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Use of nanoemulsion liquid chromatography (NELC) for the analysis of inhaled drugs. Investigation into the application of oil-in-water nanoemulsion as mobile phase for determination of inhaled drugs in dosage forms and in clinical samples.Althanyan, Mohammed S. January 2011 (has links)
There has been very little research into the bioanalytical application of Microemulsion High Performance Liquid Chromatography (MELC), a recently established technique for separating an active pharmaceutical ingredient from its related substances and for determining the quantity of active drug in a dose. Also, the technique is not good at separating hydrophilic drugs of very similar chemical structures.
Different phase diagrams of oil (octane or ethyl acetate), co-surfactant (butanol), surfactant (sodium dodecyl sulphate (SDS) or Brij-35) and buffer (Phosphate pH 3) were developed and several nanoemulsion mobile phases identified. Nanoemulsion mobile phase that is, prepared with SDS, octane, butanol and a phosphate buffer, failed to separate hydrophilic compounds with a very close chemical structure, such as terbutaline and salbutamol. A nanoemulsion mobile phase containing a non-ionic surfactant (Brij-35) with ethyl acetate, butanol and a phosphate buffer, was, however, successful in achieving a base line separation, and the method was validated for simultaneous determination of terbutaline and salbutamol in aqueous and urine samples.
An oil-in-water (O/W) NELC method was developed and validated for the determination of formoterol in an Oxis® Turbuhaler® using pre-column fluorescence derivatisation. Although the same mobile phase was extended for separation of formoterol in urine, the formoterol peak¿s overlap with endogenous peaks meant that fluorescence detection could not determine formoterol in urine samples. Solid phase extraction, concentrating the final analyte 40 times, enabled determination of a low concentration of formoterol in urine samples by UV detection. The method was validated and an acceptable assay precision %CV <4.89 inter-day and %CV <2.33 intra-day was achieved. Then after the application of O/W nanoemulsion mobile phase for HPLC was extended for the separation of lipophilic drugs. The nanoemulsion liquid chromatography (NELC) method was optimised for the determination of salmeterol and fluticasone propionate in good validation data was achieved.
This thesis shows that, in general, the performance of O/W NELC is superior to that of conventional High Performance Liquid Chromatography (HPLC) for the analysis of both hydrophilic and lipophilic drugs in inhaled dosage formulations and urine samples. It has been shown that NELC uses cheaper solvents and that analysis time is faster for aqueous and urine samples. This considerable saving in both cost and time will potentially improve efficiency within quality control.
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Engineering hematopoietic and immune cells from human pluripotent stem cells for fundamental and therapeutic applicationsJuhyung Jung (17045163) 27 September 2023 (has links)
<p dir="ltr">Hematopoietic stem cells (HSCs) originating from aorta-gonad-mesonephros (AGM) could self-renew and develop into various immune cells, such as T cells, neutrophil and natural killer (NK) cells, rendering them as a promising cell source for immunotherapy. NK cells belong to the family of the innate lymphoid cells, and are employed as one of immunotherapy to cure solid and hematological malignancies including leukemia. Neutrophils are one of the granulocytes, and they are emerging as a new therapeutic target in various cancers. Due to the lack of reliable sources for the amounts of HSCs and immune cells required for clinical infusions (~10<sup>9</sup> cells/patient), it remains as a major challenge to realize their full potential in targeted cell and immunotherapy. While substantial efforts have been made to generate native cell-like HSPCs and immune cells from human pluripotent stem cells (hPSCs), intricate molecular process governing the differentiation of HSCs and immune cells remain elusive, preventing the development of robust strategies for HSC and immune cell productions.</p><p dir="ltr">In this study, we first demonstrated that critical role of temporally regulating Wnt signaling in initiating AGM-like hematopoiesis across 11 hPSC lines. By inhibiting TGFβ at the stage of aorta-like CD34+SOX17<sup>+</sup> hemogenic endothelium, which led to the downregulation of Wnt signaling, we established a chemically defined, feeder-free culture system that efficiently produced robust AGM-like hematopoietic cells. Furthermore, we investigated how hypoxia affects the <i>in vitro</i> hPSC differentiation into HSPCs, which resulted in a hypoxia-enhanced HSPC differentiation platform.</p><p dir="ltr">Next, the temporal roles of transcription factors (TFs), including <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, in regulating and promoting NK cell differentiation from hPSCs are determined. <i>NFIL3</i> and <i>SPI1</i> have been reported to influence the early stages of NK cell development, while <i>ID2</i> has an impact on the generation of NK cells throughout the early and intermediate stage. We genetically modified hPSCs with doxycycline-inducible expression of <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, and investigated their roles in NK cell induction from hPSCs. Among these three TFs, forced expression of <i>ID2</i> yielded the highest percentage of NK cells under a chemically defined, feeder-free monolayer culture condition, demonstrating that forced expression of NK-specific TFs improves the efficiency of NK cell differentiation from hPSCs.</p><p dir="ltr">Chimeric antigen receptor (CAR) is an artificial cell receptor expressed on immune T or NK cells that has been engineered to allow T or NK cells to re-target cancer cells by exclusively binding to a cancer-specific protein. CAR engineering has significantly improved the anti-tumor efficacy of NK cell therapy, resulting in 6 FDA-approved CAR-T therapies and many other ongoing clinical trials. Recently, a chlorotoxin (CLTX)-based CAR was developed and shown to specifically bind to a variety of heterogenous glioblastoma (GBM) cell lines. To test whether CLTX-CAR could improve the anti-tumor cytotoxicity of hPSC-derived NK cells, hPSCs were engineered with CLTX-CAR for stable and homogenous CAR expression via Cas9-mediated homologous recombination. The expression of CLTX-CAR did not affect the pluripotency and NK cell differentiation potential of hPSCs, and CLTX-CAR significantly improved the cytotoxicity of hPSC-derived NK cells against GBM cells.</p><p dir="ltr">Finally, we implemented a GBM-on-a-chip microfluidic model to interrogate the tumor microenvironment (TME). Microfluidics are an emerging device for investigating cancer biology with spatiotemporal control over signaling modulators by using a small volume. The interaction between hPSC-drived neutrophils and GBM was explored in this microfluidic device. GBM TME is very complex and involves many cell types, including neurons, microglia, immune T and NK cells. In the future, microfluidic models with isogenic cell components will be designed and implemented to better model GBM TME.</p><p dir="ltr">In summary, these discoveries confirm the pivotal role of Wnt signaling in guiding hPSCs towards hematopoietic lineages, while also highlighting <i>ID2</i> as a potent enhancer of NK cell differentiation from hPSC-derived hematopoietic progenitor cells. Additionally, CAR engineering enhances the anti-tumor capabilities of hPSC-derived NK cells. Furthermore, microfluidic models are employed to interrogate GBM TME.</p>
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HIV-1 ENV: IMPACTING HIV-1 FITNESS, ENTRY INHIBITOR DRUG SENSITIVITY, AND IN VIVO SELECTION OF A RESISTANT VIRUS TO THE MICROBICIDE PSC-RANTESDudley, Dawn M. January 2008 (has links)
No description available.
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我國因應船員執業證書國際公約修正案之研究:政策論證之分析 / On the ROC of Policy Regarding the Amendment of the Seafarer Practicing Certificate International Convertion : Analysis Policy Argument尹台生, yin,tai-shen Unknown Date (has links)
政策是公權力對大眾利益表現一種型態,為確保其一貫施政品質,公共治理的理論,逐漸重視政策的效果 - 政策論證(policy argument),尤其以全民政治為基礎的政策治理,對於整體資源的投入/產出,必須強化其自體檢核功能。
航運的操作有兩項基本的動因,一個是船,另一個是船員,兩者缺一不可,然後貨才能「暢其流」,尤其船員的因素,是航運興衰的關鍵動因。我國在邁進開發國家之列時,對於各類專業人才必須予以適當的培訓及保障,整體經濟的生產力才能提升,並能強化競爭力,邁向國際化,航運業界、政府及學界均應有此共識,在面對國際船員訓練、發證的考驗時,同心協力,參與國際海事組織(IMO)船員訓練發證政策的挑戰,突破港口國管制(PSC)的檢查,使船員在工作保障及社會福利有所依循,以符合船員訓練STCW 78/95國際公約修訂的檢查及港口國管制(PSC)的要求標準。
中國方面,因為是國際海事組織(IMO)的會員,對於船員訓練及發證,均按照STCW78/95國際公約的規定與要求,頒布一系列規章及訓練規範,而且根據中國「民法通則」規定,法律條約沒有規定的,可適用國際慣例,中華民國的船員執業證照則處於相對不確定的狀況,尚待政府的國際政策躓琢。
由於兩岸經濟依存,政治對立,各自表述,在意識形態上產生不接軌的現象,再加上國際情勢發展的不對稱影響,我國船員訓練主管單位及相關港監部門及學術組織,本於國家利益的基礎上,為謀我國航運業的發展及保障船員的工作權,除強化國內的船員訓練品質,另確有必要務實的參與國際組織相關活動及其相互間的合作,將我國現職船員訓練符合STCW 78/95國際公約之發證政策與執行規定,順利遞送國際海事組織(IMO)的秘書處,並確認發證履行報告,完成從第三走廊(軌道)列入〈白名單〉(White List)之列(如本論文第五章敘述)。
本研究肇因於國際公約的修訂,影響我國船員訓練執業證書的認可,政府從一貫的船員政策實施過程為核心,配合考試、適任評估,目標是符合國際公約STCW 78/95的標準及船員證照認證的公共治理為論證(policy argument),從質性研究的模式,並依政治學方法論的比較研究法及政策論證的步驟與國際關係(IR)的談判元素,全文以建構類型(constructive typology) 理論,作為理論架構,驗證第三軌道的模式。 / Decision-making is the pattern that shows a certain type of state authority; administrative power displays the attitude to the general interests based on effectiveness of administration. In order to guarantee its consistent administrative quality, the administration theory shows how a state manages its public policy. Also, governments undoubtedly pay much attention to the outcome of them. How those public policies are approved (i.e., policy arguments), also exemplify the fact that decision-making is based on the participation of individuals in whole society. Inputs/output model could strengthen our knowledge from the key function of physical examination.
There are two basic theories regarding the operation of shipping. One is the ship; with the other one names the seafarer. None of them can be dispensed with—can commodities be transported smoothly only through this understanding. Seafarer's role, in particular, is the key factor attributing to the shipping frequency. The Republic of China (ROC) on Taiwan has to offer proper crew training so as to guarantee all kinds of qualified personnel in a specific field are sufficient while striding forward to develop its economy. Only if the productivity could be promoted, can the ROC strengthen competitiveness, march forward to further internationalization. Shipping could reflect a certain country’s industrial level, governmental effectiveness and its educational development. As local trains’ seafarers— instead of by international authorities, this country encounters the license-issuing problem. Only through concerted efforts, can this country meet ordeals when participating in International Maritime Organization (IMO)’s training and license-issuing process, pass through the inspection that the port state controlled (PSC), make seafarers’ rights guaranteed. In addition, the ROC has to assure seafarers’ welfare, in accordance with the inspection of the international convention revising (STCW 78/95) and to the standard demanded by the port state control (PSC).
The People’s Republic of China (PRC) is a member of International Maritime Organization (IMO). The PRC obeys the regulations and request of international convention STCW78/95, the general rule of the civil law, and announces a series of training rules when training seafarers and issuing licenses. The PRC also declares that they could apply to international custom if their lacks of local regulations. In contrast, the seafarers of the Republic of China are still not for sure that they could be issues operation certificates. The ROC government is awaited to do something.
Economies along the two sides of the Taiwan Straits have become mutually interdependent, whereas their political relations are still stridently confrontational. Due to the huge gap between their international statuses, the authorities that responsible for seafarer training and harbor supervisors, as well as scholars, have to promote the training quality of our seafarers, participate caucuses and activities of various international organizations so as to assure the ROC’s sea transportation and their working opportunities. The government has to submit its 78/95 STCW regulations training post crewman to the Secretariat of the International Maritime Organization (IMO), confirm license-issuing and fulfill the report, enlist itself to the White List from the “Third Track (as noted in Chapter 5).”
This research could be attributed to the revision of the international convention, which affects the validity the ROC’s approval to the certificates in seafarer training and issuing licenses. The government designs courses from consistent policies of seafarer training, helping seafarers pass examination and evaluation and finally reach the standard of international convention named STCW 78/95. In addition, one of the government’s objectives is to comply the policy argument of governance concerning license issuing. This author tries to apply to quality research, comparative methodology, and negotiating factor in international relations (IR), and constructive typology for the purpose of verifying the operation of the “Third Track.”
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(De)legitimizing rape as a weapon of war: patriarchy, narratives and the African UnionLangeveldt, Veleska January 2014 (has links)
Magister Administrationis - MAdmin / The African continent has over the past 40 years witnessed a continued scourge of violent conflict and human rights abuses. These conflicts have significantly undermined the social, political, and economic prosperity of African citizens. Additionally, women and children are particularly affected by these conflicts. Women and children are regarded as ‘the most vulnerable’ as they often become the targets of sexual abuse by the enemy. The African Union (AU) is primarily responsible for the resolution of conflicts on the continent. It professes to be committed to the prevention of human rights abuses and the protection of African women (and children) during armed conflicts. It has thus developed an array of mechanisms, protocols, and instruments to address the exploitation and sexual abuse of women during conflict periods. These instruments include: The Constitutive Act of the AU (2000); The Solemn Declaration of Gender Equality in Africa (2003); the Protocol to the African Charter on Human and People’s Rights on the Rights of Women in Africa- ACHPRWA (2004); and the Protocol relating to the Peace and Security Council. In this research project, I consider whether the narratives used in these AU documents sufficiently and explicitly address the use of rape as a strategic weapon during armed conflicts; or whether these narratives inadvertently contribute to a culture that perpetuates war-time rape. My analysis shows that these AU documents deal with war-time rape in very vague and euphemistic terms. Although gender discrimination, sexual violence, exploitation, discrimination, and harmful practices against women are condemned, the delegitimization of rape as a weapon of war is not specifically discussed. This allows for varying interpretations of AU protocols, including interpretations which may diminish the severity of strategic rape. This has lead me to propose that the narratives used in these AU protocols and related documents draw on patriarchy, perpetuate patriarchy, and thus inadvertently perpetuates a culture that perpetuates the use of rape as a weapon of war
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L’identification de nouvelles activités chez les complexes Polycomb les lient aux structures d’ADN non-canoniquesAlecki, Célia 06 1900 (has links)
Les protéines du groupe Polycomb (PcG) sont des protéines essentielles et conservées, qui forment deux complexes principaux, PRC1 et PRC2, qui sont recrutés au niveau de la chromatine et qui répriment stablement l’expression génique. Chez Drosophila melanogaster, les complexes Polycomb sont recrutés à des éléments d’ADN appelés éléments de réponse Polycomb (PREs) pour réprimer la transcription. PREs sont des éléments mémoires permutables qui peuvent maintenir la répression ou l’expression génique. Malgré des dizaines d’années d’étude, des questions fondamentales sur le fonctionnement du système PcG subsistent. 1) Comment les protéines PcG sont recrutées aux PREs uniquement lors du contexte développemental approprié, et comment les PREs peuvent conduire à la fois à l’activation et à la répression stable. 2) Comment les complexes PcG répriment la transcription, et si cela implique de nouvelles activités biochimiques et interactions. 3) Comment la répression dépendante des PcG peut-elle être propagé à travers le cycle cellulaire. La recherche de nouvelles activités biochimiques pour les complexes PcG pouvant répondre à ces questions fait l’objet de cette thèse.
Les PREs sont transcrits en ARN qui pourraient donner la spécificité de contexte pour recruter les protéines PcG. Nous avons supposé que des R-loops puissent se former aux PREs, et être reconnues par les complexes Polycomb, ce que vous avons testé dans le chapitre 2. Nous avons identifié les séquences formant des R-loops dans des embryons et une lignée cellulaire de Drosophila melanogaster, et nous avons trouvé que ~30% des PREs forment des R-loops. Nous avons découvert que les PREs ayant formé des R-loops ont une plus forte probabilité d’être liés par les protéines PcG in vivo et in vitro. PRC2 lie des milliers d’ARN in vivo, mais aucune fonction claire n’y a été associée. En utilisant des expériences in vitro, nous avons identifié une activité d’invasion de brins pour PRC2 qui induit la formation d’hybride ARN-ADN, la partie principale d’une R-loop. Dans ce chapitre, nous avons trouvé que les PREs forment des R-loops, et sont impliquées dans le recrutement des protéines PcG qui induisent la répression génique stable. Nous avons découvert une activité d’invasion de brins pour PRC2 qui pourrait impliquer ce complexe Polycomb dans la formation de R-loops in vivo.
Dans le chapitre 3, nous avons identifié une activité similaire à celle de la topoisomérase I associée avec PRC1 et la région C-terminale de sa sous-unité PSC (PSC-CTR). PRC1 et PSC-CTR peuvent relaxer un plasmide surenroulé négativement et ajouter des supertours négatifs à un plasmide relaxé en absence de topoisomérase. Cette activité suggère que la régulation de la topologie de l’ADN puisse être un nouveau mécanisme utilisé par les complexes PcG. PRC1 peut résoudre les R-loops formées sur un ADN négativement surenroulé in vitro. Une fonction possible pour cette activité de topoisomérase peut être la régulation des R-loops, dont la stabilité dépend à la fois de la séquence d’ADN et de la topologie de l’ADN environnant, in vivo.
Dans cette thèse, nous avons identifié de nouvelles activités chez les complexes PcG : une activité d’invasion de brins pour PRC2 et une activité similaire à celle des topoisomérases pour PRC1. Ces deux activités impliquent les complexes PcG dans la formation et la résolution de R-loops. De plus, ces deux complexes peuvent reconnaitre les R-loops et sont recrutés aux PREs ayant formé ces structures. En conclusion, nous avons identifié de nouvelles activités pour les complexes Polycomb PRC1 et PRC2 qui les lient à la formation, la reconnaissance et la résolution de R-loops. / Polycomb group (PcG) proteins are conserved, essential proteins, which assemble in two main complexes, PRC1 and PRC2, which are targeted to chromatin and stably repress gene expression. In Drosophila melanogaster, Polycomb complexes are targeted to DNA elements called Polycomb response elements (PREs) to repress transcription. PREs are switchable memory elements that can maintain either gene repression or gene activation. Despite decades of study, fundamental questions about how the PcG system functions remain. These include: 1) how PcG proteins are targeted to PREs only in the appropriate developmental context, and how PREs can mediate both stable activation and repression; 2) how PcG complexes repress transcription, and whether it involves novel biochemical mechanisms and interactions; 3) how PcG repression can be propagated through cell cycles. The search for new biochemical activities for PcG complexes that may answer these questions is the topic of this thesis.
PREs are transcribed into RNAs which may give the context specificity to recruit PcG proteins. We hypothesized that R-loops may form at PREs, and be recognized by PcG complexes, which we tested in Chapter 2. We identified R-loop forming sequences in Drosophila melanogaster embryos and tissue culture cells, and found that ~30% of the PREs form R-loops. We found that PREs which have formed R-loops are more likely to be bound by PcG proteins both in vivo and in vitro. PRC2 binds to thousand RNA in vivo but no clear activity has been associated with it. Using in vitro assays, we identified a strand exchange activity for PRC2 which induces the formation of RNA-DNA hybrid, the main part of an R-loop. In this chapter, we have found that PREs form R-loops and are involved in the targeting of PcG proteins which induce stable gene repression. We have discovered an RNA strand exchange activity for PRC2 which may involve this Polycomb complex in the formation of R-loops in vivo.
In Chapter 3, we identified a type I topoisomerase-like activity associated with PRC1 and the C-terminal region of its subunit PSC (PSC-CTR). PRC1 and PSC-CTR can relax a negatively supercoiled plasmid and add negative coils to a relaxed plasmid in absence of topoisomerase. This activity suggests regulation of DNA topology may be a novel mechanism used by PcG complexes. PRC1 can resolve R-loops formed on negatively supercoiled DNA in vitro. One role for the topoisomerase-like activity may be to regulate R-loops, whose stability of depends on both the DNA sequence and the topology of the surrounding DNA, in vivo.
In this thesis, we identified new activities for Polycomb group complexes: an RNA strand exchange activity for PRC2 and a topoisomerase-like activity for PRC1. Both activities link PcG complexes to the formation and resolution of R-loops. In addition, both complexes can recognize R-loops and are recruited to PREs which have formed these structures. In conclusion, we have identified new nucleic acid-based activities for the Polycomb complexes PRC1 and PRC2, which link them to the formation, recognition and resolution of R-loops.
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An analysis of the relationship between psychosocial safety climate and the work-school-interface for non-traditional students in South AfricaApril, Kelly 12 January 2022 (has links)
The growth of a country's economy is highly dependent on the levels of skills available in that country. South Africa has a stark shortage of skilled individuals due to an inadequate and failing education system (Horwitz, 2013). As a response to this problem the South African government actively incentivises organisations to develop the skillset of their employees in order to build a strong economy, improve job creation and promote social development (Department of Higher Education and Training, 2019). It is therefore important that all stakeholders involved yield a return on this investment. It is also a global phenomenon that an increasing number of fulltime employees are also engaging in formal further studies due to the rapid changes in the labour market. For example, advancements in technology have had a major impact on traditional business models and the roles, and skills needed. These employees are referred to as non-traditional students and are the subject of this study. Research shows that trying to manage both work and school simultaneously can cause psychological strain for non-traditional students (Adebayo et al., 2008). The psychosocial safety climate (PSC) is an emerging construct which refers to the shared perceptions regarding policies, practices and procedures designed to protect the psychological health of employees (Dollard et al., 2012). This study builds on existing research in the work - school interface by investigating its application within the South African context. Participants in this research (n=127), comprised of non-traditional students (n=40) and employees who are not engaged in further studies (n=87). Correlation analysis demonstrated that PSC had a positive relationship with work school facilitation (WSF) and a negative relationship with work school conflict (WSC). It was further confirmed that job control (JC) mediated the relationship between PSC and WSF and that PSC mediated the relationship between JC and WSF. These findings show that PSC is an antecedent to the work school interface in that it promotes the positive outcomes of studying while working (work school facilitation). It further demonstrates that PSC also reduces the negative outcomes (work school conflict). This study confirmed that the working environment plays a crucial role in the work school interface and introduces PSC as a construct South African organisations should be concerned with and make a priority, based on the resources it provides employees, more especially their non-traditional students. This study's findings will add to the existing body of research and provide practical insights for enhancing the PSC application within South African organisations who have non-traditional students.
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Nurse Perceptions: The Relationship Between Patient Safety Culture, Error Reporting and Patient Safety in U.S. HospitalsHyatt, Rick D. 15 December 2020 (has links)
No description available.
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Designing a Novel Prototype for Efficient Blood Sampling and Storage: An Experimental Study on Plasma Separation CardsLundgren, Philip, Ghebreyesus, Adam January 2024 (has links)
The aim of this study is to optimize the storage and shipping processes of Plasma Separation Cards (PSC) by developing a novel prototype designed to reduce manual labore and ensure sample integrity. An experimental design was employed to address current limitations in PSC handling. These limitations include labor-intensive processes and potential sample degradation. In this approach rigorous testing was done to evaluate the prototype's performance in various environments and different materials. The research in this study include lab testing of samples stored in the prototype, assessment of different storage conditions and analysis of shipping processes. The main research question was: “How can a prototype for PSC storage be optimized to reduce manual labor in PSC handling while ensuring sample integrity during storage and shipping?”. To answer this question the prototype was tested for its effectiveness in reducing storage space and facilitating automated handling The findings of this study show that the prototype that was created can significantly reduce the required storage space while still being usable by liquid handling robots. During the study it was noted that minor misalignment issues were present due to the limitations of the type of 3D printers used. These issues could be addressed with more precise printing technologies such as Stereolithography (SLA) or Selective Laser Sintering (SLS). Both materials used in this study, PLA (Polylactic acid) and PETG (Polyethylene Terephthalate Glycol) materials were found suitable for storing PSC samples under most conditions. However, the measurements for the samples with PLA indicated limitations at 65 °C under certain conditions, making PETG a safer choice under higher temperature. The study contributes to the field by providing an innovating solution for PSC sampling that is scalable. This enhances the possibility for remote monitoring end predictive diagnostics. The prototype indicates a promising direction for future research and application and medical diagnostics by improving operational efficiency while aligning with sustainability goals. This study explores the following aspects: sample handling, storage and shipping. The practical implication includes reduced manual labor, minimized human error and increased efficiency for these aspects.
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Development and exploitation of MAGIC experimental populations in eggplant and tomato genetics and breedingArrones Olmo, Andrea 02 September 2024 (has links)
Tesis por compendio / [ES] La familia Solanaceae incluye cultivos de gran importancia económica mundial como la berenjena (Solanum melongena L.) y el tomate (S. lycopersicum L.). Aunque ambos cultivos muestran tendencias positivas en producción, rendimiento y consumo, actualmente se encuentran bajo la amenaza constante de múltiples estreses abióticos y bióticos, debido al estrechamiento y uniformidad genética de las variedades comerciales modernas de alto rendimiento. Como consecuencia de los procesos de domesticación y de los programas de mejora, habitualmente se ha producido una importante pérdida de variabilidad genética en las especies cultivadas, lo que conlleva un aumento del riesgo de pérdidas de rendimiento frente a las amenazas y una disminución de las fuentes de variabilidad para la mejora del cultivo.
Por ello, en la presente tesis doctoral, se propone el aprovechamiento del potencial oculto de los parentales silvestres mediante la construcción de poblaciones experimentales interespecíficas Multi-parent Advanced Generation InterCross (MAGIC) que combinen germoplasma cultivado, semidomesticado y silvestre para contribuir en el rescate de parte de la variabilidad perdida en berenjena y tomate y la identificación de genes candidatos y polimorfismos causales que controlen caracteres cuantitativos de interés.
En el primer capítulo de esta tesis, nos propusimos presentar la primera población MAGIC de berenjena desarrollada hasta el momento, a partir de siete accesiones de berenjena común de diferentes orígenes y una accesión de la especie silvestre S. incanum. Los ocho parentales utilizados para la construcción de esta población mostraron una amplia diversidad genética según los datos de resecuenciación genómica, fenotípica, de fruto, agronómica y de caracteres de resistencia al estrés. Esta población interespecífica fue fenotipada para diferentes caracteres y genotipada mediante la plataforma de Single Primer Enrichment Technology (SPET). Los resultados se utilizaron para la elucidación de los genes responsables de los diferentes patrones de pigmentación del fruto, incluyendo la biosíntesis de antocianinas y clorofilas uniformes, y la malla verde irregular del fruto. Comprender los mecanismos por los que se sintetizan los pigmentos de los frutos podría ser de gran utilidad para impulsar futuros programas de mejora centrados no sólo en la demanda específica de diversificación de frutos por parte de los consumidores, sino también en el desarrollo de nuevas variedades que presenten una mayor calidad nutricional y resistencia para hacer frente a los retos medioambientales que se avecinan. Esto es posible gracias a la relación de los pigmentos con las propiedades antioxidantes, la fotosíntesis y la biología del estrés de las plantas, entre otras.
En el segundo capítulo de esta tesis, se abordó el desarrollo de una población MAGIC de tomate utilizando cuatro accesiones de la especie silvestre más cercana semidomesticada S. lycopersicum var. cerasiforme, y cuatro accesiones de S. pimpinellifolium, el ancestro del tomate cultivado. Estas especies se han utilizado poco en la mejora genética del tomate y no se ha explotado todo su potencial a pesar de su gran diversidad genética, su amplia variabilidad climática y ecológica y su proximidad al tomate cultivado. Las poblaciones MAGIC anteriores se desarrollaron combinando accesiones de tomate común de tipo cherry y de fruto más grande. Sin embargo, los resultados indican que estos ocho parentales no representan la variabilidad genética andina de S. l. var. cerasiforme perdida durante el proceso de domesticación y, por lo tanto, la población propuesta es complementaria a la ya desarrollada. La población interespecífica final también se genotipó mediante un panel SPET y se realizó una prueba de concepto para comprobar el potencial de la población fenotipándola para tamaño de fruto, pigmentación de la planta, morfología de la hoja y precocidad. / [CA] La família Solanaceae inclou cultius de gran importància econòmica mundial com l'albergina (Solanum melongena L.) i la tomaca (S. lycopersicum L.). Encara que tots dos cultius mostren tendències positives en producció, rendiment i consum, actualment es troben sota l'amenaça constant de múltiples estressos abiòtics i biòtics, a causa de l'estrenyiment i uniformitat genètica de les varietats comercials modernes d'alt rendiment. A conseqüència dels processos de domesticació i dels programes de millora, habitualment s'ha produït una important pèrdua de variabilitat genètica en les espècies cultivades, la qual cosa comporta un augment del risc de pèrdues de rendiment enfront de les amenaces i una disminució de les fonts de variabilitat per a la millora del cultiu.
Per això, en la present tesi doctoral, es proposa l'aprofitament del potencial ocult dels parentals silvestres mitjançant la construcció de poblacions experimentals interespecífiques Multi-parent Advanced Generation InterCross (MAGIC) que combinen germoplasma cultivat, semidomesticat i silvestre per a contribuir en el rescat de part de la variabilitat perduda en albergina i tomaca i la identificació de gens candidats i polimorfismes causals que controlen caràcters quantitatius d'interès.
En el primer capítol d'aquesta tesi, ens vam proposar presentar la primera població MAGIC d'albergina desenvolupada fins ara, a partir de set accessions d'albergínia comuna de diferents orígens i una accessió de l'espècie silvestre S. incanum. Els huit parentals utilitzats per a la construcció d'aquesta població van mostrar una àmplia diversitat genètica segons les dades de reseqüenciació genòmica, fenotípica, de fruit, agronòmica i de caràcters de resistència a l'estrès. Aquesta població interespecífica va ser fenotipada per a diferents caràcters i genotipada mitjançant la plataforma de Single Primer Enrichment Technology (SPET). Els resultats es van utilitzar per a l'elucidació dels gens responsables dels diferents patrons de pigmentació del fruit, incloent-hi la biosíntesi d'antocianines i clorofil·les uniformes, i la malla verda irregular del fruit. Comprendre els mecanismes pels quals se sintetitzen els pigments dels fruits podria ser de gran utilitat per a impulsar futurs programes de millora centrats no sols en la demanda específica de diversificació de fruits per part dels consumidors, sinó també en el desenvolupament de noves varietats que presenten una major qualitat nutricional i resistència per a fer front als reptes mediambientals que s'aveïnen. Això és possible gràcies a la relació dels pigments amb les propietats antioxidants, la fotosíntesi i la biologia de l'estrès de les plantes, entre altres.
En el segon capítol d'aquesta tesi, es va abordar el desenvolupament d'una població MAGIC de tomaca utilitzant quatre accessions de l'espècie silvestre més pròxima semidomesticada S. lycopersicum var. cerasiforme, i quatre accessions de S. pimpinellifolium, l'ancestre de la tomaca cultivada. Aquestes espècies s'han utilitzat poc en la millora genètica de la tomaca i no s'ha explotat tot el seu potencial malgrat la seua gran diversitat genètica, la seua àmplia variabilitat climàtica i ecològica i la seua proximitat a la tomaca cultivada. Les poblacions MAGIC anteriors es van desenvolupar combinant accessions de tomaca comuna de tipus cherry i de fruit més gran. No obstant això, els resultats indiquen que aquests huit parentals no representen la variabilitat genètica andina de S. l. var. cerasiforme perduda durant el procés de domesticació i, per tant, la població proposada és complementària a la ja desenvolupada. La població interespecífica final també es va genotipar mitjançant un panell SPET i es va realitzar una prova de concepte per a comprovar el potencial de la població fenotipant-la per a grandària de fruit, pigmentació de la planta, morfologia de la fulla i precocitat. / [EN] The Solanaceae family includes crops of global economic importance such as eggplant (Solanum melongena L.) and tomato (S. lycopersicum L.). Although both crops show positive trends in production, yield, and consumption, they are currently under constant threat from multiple abiotic and biotic stresses, due to the genetic narrowing and uniformity of modern high-yielding commercial varieties. As a consequence of domestication processes and plant breeding programs, usually there has been a significant loss of genetic variability in cultivated species, leading to an increased risk of yield losses when facing threats and a decrease in sources of variability for crop improvement.
Therefore, in the present doctoral thesis, it is proposed the leverage of the hidden potential of the wild relatives by constructing inter-specific Multi-parent Advanced Generation InterCross (MAGIC) experimental populations combining cultivated, semi-domesticated, and wild germplasm to contribute to rescuing some of the lost variability in eggplant and tomato and the identification of candidate genes and causative polymorphisms controlling interesting quantitative traits.
In the first chapter of this thesis, we aimed to present the first eggplant MAGIC population so far, developed from seven accessions of common eggplant from different origins and one wild species S. incanum accession. The eight founders used for the construction of this population showed a wide genetic diversity according to genome resequencing data, phenotypic, fruit, agronomic and stress resistance traits. This inter-specific population was phenotyped for different traits and genotyped through the single primer enriched technology (SPET) platform. Results were used for the elucidation of the genes responsible for different fruit pigmentation patterns including the biosynthesis of uniform anthocyanins and chlorophylls, and the irregular fruit green netting. Understanding the mechanisms by which fruit pigments are synthesized could be extremely useful to foster future breeding programs focused not only on specific consumers' demand for fruit diversification but also on the development of novel varieties exhibiting enhanced nutritional quality and resilience to tackle the forthcoming environmental challenges. This could be possible thanks to the relationship of pigments with antioxidant properties, photosynthesis, and the stress biology of plants, among others.
In the second chapter of this thesis, we addressed the development of a tomato MAGIC population using four accessions of the semi-domesticated closest wild relative S. lycopersicum var. cerasiforme, and four accessions of the S. pimpinellifolium, which is the ancestor of the cultivated tomato. These species have been little used in breeding and their full potential has not been exploited despite their high genetic diversity, their wide climatic and ecological variability and their closeness to the cultivated tomato. Previous MAGIC populations have been developed combining cherry type and larger fruit accessions of common tomato. However, the results indicate that these eight founders do not represent the Andean genetic variability of S. l. var. cerasiforme lost during the domestication process and therefore, the proposed population is complementary to that already developed. The final inter-specific population was also genotyped through a SPET panel and a proof-of-concept for testing the potential of the population was conducted by phenotyping it for fruit size, plant pigmentation, leaf morphology, and earliness traits. / Arrones Olmo, A. (2024). Development and exploitation of MAGIC experimental populations in eggplant and tomato genetics and breeding [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/207347 / Compendio
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