Dissertations / Theses on the topic 'Seed screenings'

Tomato has been genetically modified for providing properties such as insect-resistance or delayed-ripening. Tomato seeds purchased from several bazaars and markets were screened for the presence of genetic modification by targeting NptII kanamycin resistance, Nos terminator, and 35S promoter gene regions which are the most commonly transformed gene regions in transgenic plants, and then ripening-delayed tomato seeds were tried to be identified in this study. F type truncated-PG gene and Sam-k gene were selected as the indicator of genetically modified ripening delayed tomatoes. DNAs of 25 seed samples were isolated by CTAB method and examined with several primer pairs, and the primer sets that provided consistent results were selected to conduct routine testing by PCR analysis of the samples. In screening analysis via conventional PCR, 4 samples were amplified with 35S, Nos and NptII primer sets. Among other samples, 3 of them were amplified with 35S and Nos primer sets and 2 of them were amplified only with 35S primer set. The amplification was observed with Nos, NptII and Sam-k primers in one sample and this sample was identified as 35 1 N, since the sequence result of the PCR product amplified with Sam-k primers showed high homology with the Samase gene of T3 Coliphage. F type truncated PG gene was not observed in any of the samples. Although this study demonstrates the presence of commonly used gene regions in genetically modified tomatoes, further analysis of the genetically modified ripening delayed tomato seeds via construct specificor event specific PCR techniques is needed for confirmation.

Micronutrient malnutrition is a leading worldwide health problem that affects billions of people particularly in the developing countries resulting in serious health conditions. The domestication of crops produced high yield and larger seed size but with a reduction in nutritious quality. The locus NAM or Gpc-1 affects both the seed size and nutrient content in wheat and barley. A non-functional allele of the gene increases the seed size but at the expense of protein and micronutrient content. However, this gene was found to be lost in wheat during the early domestication resulting in lower nutrient content. Therefore, the selection for high yield has lead to lower grain nutrients. Our aim of the present study is to investigate when the selection for yield occured in barley and to check the existence of the wild type allele in the 19th century of landrace barley crops. In addition, to analyse the barley grain concentration of protein, iron and zinc among the landrace and cultivars from various time periods of northern Europe. The grain nutrient concentration of Nitrogen, Iron and Zinc did not show significant difference among the investigated 80 Scandinavian barley accessions. The grain nutrient concentration did not correlate with the seed size and chlorophyll content. The polymorphism was not observed among the allelic diversity of HvNAM-1 gene indicating that the NAM-B1 gene still prevails in the 19th century barley cultivars.

Woody plant herbicide screening techniques were evaluated in an attempt to expedite the screening process and decrease amounts of herbicide active ingredient required. Rapid greenhouse screening of woody plant seedlings was performed in less than six months while rapid seed screening was performed in less than twenty days. A traditional field screen, requiring ten months, was performed for comparison purposes. Leaf area â biomass ratios were also examined for their influence on herbicide efficacy. Linear regressions were performed using traditional field screen data as the dependent variable and rapid screening technique data as the independent data.

Rapid screens using triclopyr produced more statistically significant regressions compared to those involving imazapyr. Significant regressions were produced that could predict field response of several species using both herbicides and either rapid screening technique. This indicated that rapid screening techniques could determine herbicide efficacy and/or species spectrum of control in much less time with much less herbicide. Rapid seed screens could estimate species spectrum within five days after treatment. The rapid greenhouse screen and rapid seed screen techniques can provide woody plant herbicide developers initial efficacy and spectrum of control data in a cost- and time- effective manner.

Testing showed that as woody plants mature from seedling to sapling, there is a decrease in the leaf area â total aboveground biomass ratio. The decrease in this ratio consistently decreased efficacy of both imazapyr and triclopyr at the lower active ingredient rates. Seedlings with the higher leaf area â biomass ratio had, on the average, higher efficacy response rates to herbicide treatments.
Master of Science

Soybeans are among seeds the common plant foods that contains high protein contents and high oil. The protein provides about 35 to 38 percent of the seeds calories compared to around 20 to 30 percent in other legumes and many animal products. The quality of soy protein is notable and approaches the quality of meat and milk. Unlike many other good sources of protein, soybeans are low in saturated fat and are cholesterol-free. Its proteins provide all the important amino acids, most the amounts needed by humans (NSRL, 2010). As the most consumed vegetable oil in the world, soybean oil has been used substantially in the food industry (Soystats, 2010). Its utilization is determined by its fatty acid composition, with commodity soybean oil typically 13% palmatic acid (16:0), 4% stearic acid (18:0), 20% oleic acid (18:1), 55% linoleic acid (18:2), and 8% linolenic acid (18:3). The change of fatty acid profiles to improve soybean oil quality has been a long time goal of many researchers throughout the world. Biodiesel is an up and coming trend in energy production. Breeding effort can be undertaken in order to produce a higher energy profile soybean oil. Using ethyl-methanesulfonate (EMS) mutagenesis effects on DNA, significant changes to the genes and gene network underlying the protein and oil profile can be achieved. These changes are hard to accomplish using standard breeding techniques. In addition, high amount of linolenic and stearic acid are very important for fuel and biodiesel production, but are not good for food production due to the fact that such oil is oxidized easily and the food goes rancid quickly. However, soybean oil with elevated amount of oleic acid is desirable for food, because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In order to improve the quality of soybean oil and processed foods, chemically mutagenized soybeans have been developed in this project. Seeds harvested from individual M3 and M4 plants (from 2 successive years 2012 and 2013) were analyzed for protein content, oil composition, and content. Moreover, seven phenotypic traits including oil analysis (stearic, palmitic, oleic, linolenic and linoleic), seed protein content, weight of the seeds (High yield), seeds color, stem length, germination rates, and branch architecture were collected and analyzed in this project of soybean `Forrest' mutagenized population. The result of this research showed that there were 25 significantly different lines (p< 0.05) compare to the wild type, which is useful for developing mutants with altered oil and fatty acid compositions in soybean.

Doutoramento em Biologia
The increasing human activity has been responsible by profound changes and a constinuos degradation of the soil compartment in all the European territory. Some European policies are appearing focusing soil’s protection and the management of contaminated sites, in order to recover land for other uses. To regulate the risk assessment and the management of contaminated soils, many European member states adopted soil guideline values, as for example soil screnning values (SSV).These values are particularly useful for the the first tier of the Ecological Risk Assessment (ERA) processes of contaminated sites,especially for a first screening of sites requiring a more site-specific evaluation. Hence, the approriate definition of regional SSVs will have relevant economic impacts in the management of contaminated sites. Portugal is one of European Member States that still lack these soil guideline values. In this context, this study gaves a remarkable contribution in the generation of ecotoxicological data for soil microbiological parameters, terrestrial plants and invertebrates for the derivation of SSVs for uranium (U), cadmium (Cd) and copper (Cu), using a Portuguese natural soil, representative of a dominant type of soil in the Portuguese territory. SSVs were derived based on two methods proposed by the the Technical Guidance Document for Risk Assessment of the European Commission; namely the assessment factor method (AF) and the species sensitivity distribution (SSD) method (with some adaptations). The outputs of both methods were compared and discussed. Further, this study laid the foundation for a deeper reflection about the cut-off (hazard concentration for a given percentage of species - HCps) to be estimated from the SSDs, and to be selected for the derivation of SSVs, with the adequate level of protection. It was proven that this selection may vary for different contaminants, however a clear justification should be given, in each case. The SSvs proposed in this study were for: U (151.4 mg U kg-1dw), Cd (5.6 mg Cd kg-1dw), and Cu (58.5 mg Cu kg-1dw) These values should now be tested for their descriminating power of soils with different levels of contamination. However, this studies clarifies the approach that should be followed for the derivation of SSVs for other metals and organic contaminants, and for other dominant types of Portuguese natural soils.
O aumento das atividades humanas tem sido responsável por mudanças profundas e por uma degradação contínua do compartimento solo, em todo o território Europeu. Em resposta a este problema, algumas políticas Europeias estão agora a emergir orientadas especificamente para a proteção do solo e para a gestão das áreas contaminadas, a fim de recuperar os solos degradados para outros usos. Para regulamentar a avaliação de risco e a gestão de solos contaminados, muitos Estados-Membros Europeus adoptaram valores de qualidade do solo, como por exemplo os “valores de rastreio ou triagem” (do inglês: soil screening values ou SSVs). Estes valores são particularmente úteis para a primeira etapa dos processos de avaliação de risco ecológico (ARE) de locais contaminados, especialmente para um primeiro rastreio dos locais, destinado a separar aqueles em que os riscos são claramente reduzidos daqueles que exigem uma avaliação mais específica e aprofundada para o local. Assim, a definição de SSVs regionais terá impactos económicos relevantes na gestão dos locais contaminados. Portugal é um dos Estados-Membros Europeus que ainda não definiu SSVs. Neste contexto, este estudo dá uma notável contribuição na geração de dados ecotoxicológicos para parâmetros microbiológicos do solo, plantas terrestres e invertebrados necessários para a obtenção de SSVs para urânio (U), cádmio (Cd) e cobre (Cu), utilizando um solo natural Português, representante de um tipo dominante de solo existente no território nacional. Assim, foram obtidos SSVs para os metais referidos com base em dois métodos propostos pelo Documento de Orientação Técnica para Avaliação de Riscos da Comissão Europeia, nomeadamente o método dos factores de avaliação (do inglês: assessment factors ou AF) e o método probabilístico da distribuição da sensibilidade espécies (do inglês: species sensitivity distributions ou SSDs) (com algumas adaptações). Os resultados dos dois métodos foram comparados e discutidos. Além disso, este estudo lançou as bases para uma reflexão mais profunda sobre o ponto de corte (concentração de risco para uma determinada percentagem de espécies) a ser estimado a partir das distribuições de sensibilidade das espécies (SSDs), e para ser selecionado para a obtenção de SSVs, com o nível adequado de proteção. Neste estudo foi comprovado que esta seleção pode variar para diferentes metais ou outros contaminantes, no entanto, uma justificação clara deve ser dada, em cada caso. Os SSvs propostos neste estudo foram de: U (151,4 mg U kg- 1ms ), Cd (5,6 mg Cd kg- 1ms ) e Cu ( 58,5 mg Cu kg- 1 ms) Estes valores devem agora ser testados quanto à sua capacidade para descriminar solos com diferentes níveis de contaminação. No entanto, este estudo esclarece e sugere a abordagem que deve ser seguida para a derivação de SSVs para outros metais e contaminantes orgânicos, e para outros tipos dominantes de solos naturais portugueses.

A doenÃa renal crÃnica (DRC) à assintomÃtica em seus estÃgios iniciais, portanto seu diagnÃstico precoce depende de rastreamento. Uma grande parcela de pessoas com risco para DRC nÃo à avaliada quanto à funÃÃo renal. Um questionÃrio denominado SCORED foi desenvolvido para predizer a probabilidade de um indivÃduo apresentar DRC, baseado apenas em informaÃÃes demogrÃficas e clÃnicas, sem necessidade de resultados laboratoriais. Nosso estudo teve por objetivo avaliar o desempenho do SCORED entre hipertensos e diabÃticos cadastrados em um Programa de SaÃde da FamÃlia na cidade de Meruoca, CearÃ. A amostra foi composta por 221 participantes. A taxa de filtraÃÃo glomerular < 60 mL/min foi utilizada para o diagnÃstico da DRC. Os resultados do desempenho do SCORED foram os seguintes: a pontuaÃÃo gerada pelo SCORED foi preditora independente da presenÃa de DRC (OR=1,26; IC 95%=1,10-1,59; p=0,04); o questionÃrio apresentou sensibilidade de 97% e especificidade de 23% em diagnosticar DRC, com valor preditivo positivo de 37%, valor preditivo negativo de 94% e acurÃcia de 47%. Alta sensibilidade e alto valor preditivo negativo habilitam o SCORED como instrumento de rastreamento de DRC entre hipertensos e diabÃticos. A utilizaÃÃo desse questionÃrio tem o potencial de incrementar o diagnÃstico precoce da DRC entre pacientes cadastrados em Programas de SaÃde da FamÃlia, alÃm de sua aplicabilidade na populaÃÃo geral e em campanhas educacionais.
Chronic kidney disease (CKD) is asymptomatic in its initial stages; therefore, its early diagnosis depends on screening. A large number of people at risk for CKD are not assessed for renal function. A questionnaire denominated SCORED (Screening for Occult Renal Disease) was developed to predict the probability of an individual presenting CKD, based on demographic and clinical information, without the need for laboratory results. Our study had as objective to assess the performance of SCORED among subjects with diabetes and hypertension registered in the Family Health Program in the city of Meruoca, CearÃ. The sample was made up of 221 participants. A glomerular filtration rate less than 60 mL/min was used for the diagnosis of CKD. Performance results on SCORED were the following: the score was an independent predictor for the presence of CKD (OR=1.26; 95% CI=1.10-1.59; p=0.04); the sensitivity and specificity of this questionnaire were 97% and 23% for the diagnosis of CKD, with positive predictive value of 37%, negative predictive value of 94%, and accuracy of 47%. High sensitivity and high negative predictive value qualified SCORED as a screening tool for CKD among subjects with diabetes and hypertension. The use of this questionnaire has the potential to improve early diagnosis of CKD among patients registered in the Family Health Program, other than its applicability in the general population and in educational campaigns.

Topoisomerase 2α (Topo2α) is an essential protein with DNA decatenating enzymatic properties, indispensable for chromosome decatenation and segregation. It is a target for a plethora of antitumour drugs and Topo2α protein levels have been associated with the success of treatment, but also drug resistance and secondary malignancies. Although unique in its ability to resolve catenated chromosomes, the role of Topo2α in other steps of DNA metabolism, such as DNA replication elongation and termination have been elusive. A thorough understanding of the role of Topo2α in the cell will not only allow for increased insight into the mechanisms it is involved in, but it will also shed light on proteins and pathways that can act as back-up in its absence, and therefore hopefully expand the basis on which to improve treatment options. Through a synthetic lethal interaction (SLI) screen with an siRNA library targeting 200 DNA repair and signalling genes, Topo2α emerged as being synthetic lethal to Werner protein (WRN), a RecQ helicase involved in maintaining genome integrity mainly in S phase, and the loss of which leads to Werner Syndrome (WS), a segmental progeroid syndrome. The screen was performed in WRN deficient cells, with the initial aim to find proteins that act to buffer against loss of viability, which is the central idea in the concept of synthetic lethality in the absence of WRN. The screen revealed an SLI between WRN and Topo2α and although we were unable to fully validate this, it spurred the question of Topo2α’s role in DNA replication. The findings in this thesis suggest that Topo2α is not required for DNA elongation and timely completion of S phase, and that simultaneous loss of the closely related isoform Topo2β does not affect replication, suggesting that these proteins do not act in parallel back-up pathways during replication. Interestingly, cells accumulate in the polyploid fraction after both depletion and inhibition of Topo2α, albeit with different kinetics. The mechanistic basis of this phenotype remains to be understood through further research, but it is highly interesting as aneuplidity and polyploidy are implicated in the initial stages of tumour development.

Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology. In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation. The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions. By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages. During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone. These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.

Animal testing is still the common method to test the efficacy of new drugs, but tissue engineered in vitro models are becoming more acceptable for replacing and reducing animal testing in anti-cancer drug screening by developing in vitro three-dimensional (3D) tumour models for anti-cancer drug testing. In this study, three-dimensional (3D) culture methods were developed to mimic the tumour microenvironment. 3D culturing is to seed, maintain and expand cultured cells in three-dimensional space, in contrast to the traditional two-dimensional (2D) method in which the cells attach to the bottom of culture containers as monolayers. To mimic the intercellular interplay for tumour study, cell co-culture was applied. In this thesis, perfusion culture showed a better homeostasis for 3D tumour model growth over 17 days, with a more controllable working platform and a more reliable response-dose correlation for data interpretation. In the Matrigel sandwich system, the co-culture of breast cancer cells and endothelial cells demonstrated the morphology featuring a vascular network and tumour structures, with the thickness of the three-dimensional structure around 100µm and tubule length 200-400 µm, and maintained for 10 days. The comparisons studies between Matrigel sandwich and other methods suggest that though not fully characterised, Matrigel is still a valuable scaffold choice for developing co-culture 3D tumour model. Finally, the combination of perfusion and co-culture showed the potential of applying this model in angiogenesis assay, with a drug response profile combining cell viability and morphology to mimic in vivo tumour physiology.

Chapter 1 Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. Chapter 2 Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. Chapter 3 Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. Chapter 4 Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. Chapter 5 Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB₂R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB₂R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.

A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.

Ce travail se place dans le cadre d'etudes sur les mecanismes moleculaires de regulation par les gibberellines (ga). Il a consiste en la recherche et l'etude de nouveaux genes dont l'expression est induite par les ga au cours de la germination des graines d'arabidopsis thaliana. La strategie utilisee repose sur le criblage differentiel d'une banque d'adnc. Ce crible moleculaire est base sur l'utilisation d'un inhibiteur de la biosynthese des ga qui, indirectement en bloquant la biosynthese des ga, inhibe l'expression des genes ga-regules. Les conditions d'inhibition specifique de la biosynthese des ga ont ete etablies au cours de la germination de graines d'arabidopsis thaliana de type sauvage, en utilisant l'uniconazol, un heterocycle azote de la famille des triazoles. Puis, le criblage differentiel d'une banque d'adnc de graines de 39 heures (traitees par ga et uniconazol) par une sonde complexe (graines traitees par uniconazol) a conduit a la selection de 5 adnc. L'analyse des caracteristiques structurales, des homologies de sequences et du profil d'expression au cours de la germination en particulier, en reponse aux ga, ont ete realises pour chacun d'entre-eux. Seule, l'expression d'un adnc codant pour une 3-cetoacyl-coa thiolase est induite par les ga au cours de la germination des graines d'arabidopsis thaliana. Les autres adnc correspondent a des marqueurs specifiques de la germination dont certains sont de nouvelles sequences chez a. Thaliana. Chez arabidopsis thaliana, une petite famille multigenique gasa de cinq membres, dont deux au moins sont regules par les ga, est caracterisee. Le gene gasa4 a ete isole et son expression a ete analysee au cours du developpement des graines et de la germination. Il a ete montre qu'il s'exprime tres precocemment dans la fleur puis dans la graine en germination. Par une etude en transgenie cette expression a ete localisee dans les cotyledons, et les pointes de racines primaires et laterales et egalement dans le meristeme vegetatif. De plus, la regulation de son expression est complexe et fait intervenir a la fois les gibberellines et la lumiere

碩士
國立屏東科技大學
生物科技系所
105
Hypertension is a chronic disease, and now becomes the most common disease. Although some chemically synthesized antihypertensive drugs have been developed, they are usually accompanied by side effects. It is generally believed that the hypotensive active peptides obtained from natural sources have lower side effects than chemically synthesized drugs. In this study, Cucurbita moschata seed proteins were extracted and hydrolyzed using thermolysin as digestion enzyme. Small peptides were obtained using super ultrafiltration by means of molecular weight cut-off membranes (3kDa cut-off). The small peptides were fractionated using reversed-phase high performance liquid chromatography (RP-HPLC) and the angiotensin-converting enzyme (ACE) inhibitory activities of the resulting fractions were examined by in vitro ACE inhibitory assay. Peptide identification for the most active fraction was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with database-assisted sequencing, IR-8 was identified from the fraction with the most effective ACE inhibition and the identity and activity of IR-8 were further confirmed by synthetic peptide. The results showed that the IC50 value of IR-8 was 14.2±1.6 μM. Inhibition kinetics showed that IR-8 was a competitive inhibitor. Furthermore, the inhibition type of IR-8 was determined using a pre-incubation experiment. The results showed that IR-8 had a significant decrease in IC50 after pre-incubated with ACE, which suggested that IR-8 was a prodrug. In this study, we successfully uncovered the potent ACE inhibitory peptide IR-8 from the thermolysin hydrolysate of pumpkin seeds. The antihypertensive effect of IR-8, as well as thermolysin hydrolysate of pumpkin seeds will be explored in the future.

碩士
國立屏東科技大學
生物科技系所
104
Hypertension has been a serious disease of modern civilization, although many therapeutic drugs have been developed. However, these synthetic drugs usually accompany with many side effects. With the growing health awareness, the use of natural ingredients for health purposes is getting popular. Since 1979, more and more studies have focused on blood pressure-lowering peptides derived from natural food sources, such as soybeans and milk. Compared with synthetic drugs, short-chain active peptides are easy to be absorbed by the gastrointestinal tract and show minimal side effects. . 　　Cassia obtusifolia seed（COS） has long been regarded as food and the current modern pharmacological studies have confirmed that cassia seed tea can be used as a complementary medicine to treat various eye diseases, hypertension, high cholesterol （fat） and constipation. However, the active components have not been well characterized. Therefore this study aims to screen hypertensive peptides from thermolysin digest of natural plant seeds " Cassia obtusifolia ". In this study, reverse phased high-performance liquid chromatography （RP-HPLC） and strong cation exchange chromatography （SCX） were used for orthogonal fractionation, and in vitro angiotensin-converting enzyme （ACE ） inhibitory assay was used to screen the most active fractions. The ACE inhibitory peptides in the most active RP and SCX fraction were de novo sequenced using a high-resolution mass spectrometry （Q Exactive ™ Hybrid Quadrupole-Orbitrap）. Two major peptide sequences FK-6 , I （L） YI （L）PH were identified and their identities and ACE inhibitory activities were confirmed using synthetic peptides. The IC50 of FK-6 was determined as 17.5±1.9 μM. The inhibition kinetics indicated that FK-6 is a competitive inhibitor and the study of its pre-incubation with ACE showed that the IC50 value was not significantly changed compared with that of post-incubation, which revealed that the inhibition type of FK-6 is true inhibitor. The interaction of FK-6 and ACE was simulated using molecular docking and the result showed that FK-6 can interact with several key residues in the active site of ACE, which is consistent with the result of inhibition kinetics. Moreover, the in vivo hypertensive effect of FK-6 was demonstrated using the animal model of spontaneously hypertensive rat. In this study, we concluded that thermolysin hydrolysate of COS contained a potent antihypertensive peptide which is potential for health food development.

Introduction: Previous literature indicates that foreign-born women have lower rates of cervical cancer testing and higher mortality rates when compared to U.S.-born women. Factors that influence receipt of cervical cancer screening among foreign-born women include acculturation and human papillomavirus (HPV) knowledge. Methods: In this cross-sectional study, the 2007 California Health Interview Survey (CHIS) was used to examine the impact of acculturation on cervical cancer screening and HPV knowledge and the 2000-2008 Surveillance Epidemiology and End Results (SEER) database was used to determine differences in stage of diagnosis and survival time. The study population included a total of 3,603,412 foreign-born and 6,749,557 U.S-born women in the CHIS between the age of 18 to 65 and a total of 10,733 U.S.-born and 5,069 foreign-born women in the SEER database. Logistic regression was used to examine the predictors for cervical cancer screening and Cox’s proportional hazards ratios were used to determine the effect of covariates on survival time. Kaplan-Meier survival analysis generated survival curves. Results: Acculturation levels were positively associated with ever having a Pap test, ever hearing about HPV, knowledge that HPV causes cancer and HPV does not cause AIDS, but not with current receipt of a Pap test, knowledge that HPV can be sexually transmitted and that HPV can go away without treatment. Women with low (0.38, (CI, 0.22, 0.66)) and medium (0.50, (CI, 0.39, 0.81) levels of acculturation were less likely to ever receive a Pap test and less likely to ever hear of HPV compared to highly acculturated women. Foreign-born women had a lower risk of death than U.S.-born women. Conclusions: Despite a reported lower risk of death, foreign-born women, particularly those less acculturated, may benefit from targeted interventions to increase cervical cancer screening utilization and general HPV awareness.
acase@tulane.edu