conditions. From the point of view of types of hemoglobin (Hb), the community became more diverse by passage 53, containing all three types of truncated Hb (trHbN, trHbO, and trHbP), possibly due to the need for NO scavenging and oxygen transfer enhancement. The proportion of cells that synthesize truncated Hb decreased slightly through passages 50 to 53 to 65. The percentage of cells that encode FlavoHbs decreased from 84 % to 50 % by passage 65. Overall there was an increase in Hb expression from passage 50 to passage 65. Heme b expression in the sludge culture was confirmed. Successful adaptation of the sludge culture to low DO conditions via enhancement of oxygen uptake was not obvious in this study. Yet the overall tendencies of SOUR and Hb expression suggest that it is possible to acquire a culture more efficient in oxygen uptake if a stable low aerobic environment can be maintained for an extended period.
M.S. in Biology, May 2016
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Title: LOW TEMPERATURE STORAGE METHODS FOR SKINNED MANDUCA SEXTA FLIGHT MUSCLE
Creator: Liu, Xue
Date: 2015, 2015-12
Description: The flight muscle of the Hawk moth, Manduca sexta, is a synchronous muscle and an emerging model system for structure and function studies of...
Show moreThe flight muscle of the Hawk moth, Manduca sexta, is a synchronous muscle and an emerging model system for structure and function studies of muscle. (Tu & Daniel, 2004). There are several interesting properties of Manduca sexta flight muscle. In its physiological characteristics, like mammalian skeletal and cardiac striated muscle, it is a synchronous muscle. However, it is much more similar structurally to the more widely known asynchronous insect flight muscles of Drosophila and Lethocerus. Up to now there has been no good method for storing Manduca sexta flight muscle for a long time. It is often difficult to match the time of obtaining live moths muscle and scheduled X-ray or mechanics experiment. Usually, best results are obtained by using freshly prepared muscle fibers. It would be highly desirable to find storage conditions that can maintain the structural and physiological functions in vitro for days or weeks rather than needing to be prepared fresh from living tissue for every experiment. (Yu-Shu Cheng, MS thesis IIT, 2013). Muscle proteins will degrade over a periods of days even in the presence of protease inhibitors even at low temperature. Here we aimed to find storage conditions which would work best for Manduca sexta flight muscle. In this thesis, trehalose, in combination with glycerol, was evaluated as an addition storage solution as a cryoprotectant to protect the muscle fiber from the low temperature. At very low temperature, organisms became dehydrated. Loss of liquid water from cells can cause irreversible damage, so they will not function even when rehydrated. Here we show that using both glycerol and trehalose as cryoprotectants in storage solution, the muscle fiber maintain high maximum active force for up to a month in storage.
M.S. in Biology, December 2015
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Title: ANTI-TUMOR SPLICING: RESTORING THE TUMOR SUPPRESSOR BAX IN MICROSATELLITE UNSTABLE TUMORS
Creator: Haferkamp, Bonnie
Date: 2011-11, 2011-12
Description: Microsatellite instability (MSI) is a hallmark for many tumors, especially colon, endometrial, gastric and bladder. Bax, a tumor suppressor...
Show moreMicrosatellite instability (MSI) is a hallmark for many tumors, especially colon, endometrial, gastric and bladder. Bax, a tumor suppressor and pro-death Bcl-2 family member, is frequently mutated in MSI tumors. A microsatellite mutation produces a frameshift with premature termination, leading to “Bax-negative” tumors. Although low Bax expression in tumors is often associated with poor prognosis, several studies have correlated lack of Bax in MSI tumors with improved prognosis. However, the molecular explanation for this paradox is unknown. Here we show that “Bax-negative” tumors in fact generate a novel family of anti-tumor Bax-MSI isoforms through alternative splicing. The thesis includes two parts. In Chapter One, we fully characterize one Bax-MSI isoform, BaxΔ2. We show that BaxΔ2 is detrimental to cancer cells but through a non-conventional death pathway, with differential sensitivity to chemotherapeutics. In Chapter Two, we present an entire family of Bax-MSI isoforms, and illustrate a potential molecular mechanism behind its production. We show that the frequency of Bax alternative splicing is significantly higher in MSI than non-MSI tumors, and that BaxΔ2 trans splicing requirements are ubiquitous in human cell lines. The discovery of functional Bax isoforms in Bax-mutated tumors may help explain why the apparent loss of Baxα in tumors is sometimes associated with a better prognosis. In addition, the unique sequences of Bax-MSI isoforms can serve as biomarkers for diagnostic and treatment purposes. Importantly, identification of Bax-MSI isoforms will provide a great opportunity from a genetic approach or drug design for treatment of MSI cancer.
Ph.D. in Biology, December 2011
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Title: LAM MUTANT LARVAE WITH MELANOTIC MASSES DO NOT SURVIVE WITH THEIR SIBLINGS THAT DO NOT DEVELOP MASSES
Creator: Cui, Yi
Date: 2012-04-28, 2012-05
Description: Melanotic masses are black nodules that form inside larvae and can be found in multiple mutants (Minakhina & Steward, 2006). There are two...
Show moreMelanotic masses are black nodules that form inside larvae and can be found in multiple mutants (Minakhina & Steward, 2006). There are two types of melanotic masses, the immune-related masses and the non-hemocyte-associated masses (Minakhina & Steward, 2006). Immune-related melanotic masses form in lam mutants (Markovic et.al, 2009). We now show all lam mutant larvae survived and 33% of the them developed melanotic masses when grown on fresh food, however, only 66% of them survived and none of them formed melanotic masses when living in the frassfood; the mixture of poo medium collected from wild type larvae vials. We find that frassfood lethality is not recipe specific, since the results were similar on normal yeast mashed potato medium and Nutrifly food. Our results also identified that it is bacteria in frassfood that kills lam mutant larvae with melanotic masses because the survival and melanotic mass incidence of lam mutant larvae were similar to fresh food when antibiotics were added to frassfood. However lam mutant larvae with melanotic masses are not generally sensitive to bacteria because all lam mutant larvae showed sensitivity to S. marcescens. Besides, since there is no frassfood lethality observed on Relish mutants, we propose that the humoral response was not affected in the lam mutants. The frassfood lethality to larvae with melanotic masses was also observed in neither cactus mutants nor the hopTum-l mutants, so the immune defects that caused melanotic masses in these two mutants do not lead to the frassfood lethality. Finally, we show that the frassfood lethality is not exactly the same in lam overexpressing larvae with melanotic masses and lam C overexpressing larvae with melanotic masses to the lam/Df larvae with melanotic masses. Thus it is possible that the gut immunity was affected in lam mutants with melanotic masses so that they were killed by the bacteria in frassfood.
M.S. in Biology, May 2012
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Title: SKINNING AND STORAGE METHODS FOR STRUCTURAL AND FUNCTIONAL STUDIES OF MANDUCA SEXTA FLGHT MUSCLE
Creator: Zhang, Mengjie
Date: 2012-04-22, 2012-05
Description: Like mammalian cardiac muscle, the flight muscle of the hawk-moth Manduca sexta is synchronous. However, it also has significant structural...
Show moreLike mammalian cardiac muscle, the flight muscle of the hawk-moth Manduca sexta is synchronous. However, it also has significant structural similarities with asynchronous insect flight muscle systems, such as those of Drosophila and Lethocerus. Different physiological function depends on the underlying different molecular structures. Although Drosophila and Lethocerus have been well studied, Menduca sexta is still a newly developed research model. Many different skinning and storage methods are being used worldwide for in vitro studies of a wide variety of muscle systems. Here our goal is to develop better skinning solution and storage condition which will maintain muscle structure and function as well as intact muscle. To achieve this end, several kinds of skinning solutions and storage conditions were evaluated by laser and X-ray mechanical experiments. The solution from HAMM Lab (University of Washington) showed the best ability to destroy plasma membrane, without affecting muscle interior structure and function. For two storage conditions, 4°C without glycerol and -20°C with 50% glycerol, both laser and X-ray experiments show the latter one is better, which means muscles stored at -20°C with 50% glycerol have faster and stronger reaction for high calcium solution, and also have sharper peaks in X-ray diffraction patterns. The presence of protease inhibitors is also necessary to maintain the contraction ability of muscle. Furthermore, the X-ray experiments for muscle fibers stored at -20°C with 50% glycerol and -80°C with 75% glycerol, show the expected results, which the muscles stored at latter condition show far superior X-ray diffraction patterns, especially in meridional reflections and layer lines. The lower temperature can minimize protein degradation. Notably, incubating muscles in glycerol solutions at 4°C before storing them at -80°C, and washing muscles thoroughly after stored at 75% glycerol, are critical to ensure that all of the glycerol goes in and out of muscle fibers, respectively. The significance of these findings is that it now appears to be possible to store prepared Manduca sexta flight muscles for at least eight days at -80°C with good structural preservation enabling a large class of future experiments not requiring fresh deliveries of moth from the suppliers.
M.S. in Biology, May 2012
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Title: STRAWBERRY EXTRACT ATTENUATES ELEVATED FREE FATTY ACID AND/OR GLUCOSE MEDIATED IMPAIRED INSULIN SIGNALING IN VITRO IN HUMAN SKELETAL MUSCLE CELLS
Creator: Kangath, Archana
Date: 2013, 2013-07
Description: Elevated glucose and free fatty acids (FFA) are evident in individuals with insulin resistance (IR) and are associated with oxidative stress....
Show moreElevated glucose and free fatty acids (FFA) are evident in individuals with insulin resistance (IR) and are associated with oxidative stress. Anthocyanins are suggested to have favorable effects on human health due to their ability to modulate oxidative stress and cell signaling pathways in peripheral tissues. We hypothesized that strawberry anthocyanins (Str) will improve oxidative stress-mediated impaired insulin signaling in vitro in human skeletal muscle cells (HSMCs). Oxidative stress was generated using physiologically high glucose, FFA or a combination of both for 2–6 h with and without Str (0.1–1.0 mg/mL) in vitro in HSMCs. Cell viability was not affected by any of the above treatments/time. At the end of treatments, cells were treated with insulin (100 nM) for 20 min. Phosphorylation of Akt (p-Akt/Akt) was studied using AlphaScreen® Surefire® assay as a measure of insulin signaling. Our results indicate that FFA (2 mM) or combination of Glucose (15 mM) + FFA (2 mM) significantly decreased p-Akt/Akt compared to control cells treated with phosphate buffered saline (P<0.05). ST extract restored FFA and Glucose + FFA-induced reductions in p-Akt/Akt levels (P<0.05). These in vitro data suggest a role of strawberry in improving insulin sensitivity having potentially important implications for people with IR.
M.S. in Biological Sciences, July 2013
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Title: DEGUELIN ACTION IS THROUGH REGULATION OF ERK-MAPK PATHWAY IN TRIPLE- NEGATIVE BREAST CANCER CELLS
Creator: Katta, Harshadadevi
Date: 2012-11-21, 2012-12
Description: Breast cancer is the most common cancer and the leading cause of cancer related deaths amongst women in the United States. In 15-20% of...
Show moreBreast cancer is the most common cancer and the leading cause of cancer related deaths amongst women in the United States. In 15-20% of patients with breast cancer, patients do not express steroid receptors(ER/PR) and do not have amplification of HER2. This condition is called triple negative breast cancer (TNBC). Patients with TNBC have poor prognosis. No molecular targeted therapies are available to treat TNBC. Anthracyclines and taxane based therapies are effective with limited success, and are often toxic. As TNBC cells overexpress Epidermal Growth Factor Receptor (EGFR), it provides an attractive molecular tool for the targeted therapy. Deguelin, a rotenoid isolated from an African plant, Mundulea sericea has been shown to inhibit growth of various experimental cancers. Deguelin is a well known inhibitor of pAKT, a downstream target of EGFR. In this study we evaluated the effect of deguelin and its mechanism of anticancer action in triple negative human breast cancer cell lines. Deguelin inhibited cell proliferation of MDA-MB-231, MDA-MB-468, BT-549, and BT- 20 in a dose and time-dependent manner. Western blots and immunofluorescence analyses suggested that the effect of Deguelin is mediated through inhibition of Receptor Tyrosine Kinases (EGFR, c-Met) and its downstream molecular targets, Our results show that deguelin treatment reduces the expression of pERK (p44/42) (Thr202/Tyr204), pAKT (ser473), c-Myc, pSTAT3 (Tyr705) and survivin in a dose and time dependent manner in MDA-MB-231, MDA-MB-468 human breast cancer cells. In conclusion, results from this study suggest that Deguelin may be of potential therapeutic value in treatment of triple negative breast cancers.
M.S. in Biology, December 2012
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Title: Behavior of E. Coli 0157:H7 in Packaged Spinach after Exposure to Sanitizing Solution
Creator: Lu, Yin
Date: 2011-12-13, 2011-12
Description: Leafy greens have been associated with multiple outbreaks of Escherichia coli O157:H7 (EC) infection in recent years. More information is...
Show moreLeafy greens have been associated with multiple outbreaks of Escherichia coli O157:H7 (EC) infection in recent years. More information is needed on EC survival during post-harvest processes, and our study aimed at evaluating survival in packaged spinach after treatment of cells with sodium hypochlorite. A green fluorescent protein-expressing strain of EC was exposed to sodium hypochlorite in two different ways, i.e., exposed in solution or on the spinach leaves. For exposure in the solution, EC was inoculated into 1 ppm sodium hypochlorite for 30 seconds, then the cells were inoculated onto the spinach leaves. For exposure on the spinach leaves, EC was first inoculated onto leaves; then the inoculated spinach leaves were dipped into 100 ppm sodium hypochlorite. After inoculation, the spinach was packaged to obtain high-oxygen (20%O2/3%CO2) and low-oxygen (0%O2/15%CO2) atmospheres, respectively. The packages were stored at 4°C or 15°C, and EC populations were quantified at six sampling points during 14 days of storage. The data were analyzed by DMFit and Microfit to demonstrate the growth curve and kinetics. In general, there were no apparent differences in EC behavior between the chlorine treated and control groups for most of the conditions. However, in certain conditions, the chlorine-treated cells showed a faster decrease and/or slower increase in population than the control packages. Generally, low temperature and high oxygen conditions led to lower survival than high temperature and low oxygen. For EC exposed in solution, the EC population showed a greater reduction and faster decrease in the high-oxygen than low-oxygen packages at 4°C. At 15°C, the lowoxygen condition resulted in a faster growth rate (+0.43 log CFU/ml/day) than high oxygen packages (+0.17 log CFU/ml/day). Also, in the 4°C high-oxygen condition, the chlorine-treated cells showed a greater population decrease (-0.95 log CFU/ml) than the control groups (-0.57 log CFU/ml). For EC exposed on the spinach, the low-oxygen packages showed greater population levels and faster growth rates compared to the highoxygen packages at 15°C. At 4°C, chlorine treated groups and control groups showed no apparent differences for both low- and high- oxygen packages. At 15°C, the chlorine treated packages showed less increase in population (+0.79 log CFU/ml) than the controls (+1.58 log CFU/ml) in low-oxygen; and the control groups increased at a faster growth rate (+0.57 log CFU/ml/day) than the chlorine treated ones (+0.25 log CFU/ml/day).
M.S. in Food Safety and Technology, December 2011
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Title: ISOLATION AND KINETIC COMPARISON OF INDIRECT FLIGHT MUSCLE MYOSIN IN MANDUCA SEXTA
Creator: Liu, Yang
Date: 2016, 2016-05
Description: The indirect flight muscles (IFM) of insects are nature’s versatile engines highly specialized to produce power for flight. Myosin, the motor...
Show moreThe indirect flight muscles (IFM) of insects are nature’s versatile engines highly specialized to produce power for flight. Myosin, the motor protein, plays an important role in this process, supporting the rapid wingbeat frequency and converting chemical energy into mechanical force. A novel efficient protocol for preparing functional myosin from Manduca sexta flight muscle is described in this study. We tested the biochemical properties of myosin from the dorsal subunit and ventral subunit of the dominant flight muscle. High salt solubilization and low salt precipitation was used to extract myosin from the homogenate of moth split thorax muscles since low ionic strength allows filament formation and precipitation of myosin while high ionic strength helps myosin solubility. Coomassie Blue stained 10% SDS PAGE analysis showed the purity was 50% and the result was confirmed by western blot with anti-myosin antibody MAC 147. This revealed the final pellet of purification was myosin and was of moderate purity and homogeneity, showing that this method was feasible for isolating myosin from this species. The focus of this study was to compare the catalytic efficiency of extracted ventral DLM1 myosin and dorsal DLM1 myosin in vitro, as myosin was the dominant catalytic protein which hydrolyze ATP to utilize energy in muscle contraction. The rate of an identical ATP hydrolysis reaction was measured catalyzed by myosin extracted from DLM1D and DLM1V, and the activation energy (Ea) was calculated using Arrhenius equation. The reaction catalyzed by dorsal myosin exhibited higher Ea than that catalyzed by ventral myosin, which meant a lower catalytic efficiency of DLM1D myosin. This indicated catalytic heterogeneity of DLM1D and DLM1V myosin in a single muscle. Additionally, a significant within muscle Ea difference of ATP hydrolysis could be partially responsible for the known temperature gradient, within the DLM1 of Manduca demonstrated previously(Nicole T George & Daniel, 2011), considering that the hydrolysis of ATP is responsible for the heat generated during muscle contraction as well as muscle force and lengthening.
M.S. in Biology, May 2016
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