Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a highly penetrant autosomal dominant genetic disorder where mutations in the tumor suppressor gene NF1 leads to decreased neurofibromin. The most debilitating manifestation is the presence of complex multilineage Schwann cell-derived plexiform neurofibromas (PN). Historically, little clinical success has been achieved targeting PN through surgery or chemotherapies. I performed an shRNA library screen of patient-derived Schwann cell lines to identify novel therapeutic targets to disrupt PN formation and progression. An shRNA library screen of human kinases and Rho-GTPases was performed in NF1-/- and paired NF1 competent immortalized Schwann cell lines. Following sequencing, candidates were identified. We previously developed a novel mouse model of NF1 wherein a neural crest specific Postncre targeted loxp-flanked Nf1 that replicated the PN found in patients. Additional cohorts of mice were generated with biallelic deletion of Rac1 (Nf1f/fRac1f/f Postn-Cre+; DKO ). Mice were aged for 9 months and peripheral nerves were harvested and fixed in formalin. Peripheral nerve size was measured and tumors were identified through blinded analysis of hematoxylin and eosin and Masson’s Trichrome (collagen) stained slides. Rho family members, including RAC1, were identified as candidates through an shRNA library screen. Genetic disruption of Rac1 in the Schwann cell lineage resulted in the prevention of tumor formation in DKO mice, as observed by peripheral nerve size and histological analysis. I observed an average of 14.8 +/- 2.65 tumors per mouse in the Nf1f/f Postnviii Cre+ cohort compared to 0 tumors in the DKO (p<0.0001). Following an shRNA library screen, RAC1 was identified as a candidate to modulate PN formation. Biallelic deletion of Rac1 in vivo prevented PN formation. I demonstrate that a candidate identified in an shRNA library screen can translate to an biological effect in a mouse model of PN.

Genetically modified (GM) food could be a solution to secure the world’s food supply, whichis in jeopardy due to the ongoing climate crisis. Thus, it is of great importance that consumerswould be willing to consume this type of food. To examine which factors influence consumerbehavior toward GM food, an extension of the theory of planned behavior was applied anddecisive factors were evaluated. The derived hypotheses and the respective influencing factorsare based on the research of Prati et al. (2012) as well as the questions that were used in theonline questionnaire. Through this online questionnaire with 117 participants, the hypotheseswere tested and factors that influence the intention of consumers to consume geneticallymodified food were identified. It was found that the following factors are relevant: Perceivedbenefits, Subjective norm, and Attitude. Of these, the factor Perceived benefits has the greatestinfluence on intention. Factors that do not play a role are Perceived control and, contrary toexpectations, Perceived risks.Another factor that was tested beyond the scope of the theory was the relevance of thenationality of participants on their intention to consume genetically modified food. However,it was found that nationality does not influence the intention. Concluding, the findings revealthat consumers can be especially influenced by their perception of benefits.

No description available.

BACKGROUND: Aberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive. METHODS: Using a well-established Sleeping Beauty (SB) transposon strategy, we injected human and cDNA together with a short hairpin siRNA against (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs. RESULTS: Mixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres' proliferation and MET/MAPK/AKT signaling. CONCLUSIONS: Overexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plantbased production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β- mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed the site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds showed inheritance of transgenes into the progeny without Mendelian segregation. Expression of the endo-β-mannanase gene for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplastderived mannanase had higher temperature stability (40 °C to 70 °C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6-7 fold iv higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the paper, oil, pharmaceutical, coffee and detergent industries.

In the past fifty years there has been an incredible amount of change made to the agrarian system of the United States. New discoveries in the realm of biotechnology led to the adoption of genetically modified organisms (GMOs) in agriculture, and transformed the industry. Due to regulatory policies set during the nineteen-eighties this technology was able to benefit from widespread commercialization. Today, we see the effects of this approach and are entering into a highly volatile political climate in regard to GMOs. This paper aims to provide an analysis of the regulatory system in place and the discrepancies that exist in US policy. The factors evaluated through this thesis include the current US regulatory approach, advancements in biotechnology, and a comparative perspective on US and EU systems. In each of these reviews it is also relevant to mention consumer opinion on GMOs and the role of interest groups. It is important for every American consumer to understand the politics and technology behind their meals. Through the analysis of recent judicial decisions and the enactment of new laws this thesis explains how the use of GMOs in agriculture is causing an unprecedented change to the political structures in place.

No description available.

<p> Exercise intensity is one of the major factors determining the utilization of carbohydrates (CHO) and lipids in mammalian skeletal muscle. Using indirect calorimetry, we determined maximal oxygen uptake (VO2max) and whole-body rates of CHO and lipid oxidation in rats selectively bred for high and low running capacity (HCR's and LCR's) during exercise at 50, 60, 70 and 80%VO2max. Previous studies have revealed a pattern of selection where mammals with different aerobic capacities use the same proportions of lipids and CHO when exercising at the same relative exercise intensity and as intensity increases, CHO use increases and lipid use decreases. The present results showed that the HCR's had a VO2max and distance run to exhaustion that was 1.3 and 4.0 times greater than the LCR's respectively. Also, both groups of rats followed the pattern of fuel selection seen in previous studies where the same proportions (in%) of lipids and CHO are used at the same relative exercise intensity. On an absolute scale, the HCR's used more lipids and CHO than the LCR's at all exercise intensities but the results were not always statistically significant. We also determined the exercise intensity that elicited the greatest lipid use to be 60% VO2max in both groups.</p> <p> In order to explain these patterns of fuel selection, metabolic indicators, metabolites and enzymes, in skeletal muscle were measured at rest and post exercise for one hour at 60%VO2max. Specifically, ATP and phosphocreatine (PCr) metabolite concentrations were determined in the medial and lateral gastrocnemius, extensor digitorum longus (EDL), tibialis anterior (TA), and soleus muscle. The medial gastrocnemius and soleus were analyzed (pre and post exercise samples were combined) for their oxidative and glycolytic enzyme activity by measuring citrate synthase (CS), cytochrome oxidase (COX), β-hydroxyacyl CoA dehydrogenase (HOAD), and lactate dehydrogenase (LDH) . PCr and ATP concentrations did not change pre and post exercise and between the HCR's and LCR's except for the EDL where there was a significant decrease (P<0.05) in both metabolites after exercise in both groups of rats. For the enzyme measurements, CS and COX activities were higher (P<0.05) in the HCR's for the soleus and HOAD activities were also higher in the HCR's medial gastrocnemius compared to the LCR's. We concluded that the HCR's have a greater oxidative capacity as shown by their greater aerobic and endurance capacity (VO2max and distance to exhaustion), their ability to oxidize a greater absolute amount of lipids and CHO's at the same relative exercise intensity, and their higher activities of oxidative enzymes in the soleus (CS and COX) and medial gastrocnemius (HOAD). Future research into the mechanisms involved in explaining these patterns of fuel selection may include examining fatty acid transport proteins, fatty acid and CHO availability, fiber types, and catecholamines.</p> / Thesis / Master of Science (MSc)

O melhoramento genético clássico de sementes milho (Zea mays L.) permitiu desenvolver inúmeras variedades, incluindo o milho com qualidade proteica melhorada (Quality Protein Maize, QPM), que visava aumentar os teores proteicos e as propriedades nutricionais. Por outro lado, novas variedades comerciais foram obtidas por vegetais geneticamente modificados (GM), com foco em parâmetros agronômicos. Em ambos os casos, a segurança dessas variedades para uso como alimento é uma das principais preocupações dos desenvolvedores e dos órgãos de regulamentação. A Equivalência Substancial é a base do sistema de avaliação da segurança de culturas geneticamente modificadas, no entanto alterações na expressão de proteínas não são devidamente analisadas e esclarecidas. As abordagens proteômicas complementam as técnicas de avaliação de biossegurança para alimentos GM, bem como permitem investigar possíveis efeitos indesejáveis derivados do melhoramento clássico. Os objetivos do presente estudo foram caracterizar e comparar os perfis proteicos de variedades de milhos convencionais melhorados (QPM) e geneticamente modificados (GMs), contra suas respectivas linhas convencionais utilizando técnicas proteômicas como eletroforese bidimensional (2-DE) e bottom up shotgun (gel-free). Num primeiro estudo, foram utilizadas três amostras de milho, sendo duas variedades convencionais com QPM (QP1 e QP2) e uma variedade convencional normal (CN). No segundo estudo, foram analisadas duas cultivares de milho GM (GM1 e GM2) e seus respectivos convencionais genitores (CG1 e CG2). As composições químicas de todas as amostras também foram avaliadas quanto a Equivalência Substancial. O extrato bruto proteico foi submetido à análise de eletroforese unidimensional (1-DE), bidimensional (2-DE) e bottom up shotgun (gel-free). As imagens dos mapas proteicos foram analisadas pelo software Image Master 2D Platinum 7.0 (GE). Os spots diferencialmente expressos e selecionados foram sequenciados por MS. Pela composição química das principais frações das amostras de milho foi possível identificar a equivalência substancial entre as amostras convencionais e GMs, bem como QPMs e sua convencional dentro das faixas de variabilidade esperadas da espécie. Nos géis 1-DE foram observadas bandas proteicas com perfis similares entre os grupos de amostras avaliadas para ambos estudos. Nas imagens dos géis 2-DE não houveram alterações extremas entre as amostras de milhos GMs e seus respectivos convencionais genitores (CGs), mas apenas diferenças na intensidade dos spots proteicos. As variedades QPMs e CN apresentaram diferenças devido à distribuição dos spots. Os mapas proteicos das amostras CG1 x GM1 e CG2 x GM2 apresentaram maior semelhança com porcentagens de matchings superiores a 70 %, enquanto as porcentagens de matchings entre variedades diferentes (QPMs e CN) foram menores. No total foram identificadas 219 proteínas das amostras CGs x GMs e QPMs x CN, classificadas quanto aos seus processos biológicos e função molecular. Em conclusão, foram encontradas diferenças entre os cultivares GMs e CGs, indicando uma variação normal entre variedades de milho, que não comprometem a segurança alimentar das amostras estudadas. Quanto às amostras com QPM e CN as diferenças encontradas são devido à sua distância nas linhagens ou germoplasma. / The classic genetic breeding of corn seeds (Zea mays) has enabled the development of many varieties, including corn with improved protein quality (Quality Protein Maize, QPM), which aimed to increase protein levels and nutritional properties. On the other hand, new commercial varieties have been obtained out of genetically modified (GM) vegetables, with a focus in agronomic parameters. In both cases, the safety of these varieties for food use is one of the main concerns for the developers and for the regulatory agencies. Substantial Equivalence is the basis of the safety evaluation system for genetically modified crops, however, alterations in the protein expressions are not been properly analyzed and clarified. The protein approaches complement the techniques of biosafety evaluation for GM foods, as well as allow for possible undesirable effects derived from classic improvement to be investigated. The goals of the current studies were to characterize and compare the protein profiles of the different varieties of conventionally improved (QPM) and genetically modified (GM) corn, against their respective conventional lines using proteomic techniques, such as, two-dimensional electrophoresis (2-DE), bottom up shotgun (gel-free) and masses spectrometry (MS). In a first instance of the study, three samples of corn were used, two of conventional varieties with QPM (QP1 and QP2) and one conventional normal variety (CN). In a second instance of the study, two cultures of GM corn (GM1 and GM2) were analyzed and their respective conventional genitors (CG1 and CG2). The chemical compositions of all the samples were also evaluated for their Substantial Equivalence. The protein raw extract was submitted to analysis of one-dimensional (1-DE), two-dimensional (2-DE) electrophoresis, and bottom up shotgun (gel-free). The protein image maps were analyzed by the Image Master 2D Platinum 7.0 (GE) software. The spots which were expressed and selected differentially were sequenced by MS. By the chemical composition of the main fractions of the samples of corn, it was possible to identify the substantial equivalence between the conventional samples and GMs, likewise with OPMs and their conventional in the ranges of variability which were expected for the species. On the 1-DE gel, it was observed protein bands with similar profiles amongst the groups of evaluated samples for both studies. In the images of the 2-DE gel, there were no alterations between the GM corn and their respective conventional genitors (CGs), but only differences in intensity of the protein spots. The OPM and CN varieties presented differences due to the distribution of the spots. The protein maps of samples CG1 vs. GM1 and CG2 vs. GM2 presented greater similarities with the percentages of matchings superior to 70%, while the percentage of matchings among different varieties (QPMs and CN) were smaller. In total, there were 219 proteins identified in the samples CGs vs. GMs and QPMs vs. CN, classified by the biologic processes and molecular function. In conclusion, there were found differences between the cultures of GMs and CGs, indicating a normal variation among the corn varieties, which do not affect the food security of the studied samples. As per the samples with QPM and CN, the differences found were due to the line distances or germplasm.

Magistro baigiamajame darbe atskleista, kad daugelis respondentų yra neapsisprendę dėl savo pozicijos GMO gamybos bei naudojimo atžvilgiu, tačiau beveik trečdalis asmenų GMO gamybai bei naudojimui nepritaria. Dauguma gyventojų mano, kad GMO ne tik kenkia žmogaus sveikatai, bet ir yra pavojingi, taip pat kenkia aplinkai, o GMO turinčių produktų naudojimas gali negrįžtamai pakeisti genetinį žmogaus kodą ir augalų bei gyvūnų populiacijose gali sukelti negrįžtamus mutacinius pokyčius. Vis dėlto GMO produkciją kai kurie gyventojai linkę naudoti dėl mažesnės jų kainos ir ilgesnio vartojimo galiojimo termino. Nors kai kurie respondentai ir buvo įsitikinę GMO teikiama nauda bei tolimesniu GMO gamybos bei naudojimo plitimu ateityje, tačiau beveik visi respondentai buvo įsitikinę ir tuo, jog GMO gamyba bei naudojimu labiausiai suinteresuotos yra kuo didesnio pelno siekiančios monopolinės kampanijos. Vienareikšmiškai galima teigti, jog nors gyventojai apie GMO informacijos daugiausiai ir gauna iš televizijos laidų, pokalbių su draugais pažįstamais ar giminaičiais bei iš spaudos leidinių arba naršydami internete, tačiau tos informacijos kiekis yra nepakankamas ir daugiau kaip pusė respondentų norėtų, jog informacijos apie GMO būtų pateikiama daugiau. / The Master’s final paper on GMO discovered that the majority of respondents have no strong opinion about production and use of GMO, but one third of responses on production and use of GMO were negative. Many people consider that GMO not only negatively affect human health, but also damage environment. Moreover, eating genetically-modified food can irreversibly change human’s genetic code as well as irreversible mutations in plants’ and animals’ populations. However, some people tend to use GM foods because they are cheaper and can be stored longer than other foods. Even though some of the respondents see benefits of GMO production and usage, almost all of the respondents claim that genetically engineered products interest mostly large companies that are seeking to profit from GMO. The most obvious finding to emerge from this study is that people learn about GMO from mass media and communicating with friends and relatives mostly, but they also admit the lack of credible information on GMO. More than a half of the respondents would like to receive more information about genetically modified organisms.