Browsing by Subject "proteins"
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Item Bioinformatics Analysis and Annotation of Microtubule Binding and Associated Proteins (MAPs) - Creating a Database of MAPs(2005-08) Shenoy, Narmada; Guenther, BrianMicrotubules have many roles in the cytoskeletal infrastructure. This infrastructure underlies vital processes of cellular life such as motility, division, morphology, and intracellular organization and transport. These different roles are carried out by the creation of different microtubule (MT) systems (such as basal bodies, centrioles, flagellum, kinetochores, and mitotic spindles). The changing roles require the cytoskeleton to be both dynamic and static in nature. Guiding these processes are a network of proteins that direct cellular behavior through their ability to bind microtubules (MTs) in a spatial- and temporal-specific manner. The identification and characterization of the suite of microtubule binding and associated proteins (MAPs) involved in MT systems is important for the understanding of the biological form and function of each MT system. This research involved the analysis and annotation of four MAPs – Ensconsin in Humans, Hook (homolog 3) in Humans, Protein Regulator of Cytokinesis 1 (PRC1) in Humans and Anaphase Spindle Elongation protein (ASE1) in yeast. A bioinformatics approach was used for the annotation and analysis. A protocol for analysis and annotation of MAPs was developed. During the process, some limitations in using bioinformatics tools and procedures were encountered. These limitations were overcome, the initial protocol was improved on and a modified protocol of analysis was developed. A database was designed and built to hold annotated information on the MAPs. We seek to disseminate this database and its functionalities as a web resource to the scientific community. It will provide an excellent forum for researchers to obtain relevant information on MT binding and associated proteins (MAPs). Infection by parasitic protozoa causes incalculable morbidity and mortality to humans and agricultural animals. In this research, we have also focused on MAPs in parasitic organisms of the Apicomplexan and Trypanosomatid genera. The protocol for analysis incorporates steps to analyze MAPs from these organisms as well. Malaria (a potentially life threatening disease) is caused by Plasmodium, an Apicomplexan parasite. This parasite is transmitted to people by the female Anopheles mosquito, which feeds on human blood. African Sleeping Sickness is an acute disease 8 caused by Trypanosoma brucei that typically leads to death within weeks or months if not treated. Microtubule-associated proteins (MAPs) and their alteration of the unique microtubule (MT) systems play major roles in these organisms throughout their life cycle and are required for their pathogenic mechanisms. Each parasite contains unique MT systems that will test our annotation process as well as prepare the DB for addition of other novel MT systems, such as those contained with plants. Additionally, these single cell organisms have a multistage life cycle that provide similar annotation challenges to those encountered when one considers multi-cellular organisms. Therefore, a researcher working on any MT system within the database will find useful information regardless of the organism that they are studying. This will leave us with a sub-set of MAPs from parasitic organisms in our database that are potential drug-targets.Item Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis(Springer Nature, 2019-03-08) Wang, Junmei; Fry, Colin M. E.; Walker, Chandler L.; Biomedical Sciences and Comprehensive Care, School of DentistryAmyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = -0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.Item Counting Photobleach Steps and the Dynamics of Bacterial Predators(Office of the Vice Chancellor for Research, 2016-04-08) Jashnsaz, Hossein; Tsekouras, Konstantinos; Al Juboori, Mohammed; Weistuch, Corey; Miller, Nick; Nguyen, Tyler; McCoy, Bryan; Perkins, Stephanie; Anderson, Gregory; Presse, StevePhotobleach (PB) counting is used to enumerate proteins by monitoring how the light intensity in some regions decreases by quanta as individual fluorophores photobleach. While it is straightforward in theory, PB counting is often difficult because fluorescence traces are noisy. In this work, we quantify the sources of noise that arise during photobleach counting to construct a principled likelihood function of observing the data given a model. Noise in the signal could arise from background fluorescence, variable fluorophore emission, and fluorophore blinking. In addition, in a completely different direction, we explore the role of hydrodynamic interactions on the dynamics of bacterial predators. Our study shows that Bdellovibrio (BV) - a model predatory bacterium - is susceptible to self-generated hydrodynamic forces. Near surfaces and defects, these hydrodynamic interactions co-localize BV with its prey, and this may enhance BV’s hunting efficiency.Item The Effect of Molecular Weight on Passage of Proteins Through the Blood-Aqueous Barrier(ARVO, 2019-04) Ragg, Susanne; Key, Melissa; Rankin, Fernanda; WuDunn, Darrell; Biostatistics, School of Public HealthPurpose: To determine the effect of molecular weight (MW) on the concentration of plasma-derived proteins in aqueous humor and to estimate the plasma-derived and eye-derived fractions for each protein. Methods: Aqueous humor and plasma samples were obtained during cataract surgery on an institutional review board–approved protocol. Protein concentrations were determined by ELISA and quantitative antibody microarrays. A total of 93 proteins were studied, with most proteins analyzed using 27 to 116 aqueous and 6 to 30 plasma samples. Results: Plasma proteins without evidence of intraocular expression by sequence tags were used to fit a logarithmic model relating aqueous-plasma ratio (AH:PL) to MW. The log(AH:PL) appears to be well predicted by the log(MW) (P < 0.0001), with smaller proteins such as cystatin C (13 kDa) having a higher AH:PL (1:6) than larger proteins such as albumin (66 kDa, 1:300) and complement component 5 (188 kDa, 1:2500). The logarithmic model was used to calculate the eye-derived intraocular fraction (IOF) for each protein. Based on the IOF, 66 proteins could be categorized as plasma-derived (IOF<20), whereas 10 proteins were primarily derived from eye tissue (IOF >80), and 17 proteins had contribution from both plasma and eye tissue (IOF 20–80). Conclusions: Protein concentration of plasma-derived proteins in aqueous is nonlinearly dependent on MW in favor of smaller proteins. Our study demonstrates that for proper interpretation of results, proteomic studies evaluating changes in aqueous humor protein levels should take into account the plasma and eye-derived fractions.Item Effects of biscuit-type feeding supplementation on the neurocognitive outcomes of HIV-affected school-age children: a randomized, double-blind, controlled intervention trial in Kenya(Compuscript, 2017-12-01) Khee Loo, Kek; Rizzo, Shemra; Chen, Qiaolin; Weiss, Robert E.; Sugar, Catherine A.; Ettyang, Grace; Ernst, Judith; Samari, Goleen; Neumann, Charlotte G.; Health Sciences, School of Health and Rehabilitation SciencesObjective: To determine if meat or soy protein dietary supplementation will enhance the neurocognitive performance of HIV-affected children at-risk of malnutrition and food insecurity. Methods: A randomized, double-blind, controlled intervention trial evaluated the effect of nutritional supplementation on the neurocognitive outcomes of 49 HIV-affected school-age children in western Kenya. The intervention consisted in providing the mother, target child, and siblings with one of three isocaloric biscuit-type supplements – soy, wheat, or beef – on 5 days per week for 18 months. Neurocognitive outcomes of the target children were assessed by a battery of eight measures and followed up longitudinally for up to 24 months. Results: Mixed effects modeling demonstrated significant differences in the rates of increase over time among all three groups (F test degrees of freedom of 2, P<0.05) for Raven’s progressive matrices performance, but not for verbal meaning, arithmetic, digit span backward, forward, and total, embedded figure test, and Beery visual–motor integration scores. Conclusion: HIV-affected school-age children provided with soy protein supplementation showed greater improvement in nonverbal cognitive (fluid intelligence) performance compared with peers who received isocaloric beef or wheat biscuits. Soy nutrients may have an enhancing effect on neurocognitive skills in HIV-affected school-age childrenItem Integrative Computational Genomics Based Approaches to Uncover the Tissue-Specific Regulatory Networks in Development and Disease(2020-03) Srivastava, Rajneesh; Janga, Sarath Chandra; Liu, Xiaowen; Marrs, James A.; Kaplan, Mark H.Regulatory protein families such as transcription factors (TFs) and RNA Binding Proteins (RBPs) are increasingly being appreciated for their role in regulating the respective targeted genomic/transcriptomic elements resulting in dynamic transcriptional (TRNs) and post-transcriptional regulatory networks (PTRNs) in higher eukaryotes. The mechanistic understanding of these two regulatory network types require a high resolution tissue-specific functional annotation of both the proteins as well as their target sites. This dissertation addresses the need to uncover the tissue-specific regulatory networks in development and disease. This work establishes multiple computational genomics based approaches to further enhance our understanding of regulatory circuits and decipher the associated mechanisms at several layers of biological processes. This study potentially contributes to the research community by providing valuable resources including novel methods, web interfaces and software which transforms our ability to build high-quality regulatory binding maps of RBPs and TFs in a tissue specific manner using multi-omics datasets. The study deciphered the broad spectrum of temporal and evolutionary dynamics of the transcriptome and their regulation at transcriptional and post transcriptional levels. It also advances our ability to functionally annotate hundreds of RBPs and their RNA binding sites across tissues in the human genome which help in decoding the role of RBPs in the context of disease phenotype, networks, and pathways. The approaches developed in this dissertation is scalable and adaptable to further investigate the tissue specific regulators in any biological systems. Overall, this study contributes towards accelerating the progress in molecular diagnostics and drug target identification using regulatory network analysis method in disease and pathophysiology.Item A Novel Western Multi-blotting Device(Office of the Vice Chancellor for Research, 2012-04-13) Wanis, Mina; Blair, Matthew; Chien, Stanley; Yokota, HirokiBlotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve 5 blotting from a single gel and reduce time and cost for protein analysis.Item Rare coding variants and X-linked loci associated with age at menarche(Nature Publishing Group, 2015-08-04) Lunetta, Kathryn L.; Day, Felix R.; Sulem, Patrick; Ruth, Katherine S.; Tung, Joyce Y.; Hinds, David A.; Esko, Tõnu; Elks, Cathy E.; Altmaier, Elisabeth; He, Chunyan; Huffman, Jennifer E.; Mihailov, Evelin; Porcu, Eleonora; Robino, Antonietta; Rose, Lynda M.; Schick, Ursula M.; Stolk, Lisette; Teumer, Alexander; Thompson, Deborah J.; Traglia, Michela; Wang, Carol A.; Yerges-Armstrong, Laura M.; Antoniou, Antonis C.; Barbieri, Caterina; Coviello, Andrea D.; Cucca, Francesco; Demerath, Ellen W.; Dunning, Alison M.; Gandin, Ilaria; Grove, Megan L.; Gudbjartsson, Daniel F.; Hocking, Lynne J.; Hofman, Albert; Huang, Jinyan; Jackson, Rebecca D.; Karasik, David; Kriebel, Jennifer; Lange, Ethan M.; Lange, Leslie A.; Langenberg, Claudia; Li, Xin; Luan, Jian'an; Mägi, Reedik; Morrison, Alanna C.; Padmanabhan, Sandosh; Pirie, Ailith; Polasek, Ozren; Porteous, David; Reiner, Alex P.; Rivadeneira, Fernando; Rudan, Igor; Sala, Cinzia F.; Schlessinger, David; Scott, Robert A.; Stöckl, Doris; Visser, Jenny A.; Völker, Uwe; Vozzi, Diego; Wilson, James G.; Zygmunt, Marek; Boerwinkle, Eric; Buring, Julie E.; Crisponi, Laura; Easton, Douglas F.; Hayward, Caroline; Hu, Frank B.; Liu, Simin; Metspalu, Andres; Pennell, Craig E.; Ridker, Paul M.; Strauch, Konstantin; Streeten, Elizabeth A.; Toniolo, Daniela; Uitterlinden, André G.; Ulivi, Sheila; Völzke, Henry; Wareham, Nicholas J.; Wellons, Melissa; Franceschini, Nora; Chasman, Daniel I.; Thorsteinsdottir, Unnur; Murray, Anna; Stefansson, Kari; Murabito, Joanne M.; Ong, Ken K.; Perry, John R. B.; Department of Epidemiology, Richard M. Fairbanks School of Public HealthMore than 100 loci have been identified for age at menarche by genome-wide association studies; however, collectively these explain only ~3% of the trait variance. Here we test two overlooked sources of variation in 192,974 European ancestry women: low-frequency protein-coding variants and X-chromosome variants. Five missense/nonsense variants (in ALMS1/LAMB2/TNRC6A/TACR3/PRKAG1) are associated with age at menarche (minor allele frequencies 0.08–4.6%; effect sizes 0.08–1.25 years per allele; P<5 × 10−8). In addition, we identify common X-chromosome loci at IGSF1 (rs762080, P=9.4 × 10−13) and FAAH2 (rs5914101, P=4.9 × 10−10). Highlighted genes implicate cellular energy homeostasis, post-transcriptional gene silencing and fatty-acid amide signalling. A frequently reported mutation in TACR3 for idiopathic hypogonatrophic hypogonadism (p.W275X) is associated with 1.25-year-later menarche (P=2.8 × 10−11), illustrating the utility of population studies to estimate the penetrance of reportedly pathogenic mutations. Collectively, these novel variants explain ~0.5% variance, indicating that these overlooked sources of variation do not substantially explain the ‘missing heritability’ of this complex trait.