Thesis (Ph. D.)--University of Wisconsin--Madison, 1971. / Typescript. Vita. Description based on print version record. Includes bibliographical references (leaves 92-107).

The rising demand for arable land, to meet the competing needs of food and energy for a growing population, will soon become unmanageable. There is therefore a pressing need to increase the efficiency with which these demands are met, or find alternative ways of meeting them. In this work, E. coli transformed with a copy of its own ADP-glucose pyrophosphorylase gene (glgC), under a lac promoter, was found to synthesise large inclusion bodies when grown in media supplemented with lactose and IPTG. Analysis of these inclusion bodies suggests that they are formed of polysaccharide, giving the cell a significantly higher total sugar content than a control grown under the same conditions. The inclusion bodies were found to react strongly with iodine, turning a blackish brown colour normally associated with iodine-starch reactions. Results also indicate that the bacteria are unable to digest these inclusion bodies once they have been formed. These findings suggest the presence of long α-helices, which would both bind with iodine and prohibit enzymatic digestion. It was therefore hypothesised that the extra GlgC enzymes were allowing glucan chains to extend at a faster rate during glycogen synthesis, leading to unbranched regions that were long enough to wind themselves into helices. The subsequent introduction of a copy of the E. coli branching enzyme gene (glgB), under the same lac promoter, was therefore expected to abolish the inclusion body phenotype, by allowing the branching of the polysaccharide to keep speed with the synthesis of its linear chains. This was indeed found to be the case. However, cells transformed with both additional gene copies were also found to accumulate a significantly higher total sugar content again: more than twice that of cells transformed with glgC alone and more than seven times that of a control, when grown under conditions designed to optimise polysaccharide synthesis. These transformants were also observed to grow to higher cell densities that a control, in various growth media. The results of both these transformations could be significant in meeting the demands of our growing society. In particular, the use of cyanobacterial glycogen as a carbon source for biofuels has recently been gaining interest, and the work presented here may well be applicable in this field, providing the possibility to significantly increase yields. Lastly, the effects of Isoamylases and Granule Bound Starch Synthase, taken from two starch producing organisms – Zea mays and Ostreococcus tauri – were investigated in an E. coli host. Results were inconclusive, but suggest many avenues for continuing the work.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-08Bitstream added on 2014-06-13T20:29:46Z : No. of bitstreams: 1 savassa_sm_me_araiq_parcial.pdf: 560908 bytes, checksum: 7322d6c1907899887286e7c9514807e4 (MD5) Bitstreams deleted on 2015-07-02T12:36:14Z: savassa_sm_me_araiq_parcial.pdf,. Added 1 bitstream(s) on 2015-07-02T12:37:34Z : No. of bitstreams: 1 000719185_20180101.pdf: 541449 bytes, checksum: 71b832c9a74978e0b6356b081a51d520 (MD5) Bitstreams deleted on 2018-01-02T17:04:40Z: 000719185_20180101.pdf,. Added 1 bitstream(s) on 2018-01-02T17:05:45Z : No. of bitstreams: 1 000719185.pdf: 3296921 bytes, checksum: 3209de8217c724ff260135d1572d7752 (MD5) / O fungo filamentoso Neurospora crassa é um organismo modelo muito utilizado para estudos de diversos aspectos da biologia dos eucariotos. Nosso laboratório tem utilizado este fungo para o estudo dos mecanismos moleculares e bioquímicos da regulação do metabolismo de glicogênio. A presente proposta de trabalho é uma consequência de experimentos anteriores realizados com o objetivo de identificar proteínas (fatores de transcrição ou não) que se ligam à região promotora do gene gsn, o qual codifica a enzima glicogênio sintase, regulatória do processo de síntese do carboidrato. Esses estudos combinaram experimentos de ensaios de retardamento em gel utilizando fragmentos do promotor gsn e proteínas do extrato total do fungo, acoplados à análise proteômica e identificação das proteínas por espectrometria de massas. Os experimentos resultaram na identificação de algumas proteínas do fungo, as quais podem ou não estar envolvidas na regulação da expressão do gene. Alguns estudos preliminares com estas proteínas foram anteriormente realizados no laboratório e apontaram um provável papel das mesmas na regulação do metabolismo do carboidrato em N. crassa. Duas dessas proteínas, as codificadas pelas ORFs NCU3482 e NCU06679 foram objeto de estudo neste trabalho. Portanto, o objetivo deste trabalho foi realizar a caracterização das linhagens mutantes nestas ORFs, além da produção e purificação das proteínas na forma recombinante. Foram realizadas análises morfológicas da linhagem mutante na ORF NCU06679, tais como: crescimento colonial e radial, crescimento linear e análise microscópica das extremidades das hifas. Esses experimentos foram realizados em comparação com a linhagem selvagem do fungo e, mostraram esta proteína está envolvida no processo de desenvolvimento do... / The fungus Neurospora crassa has been widely used as a model organism for fundamental aspects of eukaryotic biology. We have been studying the biochemical and molecular mechanisms involved in glycogen metabolism regulation in this fungus. The present work is a consequence of previous experiments performed in the laboratory to identify proteins that bind to the promoter region of the gsn gene which encodes glycogen synthase, the regulatory enzyme in glycogen synthesis. Previous studies were performed by using a combination of DNA gel shift assay coupled to proteomic analysis, followed by identification of proteins by mass spectrometry. The assays resulted in the identification of proteins likely involved in the regulation of gene expression. Preliminary studies with these proteins have previously been carried out and suggested that they might have a role in the regulation of glycogen metabolism in N. crassa. Two of them, the ORFs NCU3482 and NCU06679 gene products were object of study in this work. The main objective was to characterize the mutant strains in both proteins and to produce and purify the recombinant proteins. Morphological analyzes were performed in the ORF NCU06679 mutant strain such as colony and radial linear growth and microscopic examination of the ends of the hyphae. These experiments showed that this protein is involved in the fungus development since growth and ability to conidiate were deficient when compared to the wild-type strain. The expression of gsn and gpn (encodes glycogen phosphorylase, the regulatory enzyme in glycogen degradation) genes were analyzed by qPCR and the results showed differences in gene expression of both genes during vegetative growth of the NCU06679 mutant strain when compared to the wild-type strain. The protein encoded by ORF NCU06679 was produced as a recombinant protein and the purification... (Complete abstract click electronic access below)

The aim of this research was to investigate the influence of rehydration with carbohydrate-electrolyte solutions, during a short-tern recovery period, on hydration status, physiological responses, and subsequent endurance capacity. The first study (Chapter 4) examined whether prescribed or ad libitum rehydration with a carbohydrate-electrolyte solution (CHO-E), during 4 h recovery from prolonged, submaximal running would influence the subsequent endurance capacity. Five women and two men performed the "recovery" protocol consisting of a 90 min run at 70%VO2 max on a level treadmill (TI) followed by 4 h rehydration-recovery (REC), and then an open-ended run to exhaustion at 70%VD2 max (1'2) as a measure of their endurance capacity, on two occasions, at least 7 days apart. During the REC, subjects were allowed to drink a 6.9% CHO-E ad libitum (AL) on one occasion. On the other occasion, the volume of the same fluid was prescribed (PI) from calculations of the body mass lost during TI. During T2, in the PI trial, the run time to exhaustion was 16% longer (P < 0.05) than during T2 in the AL nial (69.9 ± 9.1 vs. 60.2 ± 10.2 min). Thus, ingestion of a prescribed volume of CHO-E after prolonged exercise, calculated to replace the body fluid losses, restored endurance capacity to a greater extent than ad libitum rehydration during the REC. The second study (Chapter 5) investigated the influence of ingesting 50 g of carbohydrate (CHO) immediately after exercise, either with subsequent serial CHO feeding or water ingestion during the REC from prolonged, submaximal running on rehydration and subsequent endurance capacity. Eight male subjects performed the "recovery" protocol [i.e. 90 min run at 70% V02 max (TI), 4 h rehydration-recovery (REC), and open-ended run at 70% V02 max (T2)] on two occasions. During the REC, subjects ingested a prescribed volume of fluid equal to the body mass lost during TI in both conditions. Subjects ingested 50 g of CHO from a 6.9% CHO-E 15 min after TI on both occasions as their first prescribed fluid intake. Thereafter, subjects drank either the same solution (CE) or water CW) at each hour after TI during the REC. During T2, the run time to exhaustion was 54.2 ± 9.2 min in the CE trial and 52.2 ± 6.2 min in the W trial, respectively (NS). The volume of fluid retained expressed as a percentage of the volume ingested (% rehydration) during the CE trial was greater than that of the W trial (CE: 73.5 ± 4.2% vs. W: 63.0 ± 5.7%; P < 0.05). Serial CHO feeding during the REC was associated with increased CHO oxidation and suppressed fat oxidation during subsequent exercise. Thus, ingesting -150 g of CHO in a 6.9% CHO-E over a 4 h period following prolonged running is more effective in terms of rehydration compared to the same volume of fluid containing only 50 g of CHO and water, but does not have a greater effect on subsequent endurance capacity. The third study (Chapter 6) investigated the effects of rehydration per se and CHO ingestion, during the REC, on subsequent endurance capacity. Nine male subjects performed the "recovery" protocol on two occasions. During the REC, subjects drank either a 6.9% CHO-E (CE) or a CHO-free sweetened placebo (PL) every 30 min after Tl up to the beginning of the 4 h of the REC. Volumes prescribed (ml) were equal to 200% of the body mass lost during Tl. However, the total volume of fluid ingested during the REC was only 170.8 ± 12.6% and 172.6 ± 13.8% of the body mass lost after Tl (NS). During T2, in the CE trial, the run time to exhaustion was 54% longer (P < 0.01) than during T2 in the PL trial (69.3 ± 5.5 vs. 45.0 ± 4.2 min). After the REC, subjects were in positive fluid balance by 423 ± 215 ml in the CE trial and 446 ± 239 ml in the PL trial (NS). Thus, positive fluid balance can be achieved by ingesting a prescribed volume of either a 6.9% CHO-E or a placebo solution over the REC, calculated to replace approximately 170% of the body fluid loss. Despite this similar hydration status after the recovery in both conditions, ingesting a CHO-E is more effective in restoring endurance capacity compared to the same volume of placebo solution. The fourth study (Chapter 7) was intended to examine, and verify, the effects of ingesting different amounts of CHO in the form of a CHO-E during the REC on rehydration and subsequent endurance capacity. Nine male subjects performed the "recovery" protocol on two occasions. During the REC, a fixed volume of fluid equivalent to 150% of the body mass lost during Tl was consumed. Subjects ingested 50 g of CHO from a 6.5% CHO-E 30 min after Tl on both occasions as their first prescribed fluid intake. Thereafter, subjects ingested either the same solution (CE) or a CHO-free sweetened placebo (PL) every 30 min up to the beginning of the 4 h of the REC. During T2, the run times were 56.9 ± 8.1 min in the CE trial and 65.4 ± 7.8 min in the PL trial (NS). After the REC, subjects were almost equally euhydrated (CE: 0 ± 184 ml; PL: -27 ± 120 ml) in both conditions (NS). Serial CHO feeding over the REC was accompanied by enhanced CHO oxidation and suppressed fat oxidation. In conclusion, ingesting a placebo solution containing 50 g of CHO and placebo over a 4 h period following prolonged running, calculated to replace 150% of the body fluid loss, is equally effective in achieving approximate euhydration and restoring endurance capacity compared to the same volume of CHO-E containing -167 g of CHO. The studies reported in this thesis suggest that in order to achieve euhydration during recovery, a volume of fluid substantially larger (~ 150%) than that lost must be ingested. The provision of additional CHO (-150 to 170 g) would be expected to restore the body's CHO stores to a greater extent than a smaller amount of CHO (50 g) during the REC and, thereby, improve the subsequent endurance capacity. However, this was not the case. It appears that the ingestion of large amounts of CHO, during the REC, resulted in disturbances in fat and CHO metabolism which prevented an improvement in endurance capacity during T2, after consumption of the additional CHO.

Six mares and six geldings were used to determine the effect of forage quality on muscle glycogen utilization by exercised horses. Horses were fed 1.5% BW/d of high quality (65.6% NDF; 41.1% ADF) or low quality (74.6% NDF; 51.2% ADF) hay and a concentrate resulting in three diets: high quality hay and concentrate balanced to meet energy requirements (HQ); low quality hay and concentrate balanced to meet energy requirements; and low quality hay with the same amount of concentrate as the HQ diet. The effect of hay quality on serum insulin, serum glucose, whole blood lactate, and respiratory parameters were also determined. Results suggested varied metabolic responses to exercise between sexes and a possible glycogen sparing effect when low quality forage was consumed.

During the first few hours of recovery from prolonged exercise, co-ingestion of protein (PRO) with carbohydrate (CHO) will increase the rate of muscle glycogen resynthesis if the rate of CHO intake is sub-optimal and/or when the feeding intervals are > 1 h apart (Burke et al.. 2004). It remains controversial whether the higher rate of glycogen resynthesis is attributable to a PRO-mediated increase in insulin release or simply the result of higher energy intake (Jentjens et al., 2001). The optimal rate of CHO ingestion necessary to maximize glycogen resynthesis remains unknown, although some studies have recommended a peak CHO intake of >1.2 g/kg/h. PURPOSE: We examined whether the addition of PRO or “extra CHO” to a drink that provided 1.2 g CHO/kg/h would increase muscle glycogen resynthesis during recovery from prolonged exercise. METHODS: Six men (22± 1 yr; V02|,eilk= 48±8 ml/kg/min) were studied during a 4-h recovery period on 3 separate occasions after a standardized 2-h exercise protocol designed to substantially lower muscle glycogen. Subjects randomly consumed 1 of 3 drinks during recovery from each trial: 1.2 gCHO/kg/h (CHO), 1.6 gCHO/kg/h (CHO/CHO) or 1.2 gCHO + 0.4 gPRO/kg/h (CHO/PRO). Drinks were consumed immediately post-exercise and at 15 min intervals for 3 hrs. RESULTS: Biopsies obtained at 0 and 4 hrs post-exercise revealed no difference in muscle glycogen resynthesis rates between trials (CHO: 22.7±6.6; CHO/CHO: 25.0±3.0; CHO/PRO: 24.6±4.2 mmol/kg dry wt/h) despite differences in energy intake between trials. The ingestion of additional CHO or PRO did not induce changes in blood [insulin ] and blood [glucose] compared to a 1.2 gCHO/kg/h beverage. Muscle [lactate] increased from immediately post-exercise to 4 hrs into recovery in all 3 trials (main effect for time. P<0.05). All subjects reported some degree of gastro-intestinal(Gl) distress after 3 and 4 hrs of recovery but there were no differences between treatments (main effect for time, P<0.05 vs. 0 hrs). CONCLUSION: Ingesting 1.2 gCHO/kg/h met or exceeded the threshold necessary to optimize muscle glycogen synthesis during the first 4 hrs of recovery from prolonged, strenuous exercise in recreationally active men. The ingestion of >1.2 gCHO/kg/h at a concentration of -20% solution induced GI distress in some individuals and may hamper subsequent same-day performance. / Thesis / Master of Science (MS)

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen, a branched polymer of glucose, acts as an intracellular carbon and energy reserve in many tissues and cell types. The breakdown of glycogen by hormonally regulated degradation involving the coordinated action of glycogen phosphorylase and debranching enzyme has been well studied. However, the importance of lysosomal disposal of glycogen has been underscored by a glycogen storage disorder, Pompe disease. This disease destroys tissues by over-accumulating glycogen in lysosomes due to a genetic defect in the lysosomal acid α-glucosidase. Details of the intracellular trafficking of glycogen are not well understood. Starch-binding domain-containing protein 1 (Stbd1) is a protein of previously unknown function with predicted hydrophobic N-terminus and C-terminal CBM20 carbohydrate binding domain. The protein is highly expressed in the liver and muscle, the major repositories of glycogen. Stbd1 binds to glycogen in vitro and in vivo with a preference for less branched and more phosphorylated polysaccharides. In animal models, the protein level of Stbd1 correlates with the genetic depletion of glycogen. Endogenous Stbd1 is found in perinuclear compartments in cultured mouse and rat cells. When over-expressed in cells, Stbd1 accumulates and coincides with glycogen and GABARAPL1, the autophagy protein. They form enlarged perinuclear structures which are abolished by removing the hydrophobic N-terminus of Stbd1. Stbd1, with point mutations in the CBM20 domain, retains the perinuclear localization but without concentration of glycogen in this compartment. In cells that are stably over-expressing glycogen synthase, glycogen exists as large perinuclear deposits, where Stbd1 can also be present. Removing glucose from the culture leads to a breakdown of the massive glycogen accumulation into numerous smaller and scattered deposits which are still positive for Stbd1. Furthermore, the autophagy protein GABARAPL1 co-immunoprecipates and co-localizes with Stbd1 when co-expressed in cells. Point mutation or deletion of the autophagy protein interacting region on Stbd1 eliminates the interaction and co-localization with GABARAPL1 but not the characteristic perinuclear distribution of Stbd1. We propose that Stbd1 is involved in glycogen metabolism. In particular, it participates in the vesicular transfer of glycogen to the lysosome with the recruitment of autophagy related proteins GABARAPL1 and/or GABARAP, as these vesicles mature prior to lysosomal fusion.

No description available.

<p>Chlamydia trachomatis, a pathogen responsible for major diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to molecular genetic manipulation. The development of a system(s) for genetic analysis would significantly accelerate our ability to identify genes that enable Chlamydia to establish infection, survive within its host, and cause disease. This thesis describes two methods used to assess gene function in Chlamydia and to provide insights into its biology and pathogenesis. The first method described is based on specific inhibitors and is used to probe the role of lipooligosaccharide (LOS), a main lipid components of bacterial outer membranes. Using this approach, we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, blocks the synthesis of LOS in C. trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of anti-chlamydial agents.</p><p> The second part of this thesis describes the development of a system with which to perform forward genetics in C. trachomatis. Forward genetics approaches set out to identify the gene or set of genes that contributes to a specific biological process and usually entails generating random mutations in a large number of organisms, isolating mutants with an aberrant phenotype, and identifying the alleles associated with the mutant phenotype. In this approach, chemical mutagenesis is coupled with whole genome sequencing (WGS) and a system for DNA exchange within infected cells to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. We identified mutants with altered glycogen metabolism, including an attenuated strain defective for Type II secretion. The coupling of chemically induced gene variations and WGS to establish genotype-phenotype associations should be broadly applicable to the growing list of microorganisms intractable to traditional genetic mutational analysis.</p> / Dissertation

Exercise performance was examined after ingestion of 45 g of liquid carbohydrate (LCHO), solid carbohydrate (SCHO), or water (W) 5 min before 1 h of intense exercise.An (CHO) meal (200 g) taken 4 h prior to exercise was combined with a solid CHO feeding (M+SCHO). Ten well-trained male cyclists (4.35 + 0.11 1/min V02 max) performed 45 min of cycling at a work load requiring an average oxygen uptake of 77.1% V02 max followed by a 15 min performance ride. Serum glucose values were elevated by 18% and 24% during SCHO and LCHO, respectively, as compared to W. Blood glycerol concentrations were significantly (p<0.05) lower during M+SCHO and significantly higher during W at 40, 50 and 60 min of exercise as compared to all other trials. Muscle biopsies at 0 and 45 min revealed no significant differences in muscle glycogen concentration or total use between the 4 trials. Total work produced during the final 15 min of exercise was significantly greater during M+SCHO (194735 + 9448 NM) as compared to all other trials and significantly greater during LCHO and SCHO (175204 + 11780 and 176013 + 10465 NM, respectively) as compared to W (159143 + 11407 NM). Carbohydrate oxidation was highly correlated (r=0.94) with work production. These results suggest that exercise performance is enhanced with the ingestion of 45 g of CHO 5 min prior to one hour of intense cycling. Further improvements in performance were found when exercise was preceded by a CHO meal 4 h prior to exercise in combination with a solid CHO feeding given immediately before exercise.