Observing GLUT4 Translocation in Live L6 Cells Using Quantum Dots

The glucose transporter 4 (GLUT4) plays a key role in maintaining whole body glucose homeostasis. Tracking GLUT4 in space and time can provide new insights for understanding the mechanisms of insulin-regulated GLUT4 translocation. Organic dyes and fluorescent proteins were used in previous studies for investigating the traffic of GLUT4 in skeletal muscle cells and adipocytes. Because of their relative weak fluorescent signal against strong cellular autofluorescence background and their fast photobleaching rate, most studies only focused on particular segments of GLUT4 traffic. In this study, we have developed a new method for observing the translocation of GLUT4 targeted with photostable and bright quantum dots (QDs) in live L6 cells. QDs were targeted to GLUT4myc specifically and internalized with GLUT4myc through receptor-mediated endocytosis. Compared with traditional fluorescence dyes and fluorescent proteins, QDs with high brightness and extremely photostability are suitable for long-term single particle tracking, so individual GLUT4-QD complex can be easily detected and tracked for long periods of time. This newly described method will be a powerful tool for observing the translocation of GLUT4 in live L6 cells

Developing a quantum dot nanotracker for endophytic bacteria translocation in plants

Magister Scientiae - MSc (Biotechnology)Endophytes are bacteria that have been shown in previous studies to promote plant growth without eliciting any sign of infection or plant disease, as well as promote bio-fortification and has thus gained much attention in agriculture. However, due to the limited confirmatory visual evidence of the in planta presence of bacteria, the significance of this plant-endophyte interaction has not been comprehensively understood. The aim of this study was to synthesise quantum dots to track the translocation of endophytes in a plant. Conventional methods track endophytes by transformation of green fluorescent protein which has the problem of fluorophore bleaching and blinking reducing detectability. Quantum dots (QDs) offer distinct advantages over organic dyes and fluorescent proteins for biological imaging applications because of their brightness, photostability, tunability and their extremely small size ensures little to no interference with the normal functions of the bacterium. This project required the synthesis of cadmium telluride QDs to be capped with mercaptopropionic acid thus making them biocompatible and the subsequent characterization techniques were performed. CdTe and Carbon QDs were transformed into the endophytic bacteria and were thereafter used to treat Brassica napus L plants. Physiological and biochemical assays were done to detect the effect of QDs on the plant and bacteria, respectively

Program and Abstracts of the Annual Meeting of the Georgia Academy of Science, 2013

The annual meeting of the Georgia Academy of Science took place March 29-30, 2013, at Valdosta State University, Valdosta, Georgia. Presentations were provided by members of the Academy who represented the following sections: I. Biological Sciences II Chemistry III. Earth & Atmospheric Sciences IV. Physics, Mathematics, Computer Science, Engineering & Technology V. Biomedical Sciences VI. Philosophy & History of Science VII. Science Education VIII. Anthropology

Immune-Mediated Regulation of Glucose Uptake in Human Adipocytes

I have investigated the potential role of Toll-Like Receptors (TLRs) in mediating adipose inflammation in obesity. TLRs are a family of pattern recognition receptors that play a key role in host defence and are also implicated in chronic inflammatory disorders. The finding that TLR4-deficient mice are protected against obesity-induced diabetes led me to hypothesise that TLR expression on adipocytes could play a role in obesity-induced adipose inflammation and metabolic dysfunction. The first chapter investigates the expression and function of TLRs in in vitro differentiated human subcutaneous adipocytes. I found that stimulation with ligands for TLR2, TLR3 and TLR4 but not the other TLRs, induces the expression of pro-inflammatory cytokines. I also explored the use of the TLR adapter molecules MyD88, Mal and TRIF by different TLRs. The second chapter examines whether TLR activation in adipocytes has an effect on glucose uptake. I established a 3H-2-deoxy-D-glucose (2DOG) uptake assay which led to an interesting yet unexpected observation: Stimulation with TLR3 and TLR4 ligands led to a decrease in insulin-stimulated glucose uptake but at the same time, insulin-independent glucose uptake was increased. I showed that these observations are at least partly due to altered expression of different glucose transporter (GLUT) isoforms. As the effects were seen only after prolonged TLR stimulation, I speculated that this was mediated via a secondary secreted factor. The third chapter is based on a cytokine and adipokine array, which I performed to identify cytokines that may be responsible for the effects described in Chapter 2. The secretion of several cytokines/chemokines with diverse pro-inflammatory functions was observed following stimulation with TLR3 and TLR4 ligands. The contribution of some of these factors to altered glucose handling was investigated. Whilst a contribution for ENA-78 was ruled out, I present evidence that IL-1 can contribute to this

Hepatic Mitochondrial Renin-Angiotensin Systems

Introduction: The circulating renin-angiotensin system (RAS) was originally described as a key endocrine regulator of intravascular homeostasis; however, the existence of a local (tissue) RAS has become increasingly reported in a variety of tissues including liver. RAS components have now also been detected in rat heart, brain and smooth muscle cell mitochondria as well as within intramitochondrial dense bodies of rat adrenal tissue. Further, reduced RAS levels have been associated with improved endurance performance and fatigue resistance in human skeletal muscle, suggesting that low RAS activity is associated with metabolic efficiency, potentially via RAS action upon, or within, mitochondria. However, such investigation has often relied heavily upon qualitative techniques (e.g. Western blotting, immunofluorescence and electron microscopy), which contain inherent limitations in that they completely rely upon the limited specificity of antibodies to demonstrate the existence of intra-mitochondrial RAS components. Methods: The presence of RAS components within the mitochondria of rat hepatic tissue and liver cell-lines was investigated via sub-fractionation of rat liver tissue and cell-lines, followed by Western blotting, as well as via immunofluorescence and confocal microscopy, and electron microscopy. The mitochondrial effects of stimulating or antagonizing hepatic RAS were assessed via functional fluorescence microscopy (for assessment of NADH, calcium and mitochondrial membrane potential) and measurement of oxygen consumption within live cells of a liver cell-line. Results: Western blotting, immunofluorescence and electron microscopy suggested the presence of RAS components within mitochondria; however, there was a lack of results consistency between techniques and the staining patterns were largely non-specific. Western blotting further demonstrated the presence of a prominent 55 kDa band, when immunostaining a mitochondrial fraction with (angiotensin-converting enzyme) ACE Cterminal antibody (usual size 180 kDa). This was further explored via isolation of the 55 kDa molecule and mass spectrometry to yield results consistent with non-specific staining only. Addition of RAS agonists or antagonists to live liver cell-lines demonstrated no consistent results, except at supra-physiological levels, where RAS antagonists improved oxygen consumption. Conclusions: Such data suggest that the previous descriptions of RAS components within mitochondria are likely to be secondary to methodological flaws, particularly the reliance upon single antibodies, which have subsequently been shown to have poor specificity. Thus, the effect of ang II on liver mitochondria is unlikely to be direct and any such action is likely to occur via one of several intracellular pathways, regulation of gene expression or mitochondrial biogenesis

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Synthesis, Characterization and In-vivo Testing of Photoactivatable Insulin Depots for Continuously Variable and Minimally Invasive Insulin Delivery

Title from PDF of title page viewed May 15, 2020Dissertation advisor: Simon H. FriedmanVitaIncludes bibliographical references (pages 275-290)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2019Proteins are macromolecules involved in a diverse array of functions. Mutations or abnormal levels of proteins are indicated in several diseases. Despite showing early promise, the translation of protein therapeutics into the clinics has been challenging. The stability of these macromolecules, their delivery, and penetration inside the cells have been the main hurdles limiting their true potential. In the dissertation, various strategies to overcome such protein delivery challenges are discussed. Insulin is a lifesaving peptide for millions of diabetics around the world. Despite significant progress in insulin therapies, the quality of life in diabetics is constrained by the burden of multiple daily injections, invasive nature of therapy and inability to control the blood glucose tightly. To address these concerns, we constructed a photoactivatable insulin depot (PAD). In the approach, an insoluble depot of modified insulin was created by linking insulin covalently to photolabile caging moieties. Transcutaneous irradiation breaks the bond to release insulin from the depot into the systemic circulation. Chapter 3 describes the first successful testing on our PAD technology in diabetic animal models. In Chapters 2 and 4, I describe second-generation materials incorporating more efficient photolabile groups utilizing visible light wavelengths and PAD material with greater insulin loading. These changes improved the overall performance by several folds when tested in-vivo. Chapter 5 discusses the strategies addressed to deliver siRNA inside cells for effective light-activated RNA interference (LARI). LARI can be used for studying biology and cellular processes. Once administered, proteins are prone to degradation by ubiquitous proteases, limiting their circulation time and therapeutic effect significantly. Chapter 6 discusses prodrug strategies to temporarily modify proteins to shield them against proteases. We envisioned cross-linking amino acid residues on the surface via small crosslinkers. The tight bridges would hinder proteases from binding to proteins and unwinding the helices preventing their proteolysis. We also attempted integration of this approach to achieve intracellular protein delivery which is another obstacle in protein delivery. Here, the cross linking was performed via disulfide linkages. The disulfide groups would be reduced once inside the cells, yielding native proteins.Introduction: photoactivatable insulin depot -- Synthesis of insulin macro polymer -- In-vivo testing of first-generation pad material -- Synthesis and testing of advanced second-generation material -- Light activated SiRNA nanoparticles -- Intracellular protein delivery using protein prodrug

Glucocorticoids

As one class of the most important steroid hormones, glucocorticoids have long been recognised and their therapeutic benefits have been widely used in clinical treatment, especially in anti-inflammation cases. Glucocorticoids regulate various processes in the body including the mobilization of energy stores, immune functions, gene expression, and maintenance of the homeostasis as well as the stress response, this is not surprising that the concept of "glucocorticoids" is mentioned in almost all medical text books that focus on specific organs or systems such as the cardiovascular system, the immune system, and the neuroendocrine system. The book of Glucocorticoids - New Recognition of Our Familiar Friend aims to introduce the latest findings relating to glucocorticoids, either freshly from the laboratory or from clinical case studies, and to open up a new angle of looking at the issue of balancing the therapeutic benefits and side effects brought up by glucocorticoids