602 research outputs found
The KINGS mouse as a model of beta cell endoplasmic reticulum (ER) stress and sex differences in diabetes.
Background: The KINGS mouse is a novel model of beta cell endoplasmic reticulum (ER) stress which shows stark sex differences in diabetes, with males developing overt and progressive hyperglycaemia whilst females are protected. Beta cell ER stress has been implicated in many types of diabetes and underpins numerous factors known to drive beta cell failure. Sex differences also exist in diabetes in humans with premenopausal women having a lower diabetes incidence compared to men. Further characterisation of the KINGS mice may provide valuable insight into these phenomena. Aims: The objectives of this thesis were to 1) further characterise beta cell ER stress and associated cellular response in the KINGS mice, 2) investigate the influence of sex hormones and beta cell ER stress manipulation on glycaemic control in the KINGS mice and 3) investigate whether diabetes development can be prevented in the male KINGS mice. Methods: Western blotting and immunofluorescent staining were used to investigate the expression of ER stress and unfolded protein response (UPR) markers in KINGS islets, as well as beta cell turnover and mass. To determine the influence of oestradiol on the KINGS phenotype, endogenous oestradiol was removed from female mice via ovariectomy, and exogenous oestradiol was delivered to male KINGS mice through implantation of oestradiol- containing capsules. A western diet was used to exacerbate beta cell ER stress in female KINGS mice, whilst liraglutide administration, TUDCA administration and removal of endogenous testosterone (via orchidectomy) was used in an attempt to reduce ER stress and prevent diabetes in the male KINGS mice. For all in vivo studies, glycaemic control was assessed through blood glucose concentration monitoring, glucose tolerance testing and insulin tolerance testing. Results: Male KINGS mice developed diabetes by 5-6 weeks of age whereas female KINGS mice were protected, in line with previous studies. Protein markers of ER stress and the UPR were observed in KINGS islets from 4 weeks of age and a sex difference was observed in expression profiles with males largely showing an increased expression of markers. Despite this, we did not observe a loss of beta cell mass in either male or female KINGS mice. However, subtle changes in beta cell proliferation and apoptosis in the male KINGS mice are suggestive of mild changes to beta cell turnover which may contribute to diabetes development. A western diet exacerbated beta cell ER stress in female KINGS mice, however this only led to a mild impairment in glycaemic control which was not as severe as that seen in male KINGS mice. This may suggest that even under conditions of further ER stress, female mice are still able to respond adaptively. Removal of endogenous oestradiol also exacerbated beta cell ER stress, however again this was only associated with a subtle impairment in glycaemic control. On the contrary, exogenous oestradiol delivery in the male KINGS mice prevented the development of overt diabetes. Treatment with liraglutide was used in an attempt to alleviate ER stress in the male KINGS mice. Although liraglutide prevented the development of diabetes and reduced blood glucose concentrations once diabetes was established, this protection only lasted during the treatment window and cessation of treatment was associated with increases in blood glucose concentrations. In addition, liraglutide had no effect on beta cell ER stress levels. Treatment with TUDCA, a chemical chaperone previously found to reduce beta cell ER stress, had no impact on blood glucose concentrations in the KINGS mice. However, removal of endogenous testosterone through orchidectomy prevented the development of overt diabetes. Conclusion: In this study we have confirmed that the KINGS mutation drives beta cell ER stress and that sex differences exist in beta cell response to this. Interestingly, an adaptive response to beta cell ER stress was still maintained in female KINGS mice when ER stress was exacerbated through a western diet. We also found that whilst oestradiol likely contributes in-part to sex differences in diabetes, it cannot be the sole mediator and other factors must be involved. Indeed, we found that endogenous testosterone removal prevented the development of diabetes in male mice. Liraglutide treatment also prevented diabetes development in male mice, however this was likely to be mediated through mechanisms unrelated to beta cell ER stress. Further study is required to investigate how testosterone removal and liraglutide protect male mice.</div
Investigation of the metabolism of rare nucleotides in plants
Nucleotides are metabolites involved in primary metabolism, and specialized
metabolism and have a regulatory role in various biochemical reactions in all forms of life. While in other organisms, the nucleotide metabolome was characterized
extensively, comparatively little is known about the cellular concentrations of
nucleotides in plants. The aim of this dissertation was to investigate the nucleotide metabolome and enzymes influencing the composition and quantities of nucleotides in plants. For this purpose, a method for the analysis of nucleotides and nucleosides in plants and algae was developed (Chapter 2.1), which comprises efficient quenching of enzymatic
activity, liquid-liquid extraction and solid phase extraction employing a weak-anionexchange resin. This method allowed the analysis of the nucleotide metabolome of plants in great depth including the quantification of low abundant deoxyribonucleotides and deoxyribonucleosides. The details of the method were summarized in an article, serving as a laboratory protocol (Chapter 2.2).
Furthermore, we contributed a review article (Chapter 2.3) that summarizes the
literature about nucleotide analysis and recent technological advances with a focus on plants and factors influencing and hindering the analysis of nucleotides in plants, i.e., a complex metabolic matrix, highly stable phosphatases and physicochemical
properties of nucleotides. To analyze the sub-cellular concentrations of metabolites, a protocol for the rapid isolation of highly pure mitochondria utilizing affinity chromatography was developed (Chapter 2.4).
The method for the purification of nucleotides furthermore contributed to the
comprehensive analysis of the nucleotide metabolome in germinating seeds and in
establishing seedlings of A. thaliana, with a focus on genes involved in the synthesis of thymidilates (Chapter 2.5) and the characterization of a novel enzyme of purine nucleotide degradation, the XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Chapter 2.6). Protein homology analysis comparing A. thaliana, S. cerevisiae, and H. sapiens led to the identification and characterization of an enzyme involved in the metabolite damage repair system of plants, the INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Chapter 2.7). It was shown that this enzyme dephosphorylates deaminated purine nucleotide triphosphates and thus prevents their incorporation into nucleic acids. Lossof-function mutants senesce early and have a constitutively increased content of salicylic acid. Also, the source of deaminated purine nucleotides in plants was investigated and it was shown that abiotic factors contribute to nucleotide damage.Nukleotide sind Metaboliten, die am Primärstoffwechsel und an spezialisierten
Stoffwechselvorgängen beteiligt sind und eine regulierende Rolle bei verschiedenen
biochemischen Reaktionen in allen Lebensformen spielen. Während bei anderen
Organismen das Nukleotidmetabolom umfassend charakterisiert wurde, ist in Pflanzen
vergleichsweise wenig über die zellulären Konzentrationen von Nukleotiden bekannt.
Ziel dieser Dissertation war es, das Nukleotidmetabolom und die Enzyme zu
untersuchen, die die Zusammensetzung und Menge der Nukleotide in Pflanzen
beeinflussen. Zu diesem Zweck wurde eine Methode zur Analyse von Nukleotiden und
Nukleosiden in Pflanzen und Algen entwickelt (Kapitel 2.1), die ein effizientes Stoppen
enzymatischer Aktivität, eine Flüssig-Flüssig-Extraktion und eine
Festphasenextraktion unter Verwendung eines schwachen Ionenaustauschers
umfasst. Mit dieser Methode konnte das Nukleotidmetabolom von Pflanzen eingehend
analysiert werden, einschließlich der Quantifizierung von Desoxyribonukleotiden und
Desoxyribonukleosiden mit geringer Abundanz. Die Einzelheiten der Methode wurden
in einem Artikel zusammengefasst, der als Laborprotokoll dient (Kapitel 2.2).
Darüber hinaus wurde ein Übersichtsartikel (Kapitel 2.3) verfasst, der die Literatur
über die Analyse von Nukleotiden und die jüngsten technologischen Fortschritte
zusammenfasst. Der Schwerpunkt lag hierbei auf Pflanzen und Faktoren, die die
Analyse von Nukleotiden in Pflanzen beeinflussen oder behindern, d. h. eine komplexe
Matrix, hochstabile Phosphatasen und physikalisch-chemische Eigenschaften von
Nukleotiden.
Um die subzellulären Konzentrationen von Metaboliten zu analysieren, wurde ein
Protokoll für die schnelle Isolierung hochreiner Mitochondrien unter Verwendung einer
Affinitätschromatographie entwickelt (Kapitel 2.4).
Die Methode zur Analyse von Nukleotiden trug außerdem zu einer umfassenden
Analyse des Nukleotidmetaboloms in keimenden Samen und in sich etablierenden
Keimlingen von A. thaliana bei, wobei der Schwerpunkt auf Genen lag, die an der
Synthese von Thymidilaten beteiligt sind (Kapitel 2.5), sowie zu der Charakterisierung
eines neuen Enzyms des Purinnukleotidabbaus, der XANTHOSINE
MONOPHOSPHATE PHOSPHATASE (Kapitel 2.6). Eine Proteinhomologieanalyse, die A. thaliana, S. cerevisiae und H. sapiens
miteinander verglich führte zur Identifizierung und Charakterisierung eines Enzyms,
das an der Reparatur von geschädigten Metaboliten in Pflanzen beteiligt ist, der
INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Kapitel 2.7). Es konnte gezeigt
werden, dass dieses Enzym desaminierte Purinnukleotidtriphosphate
dephosphoryliert und so deren Einbau in Nukleinsäuren verhindert.
Funktionsverlustmutanten altern früh und weisen einen konstitutiv erhöhten Gehalt an Salicylsäure auf. Außerdem wurde die Quelle der desaminierten Purinnukleotide in Pflanzen untersucht, und es wurde gezeigt, dass abiotische Faktoren zur
Nukleotidschädigung beitragen
Evaluation of mixed microalgae species biorefinery of Desmodesmus sp. And Scenedesmus sp. For bioproducts synthesis
Microalgae is known to produce numerous bioactive compounds for instance proteins, fatty acid, polysaccharides, enzymes, sterols, and antioxidants. Due to their valuable biochemical composition, microalgae are regarded as a very intriguing source to produce novel food products and can be utilised to improve the nutritional content of traditional foods. Additionally, microalgae are used as animal feed and additives in the cosmetics, pharmaceutical as well as nutraceutical industries. As compared to other terrestrial plants and other microorganisms, microalgae possess few advantages: (1) rapid growth rate; (2) able to grow in non-arable land and harsh cultivation conditions; (3) low nutritional requirements; (4) high productivity; and (5) reduce emission of carbon dioxide. Despite the large number of microalgae species found in nature, only a few species are identified and commercialized such as Chlorella sp., Spirulina sp. Haematococcus pluvialis, Nannochloropsis sp. and Chlamydomonas reinhardtii, which is one of the major obstacles preventing the full utilisation of microalgae-based technology.
This thesis provides information on the overall composition of mixed microalgae species, Desmodesmus sp. and Scenedesmus sp., for instance protein, carbohydrate, lipid, antioxidants, and pigment. This thesis firstly introduces the application of triphasic partitioning (TPP) in the extraction and partitioning of the biomolecules from the microalgae. The latest advancement of technology has evolved from a liquid biphasic flotation (LBF) to TPP. T-butanol and ammonium sulphate are used in TPP to precipitate desired biomolecules from the aqueous solutions with the formation of three layer. TPP is a simple, time- and cost- efficient, as well as scalable process that does not require toxic organic solvents. Lipase is abundantly produced by microbes, bacteria, fungi, yeast, mammals, and plants. Lipase is widely used in the oleochemical, detergent, dairy, leather, cosmetics, paper, cosmetics, and nutraceutical industries. Therefore, this thesis also discusses the possibility of identifying and extracting enzyme lipase from the microalgae using LBF. Several parameters (volume and concentration of solvents, weight of biomass, flotation kinetics and solvent types, etc.) have been investigated to optimize the lipase extraction from LBF.
Chlorophyll is the main pigment present in the microalgae. Thus, this work proposes the digital imaging approach to determine the chlorophyll concentration in the microalgae rapidly because the chlorophyll content has a significant impact on microalgae physiological health status as well as identifies the chlorophyll concentration in the production of by-products. Lastly, microalgae oil can be used as the feedstock for biodiesel as well as nutraceutical, pharmaceutical, and health-care products. The challenge in the lipid extraction is the co-extraction of chlorophyll into the oil, which can have serious consequences for downstream processing. Therefore, the removal of the chlorophyll from the microalgae using activated clay or sodium chlorite in the pre-treatment procedure are examined. The research achievements in these works and future opportunities are highlighted in the last chapter of the thesis
Biomaterials for Bone Tissue Engineering 2020
This book presents recent advances in the field of bone tissue engineering, including molecular insights, innovative biomaterials with regenerative properties (e.g., osteoinduction and osteoconduction), and physical stimuli to enhance bone regeneration
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