College level science is frequently taught as a recitation of facts in a lecture hall, and the students are expected to gain understanding and insight with their own study. Interactive learning is more effective than lecture based learning and more memorable for the students. Teaching with hands on models has been shown to specifically be beneficial in a college level molecular biology context. Included here is a guide for the instructor leading her through topic selection, activity development, and presentation to the class, as well as five complete and tested lesson plans with notes on alteration made and the reasons for them. / text

Cells utilize various signal transduction networks to regulate metabolism. Nevertheless, a quantitative understanding of the relationship between growth factor signaling and metabolic state at the single cell level has been lacking. The signal transduction and metabolic states could vary widely among individual cells. However, such cell-to-cell variation might be masked by the bulk measurements obtained from conventional biochemical methods. To assess the spatiotemporal dynamics of metabolism in individual intact cells, we developed genetically encoded biosensors based on fluorescent proteins. As a key redox cofactor in metabolism, NADH has been implicated in the Warburg effect, the abnormal metabolism of glucose that is a hallmark of cancer cells. To date, however, sensitive and specific detection of NADH in the cytosol of individual live cells has been difficult. We engineered a fluorescent biosensor of NADH by combining a circularly permuted green fluorescent protein variant with a bacterial NADH-binding protein Rex. The optimized biosensor Peredox reports cytosolic \(NADH:NAD^+\) ratios in individual live cells and can be calibrated with exogenous lactate and pyruvate. Notably pH resistant, this biosensor can be used in several cultured and primary cell types and in a high-content imaging format. We then examined the single cell dynamics of glycolysis and energy-sensing signaling pathways using Peredox and other fluorescent biosensors: AMPKAR, a sensor of the AMPK activity; and FOXO3-FP, a fluorescently-tagged protein domain from Forkhead transcription factor FOXO3 to report on the PI3K/Akt pathway activity. With perturbation to growth factor signaling, we observed a transient response in the cytosolic \(NADH:NAD^+\) redox state. In contrast, with partial inhibition of glycolysis by iodoacetate, individual cells varied substantially in their responses, and cytosolic \(NADH:NAD^+\) ratios oscillated between high and low states with a regular, approximately half-hour period, persisting for hours. These glycolytic NADH oscillations appeared to be cell-autonomous and coincided with the activation of the PI3K/Akt pathway but not the AMPK pathway. These results suggest a dynamic coupling between growth factor signaling and metabolic parameters. Overall, this thesis presents novel optical tools to assess metabolic dynamics – and to unravel the elaborate and complex integration of glucose metabolism and signaling pathways at the single cell level.

Epidemiology studies have established a strong link between chronic arsenic exposure and lung cancer. Currently, contribution of perturbed energy metabolism to carcinogenesis is an intensive area of research. In several human cell culture models (primary, immortal, malignant), we observed that non-cytotoxic exposure to arsenite increased extracellular acidification rate. Lactate accumulation caused by extracellular acidification, could be inhibited by 2-deoxy-D-glucose, a non-metabolized glucose analog. This established that arsenite induces aerobic glycolysis (the Warburg effect), a metabolic shift frequently observed in the acquisition of malignancy. Our studies in BEAS-2B, a non-malignant pulmonary epithelial cell line, found that the metabolic perturbation began early in the course of malignant transformation by arsenite (6 weeks). Correlated with the surge of glycolysis, we found elevated levels of HIF-1A and loss of E-Cadherin during chronic arsenite exposure. Our evidence suggests that this metabolic shift is sustained by HIF-1A (hypoxia-inducible factor 1A). We found that arsenite-exposed BEAS-2B accumulated HIF-1A protein, and underwent transcriptional up-regulation of HIF-1A-target genes. Overexpression of HIF-1A increases glycolysis 15% (vs. control), confirming that HIF-1A can modulate glycolysis in BEAS-2B. Coincident with induction of glycolysis, we observed a decrease in E-cadherin expression, indicating loss of epithelial identity. HIF-1A stable knockdown in BEAS-2B abrogated the arsenite induction of glycolysis, and indicated suppression in colony formation. These findings suggest that the hypoxia-mimetic effect of arsenite plays an important role in arsenite-induced malignant transformation. The significance of this study is that arsenite-induced alteration of energy metabolism represents the type of fundamental perturbation that could extend to many diverse effects caused by arsenic.

Fatty acid synthase (FAS) is over-expressed in many human cancers and its activity is required for cancer cell survival. To understand why FAS is over-expressed, we compared in breast cancer cells the utilization of fatty acids synthesized endogenously by FAS to those supplied exogenously in the culture medium. We found that endogenously synthesized fatty acids are esterified to the same lipid and phospholipid classes in the same proportions as those derived exogenously and that some endogenous fatty acids are excreted. Thus, FAS over-expression in cancer does not fulfill a specific requirement for endogenously synthesized fatty acids. We next investigated whether lipogenic activity mediated by FAS was, instead, involved in the maintenance of high glycolytic activity in cancer cells. By culturing breast cancer and non-cancer cells in anoxic conditions, we increased glycolysis 2-3 fold but observed no concomitant increase in lipogenesis. More research is needed to understand why FAS is over-expressed in cancer.

Šiame darbe nuodugniai ištirtos gamybinių polietilentereftalato (PET) atliekų susidarymo vietos, priežastys bei jų savybės. Pasiūlyti skirtingi cheminio perdirbimo būdai ir sąlygos kiekvienai gamybinių PET atliekų rūšiai. Vykdant gamybinių PET atliekų glikolizę etilenglikoliu, pasiekta didesnė negu 85 % bis(2-hidroksietilen)tereftalatо išeiga. Peresterinant gamybines PET atliekas dietilenglikoliu (DEG) ir naudojant funkcinius priedus glicerolį (GL) ir/arba adipo rūgštį (ADR), susintetinta serija aromatinių poliesterpoliolių (APP), besiskiriančių savo klampa ir kitomis savybėmis. Pirmą kartą nuodugniai ištirta ir matematiškai aprašyta peresterinimo reakcijos mišinyje esančių funkcinių priedų įtaka APP klampai. APP, susintetinti peresterinant gamybines PET atliekas DEG ir turintys ADR ir/arba GL fragmentų, yra mažai linkę kristalintis ir stabilūs saugant juos kambario temperatūroje. APP klampa mažai priklauso nuo metaloorganinio katalizatoriaus cheminės sudėties ir jo koncentracijos. Naudojant PET peresterinimo metu gautus APP ir diizocianato perteklių, susintetintos poliuretano-poliizocianurato (PU-PIR) putos. Putos, gautos iš APP, kuriuose yra GL ir/arba ADR fragmentų, pasižymi geromis fizikomechaninėmis savybėmis ir dideliu terminiu stabilumu, joms degant išsiskiria mažesnis šilumos ir dūmų kiekis. Atliekant degumo bandymus nustatyta, kad PU-PIR putos atitinka reikalavimus, taikomus Е klasės statybinėms konstrukcijoms ir elementams. / In this study, the generation points, reasons and properties of industrial PET waste were examined in detail. Different chemical recycling ways were suggested for each kind of industrial PET waste. Under glycolysis of industrial PET waste by ethylene glycol, the yield of the main product bis(2-hidroxyethylene) terephthalate was higher than 85 %. Several series of aromatic polyester polyols (APP) were synthesized by transesterification of industrial PET waste using diethyleneglycol (DEG) in the presence of functional additives glycerol (GL) or/and adipic acid (ADA). The effect of functional additives on transesterification process and viscosity of APP was thoroughly studied and mathematically described for the first time. APP synthesized by transesterification of industrial PET waste using DEG in the presence of ADA and/or GL fragments, had lower crystallinity and were much more stable during storage at room temperature. Viscosity of APP slightly depended on the catalyst type and its concentration. Polyurethane-polyisocyanurate (PU-PIR) foams were produced under the reaction of APP and an excess of diisocyanate. PU-PIR foams based on PET-waste-derived APP containing fragments of GL or/and ADA were characterized by excellent physical-mechanical properties, high thermal stability, low heat release and smoke production. The burning test confirmed that PU-PIR foams satisfied the requirements for class E of construction products and building elements.

Bone is refractory to most conventional biochemical Procedures. However because it is now possible to cut sections (e. g. lopm) of fresh, undemineralized adult bone, this tissue can be analyzed by suitably modified methods of quantitative cytochemistry. A new substrate for assaying hydroxyacyl dehydrogenase activity demonstrated that bone cells may use fatty acids as a major source of energy: detailed analysis of the activities of key enzymes indicated that the paradox of ‘aerobic glycolysis’ of bone could be explained by fatty acid oxidation satisfying the requirements of the Krebs' cycle and directing the conversion of pyruvate to lactate The influence of glucose 6-phosphate dehydrogenase (G6PD) activity in aerobic glycolysis has been considered. The inverse relationships between this activity and that of Na-K-ATPase led to the development of a new method for the latter, based on a new concept in cytochemistry ('hidden-capture' procedure). A major feature of fracture-healing is increased periosteal G6PD activity. The association with the vitamin K cycle has been investigated by feeding rats with dicoumarol which not only inhibited bone-formation but also G6PD activity. The stimulation of this activity in fracture-healing has been linked with ornithine decarboxylase (ODC) activity, for which a new method has been developed. Rats deficient in pyridoxal phosphate (cofactor for ODC) had decreased G6PD responses and also appeared to become osteoporotic. Studies on osteoporotic fractures in the human showed the presence of relatively large apatite crystals close to the fracture-site, and disorganized glycosaminoglycans (demonstrated by the new method of ‘induced birefringence’).

The impact of fatigue on the intermittent high intensity exercise undertaken during participation in team sports has not been extensively studied. Team sports are characterised not only by intennittent exercise, but also by the contribution of a wide range of skills. This thesis describes a series of studies conducted in a controlled environment to assess the influence of fluid ingestion and fatigue on selected soccer skills. The aim of the first study was to examine the effect of 90-min of high intensity shuttle running with and without water ingestion on a socc er-dribb ling test. The subjects were allocated to two randomly assigned trials either ingesting or abstaining from fluid intake during a 90 min intennittent exercise protocol (Loughborough Intermittent Shuttle Test: LIST). In the absence of water ingestion soccer skill deteriorated (p < 0.05) by 5% but was maintained when fluid was ingested. The principal aim of the second study was to understand further the mechanisms contributing to the deterioration observed during the LIST. Subjects completed the LIST ingesting a 6.4% carbohydrate electrolyte solution (CHO), placebo (CON) or no fluid (NON). Free fatty acids, cortisol and aldosterone responses were lower (P < 0.01) at the end of exercise during both CHO and CON in comparison to NON. There was no difference in respiratory exchange ratio between trials. Fluid ingestion did not appear to cause a shift in substrate metabolism even though there were differences in plasma FFA concentrations. The consumption of carbohydrate during exercise has been shown to increase physical performance, capacity and cognitive function. The aim of the third study was to assess the influence of a 6.4 % carbohydrate-electrolyte (CHO) placebo (CON) or no fluid (NON) on passing and dribbling soccer skills following the LIST. During the NON trial performance of the dribbling test followed a similar pattern to that in the first study and performance of the passing test decreased (p < 0.05). This reduction in performance was prevented during the CHO and CON trials. The purpose of the final study was to identify whether a rehydration strategy following the LIST would result in a recovery of skill performance. Subjects were allocated to two randomly assigned trials either ingesting a volume of fluid equivalent to 150% (L) or 9% (S) of body mass loss during the LIST, over a2h recovery period. During the recovery period serum sodium and osmolality returned to resting concentrations in the L trial but remained elevated in the S trial (P < 0.05). Despite body mass returning to resting values following the rehydration period, performance of the skills tests remained impaired. Deterioration in skill test performance may have been related to a reduction in neuromuscular control either by a reduction in muscle glycogen or by an increase in muscle damage during the no fluid trials. The mechanism responsible for the deterioration in skill performance remains to be elucidated.

Fatty acid synthase (FAS) is over-expressed in many human cancers and its activity is required for cancer cell survival. To understand why FAS is over-expressed, we compared in breast cancer cells the utilization of fatty acids synthesized endogenously by FAS to those supplied exogenously in the culture medium. We found that endogenously synthesized fatty acids are esterified to the same lipid and phospholipid classes in the same proportions as those derived exogenously and that some endogenous fatty acids are excreted. Thus, FAS over-expression in cancer does not fulfill a specific requirement for endogenously synthesized fatty acids. We next investigated whether lipogenic activity mediated by FAS was, instead, involved in the maintenance of high glycolytic activity in cancer cells. By culturing breast cancer and non-cancer cells in anoxic conditions, we increased glycolysis 2-3 fold but observed no concomitant increase in lipogenesis. More research is needed to understand why FAS is over-expressed in cancer.

In this study, the effects of glycol type, organoclay type and concentration on the final properties of nanocomposites based on recycled poly(ethylene terephthalate) was investigated. For this purpose, first recycled PET was glycolysed and after that unsaturated polyester-montmorillonite nanocomposites were synthesized by using three different types of glycols (i.e. ethylene glycol (EG), propylene glycol (PG) and diethylene glycol (DEG)). As the first step, all the compositions were prepared by Cloisite 30B type of clay, and then for comparison of clay type, nanocomposites containing 1 wt. % of Cloisite 15A and Cloisite 25A type of clay were also synthesized. Morphological and mechanical analyses were performed for the characterization of the nanocomposites. According to the results of XRD analysis, for all glycol types maximum intercalation was observed in Cloisite 30B containing samples. Exfoliated structures were obtained in the samples containing EG at 1 wt. % Cloisite 30B content and DEG at 3 wt. % Cloisite 30B content. Mechanical tests showed that, for all properties, glycol type is the most effective experimental parameter. DEG based samples are the most flexible whereas PG based samples are the least flexible. EG and DEG based samples give maximum tensile strength and tensile modulus values at 1 wt. % clay loading. Samples prepared by DEG exhibited maxima in both flexural strength and modulus at 1 wt. % clay content. With respect to the organoclay type, Cloisite 30B containing samples gave the highest compatibility with the unsaturated polyester matrix as indicated by the tensile test results. Organoclay type and content had no positive effect on the impact strength. Clay particles acted as stress concentrators and lowered the impact strength.

The mammalian ADP-dependent glucokinase is the most recent mammalian glucokinase to have been discovered, and is unique in its ability to catalyse the phosphorylation of glucose to glucose-6-phosphate using ADP as the phosphoryl donor. Up until the discovery of this enzyme, the traditional biochemical view was that the first step of glycolysis was solely catalysed by ATP-dependent hexokinases, types I-IV. The particular role played by ADP-GK in the mammalian cell and the significance of this role has not yet been determined, although it is hypothesised that the ADP-dependent glucokinase could be potentially significant in contributing to the survival of cells under low energy and hypoxic or ischemic conditions. By using ADP as the energy investment in phase one of the glycolytic cycle instead of ATP, it is predicted that glycolysis could be sustained for longer during lower energy conditions (conditions of high ADP:ATP ratios). Since the phosphorylation of glucose by ADP-GK results in the production of AMP, it may also be possible that this has a direct effect on the energy charge of the cell. The AMP produced could lead to the regulation of cellular metabolism during hypoxia and/or ischemia via the activation of the cell-energy regulator AMPK. The study of mammalian ADP-dependent glucokinase is a very new area, and prior to this no investigation of the human ADP-GK enzyme had been undertaken. The main objective of this project was to clone, express and purify the recombinant ADP-GK so it could be kinetically characterised and directly compared with the recombinant mouse kinetic characteristics, the only other mammalian ADP-GK to have been studied. Unfortunately, due to complications in the expression and purification of soluble recombinant human ADP-GK, the project did not incorporate the kinetic characterisation of the enzyme. Acquiring data on the kinetic characteristics of the human ADP-GK will, in the long term, assist in the elucidation of the metabolic role of this enzyme, so the continuation of this project would be worthwhile.