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The goal of this research was to design, prepare and study a new class of supermolecules coupling ruthenium and platinum, which would display covalent binding to DNA. Drawing upon the well-established efficacy of cis-diamminedichloroplatinum(II) (cisplatin) and the DNA-binding properties of select ruthenium polyazine complexes, the approach was to bind the cis-PtIICl2 active site of cisplatin to ruthenium light absorbers using the dpq and dpb bridging ligands (where dpq = 2,3-bis(2-pyridyl)quinoxaline, dpb = 2,3-bis(2-pyridyl) benzoquinoxaline). These complexes are potentially bifunctional, capable of DNA intercalation through the bridging ligand and covalent binding to DNA through the cis-PtCl2 site. Synthetic methods were developed to prepare the mixed-metal, bimetallic complexes [(bpy)2Ru(BL)PtCl2](CF3SO3)2 and [(phen)2Ru(BL)PtCl2](CF3SO3)2 (where bpy = 2,2¢-bipyridine, phen = 1,10-phenanthroline) in high purity and good overall yields. The DNA-binding ability of these complexes was probed by reaction with linearized plasmid DNA and subsequent analysis by native and denaturing gel electrophoresis. The known DNA binders, cisplatin and trans-{[PtCl(NH3)2]2(m-H2N(CH2)6NH2)}(NO3)2 (1,1/t,t), were examined under equivalent conditions and used as positive controls. Native gel electrophoresis was used to show that these complexes strongly bind DNA, retarding the migration of DNA through the gel in a fashion inversely proportional to the ratio of DNA base pairs (bp) to metal complex (mc). Analysis by denaturing gel electrophoresis determined that the Ru-Pt complexes bind to DNA in a fashion similar to cisplatin, forming primarily intrastrand adducts. However, these systems also appear to form interstrand adducts at a 10-fold lower metal concentration than cisplatin. In addition to affecting the migration rate, the bimetallic complexes also significantly reduced the fluorescence of DNA-intercalated ethidium bromide for the Ru-Pt reacted samples at low-DNA bp: mc ratios. This was not observed for the cisplatin and 1,1/t,t treated samples. This observation was quantitated by gel densitometry. Precipitation of the DNA by cisplatin, 1,1/t,t and all four Ru-Pt complexes was determined not to be the cause of reduced ethidium bromide fluorescence intensity. Homogenous solution fluorescence quenching studies have revealed that the Ru-Pt complexes quench the emission of ethidium bromide even in the absence of DNA, whereas cisplatin and 1,1/t,t do not. In order to compare the effects on DNA migration produced by cisplatin, 1,1/t,t and the Ru-Pt complexes, Rf values were calculated. This analysis has revealed that all four Ru-Pt complexes retard DNA migration to approximately the same degree. Calculation of theoretical DNA migration distances, based upon the molecular weight change of DNA caused by metal-complex binding, have revealed that the observed affect on DNA migration cannot be accounted for by an increase in molecular weight alone. This indicates that changes in charge and three-dimensional shape of the DNA upon binding of the Ru-Pt complexes may also contribute. / Ph. D.

La maladie sporadique de Creutzfeldt-Jakob (sCJD) est une encéphalopathie spongiforme transmissible mortelle caractérisée par une large gamme de manifestations cliniques et pathologiques. Les caractéristiques pathologiques de la SDMC dépendent en grande partie de la présence d'une forme mal repliée de protéine prion cellulaire (PrPC); Connu sous le nom de PrPSC et polymorphisme (méthionine et valine) au codon 129 du gène PRNP qui code pour PrPC. Les facteurs étiologiques exacts de la MCSJ sont encore inconnus. Le génotype Codon 129 du gène PRNP et le type de PrPSC (type 1 ou type 2) influent sur l'hétérogénéité de la maladie telle que définie par des caractéristiques pathologiques spécifiques de la région qui peuvent réguler les voies moléculaires qui conduisent au développement de phénotypes pathogènes dépendants des sous-types. Dans cette étude, nous avons étudié l'ensemble de la réglementation protéomique dans la région cerebrale du cerveau des deux sous-types les plus répandus (MM1 et VV2) des patients atteints de SDMC utilisant une électrophorèse sur gel bidimensionnelle (2DE) et une spectrométrie de masse. L'analyse de toutes les taches de protéines sur les gels 2DE avec le logiciel DECODON Delta2D a révélé vingt-cinq taches de protéines différenciées et l'identification de ces taches avec MALDI-TOF MS / MS a révélé quatre-vingts trois protéines différentiellement réglementées dans les deux sous-types dans la région du cervebelle du cerveau par les patients atteints de sCJD. Quarante protéines dans le sous-type MM1 et quarante-trois protéines dans le sous-type VV2 ont été classées. Douze protéines étaient communément réglementées dans les deux sous-types, dont cinq d'entre elles présentaient une régulation expresnelle inverse et le repos sept avait une régulation expresnelle similaire dans les deux sous-types. Les trois principaux mécanismes moléculaires cellulaires réglementés dans les deux sous-types comprennent: i) le cycle cellulaire; L'expression des gènes et la mort cellulaire, ii) - la réponse au stress cellulaire / le stress oxydatif et iii) - la transduction du signal et les fonctions synaptiques La plupart des protéines sous la classification des réponses au stress cellulaire étaient associées aux fonctions moléculaires cellulaires liées au stress oxydatif. DJ-1 qui est un capteur bien connu de stress oxydatif, a également été jugé réglementé dans la catégorie des réponses au stress cellulaire. Le DJ-1 protège les cellules contre le stress oxydatif directement en translocant au noyau pour l'activation de gènes antioxydants et indirectement en activant la voie Nrf2 / ARE. Nos résultats expérimentaux ont démontré l'activation de la voie Nrf2 / ARE dans les sous-types MM1 et VV2 de sCJD. Le DJ-1 a également montré une régulation positive significative dans son expression de l'ARNm dans les sous-types MM1 et VV2 mais l'expression des protéines uniquement dans le sous-type VV2 dans le cervelet du cerveau des patients atteints de sCJD. En outre, l'expression de la protéine DJ-1 a également été augmentée au cours des stades pré-symptomatiques et symptomatiques dans le cervelet du cerveau des modèles de souris de sCJD (MM1 et VV2) et pendant le stade clinique dans les échantillons de CSF des patients atteints de SDMC. Ces résultats suggèrent l'implication du stress oxydatif lors de la pathophysiologie de la SDMC et l'utilisation de DJ-1 comme capteur potentiel de stress oxydatif pendant la phase clinique de la sCJD.

This diploma thesis is focused on studies of changing of protein profile during barley malting. Substantial part of this work is devoted to the proteomics identification of barley proteins which change during malting and so become more stationary and they influence quality of beer (haze and foam in beer). For this experiment was used barley variety Jersey. In the theoretical part of this thesis there is information about beer, manufacturing of beer with description of important commodities for manufacturing of beer and information about barley malting and information about malting process. Next there is description of methods for separation of proteins (1D gel electrophoresis and 2D gel electrophoresis), MALDI TOF/TOF mass spectrometry and this use for the analysis and identification of proteins, the use of matrices and ways of the sample preparation. In the experimental part of this thesis there was carried out the optimisation of the dosage of sample for 1D gel electrophoresis and the optimisation of staining. The 15 % TRIS-HCl gel was the best, this gel was stained by Commassie Brilliant Blue G-250. For illustration of changes was made 2D gel electrophoresis. With help of method peptide mass fingerprinting and MS/MS protein of barley – protein Z, -amylase subtilisin inhibitor, -amylase a peroxidase were identificated. The analysis of barley extract intact proteins was carried out, this analysis was focused on changes of important barley protein LTP 1.

Assignment (MMed)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Lipoprotein X (LpX) is an abnormal cholesterol-containing particle that may be present in the serum of subjects with cholestasis, lecithin:cholesterol acyltransferase (LCAT) deficiency and parenteral nutrition. The biochemistry, metabolism, clinical significance and laboratory analysis of LpX is discussed in this study. This laboratory-based project investigated icteric samples received at the Chemical Pathology laboratory, Tygerberg Hospital, for serum predictors of LpX and the use of a modified non-denaturing polyacrylamide gradient gel electrophoresis system in the detection of LpX. The study showed that the non-denaturing polyacrylamide gradient gel electrophoresis system (2-8%) is a useful test in demonstrating LpX in icteric plasma and has potential for a screening test in LCAT deficiency. Serum concentration of conjugated bilirubin, alkaline phosphatase, gamma glutamyltransferase, free cholesterol, phospholipid, free cholesterol: total cholesterol ratio and conjugated bilirubin: total bilirubin ratio are all good predictors of LpX. The ratio of free cholesterol to total cholesterol (FC/TC > 0.6) was the best predictor of LpX. In the setting of obstructive liver disease LpX is seen in 66% of patients if total cholesterol is > 7.5 mmol/L. / AFRIKAANSE OPSOMMING: Lipoproteien X (LpX) is ‘n abnormale cholesterol-bevattende partikel wat teenwoordig mag wees in die serum van persone met cholestase, lesitien:cholesterol asieltransferase (LCAT) gebrek en parenterale voeding. Die biochemie, metabolisme, kliniese belang en laboratorium analise van LpX word bespreek in hierdie werkstuk. Hierdie laboratorium-gebaseerde projek het geelsugtige monsters ondersoek wat ontvang is by die Chemiese Patologie laboratorium, Tygerberg Hospitaal, vir serum voorspellers van LpX en die gebruik van ‘n gemodifiseerde nie-denaturerende polie-akrielamied gradiënt gel elektroforese sisteem in die demonstrasie van LpX. Die bevindinge was dat die nie-denaturerende polie-akrielamied gradient gel elektroforese sisteem (2-8%) is ‘n nuttige toets om LpX te demonstreer in geelsugtige plasma en het potensiaal as ‘n siftingstoets in LCAT gebrek. Serum konsentrasie van gekonjugeerde bilirubien, alkaliese fosfatase, gamma glutamieltransferase, vry cholesterol, fosfolipied, vry cholesterol:totale cholesterol verhouding en gekonjugeerde bilirubien:totale bilirubien verhouding is alles goeie voorspellers van LpX. Die verhouding van vry cholesterol tot totale cholesterol (VC/TC > 0.6) was die beste voorspeller van LpX. In gevalle van obstruktiewe lewersiekte word LpX gesien in 66% van pasiente as die totale cholesterol meer as 7.5 mmol/l is.

<p>Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.</p>

Cyanine dyes that absorb light in the near infrared to far red region of the electromagnetic spectrum are desirable as photosensitizers for photodynamic cancer therapy. Light of wavelengths in this range is able to deeply penetrate tissue allowing for practical in vivo use of these dyes. A series of three structurally similar pentamethine cyanine dyes that absorb light ~800 nm to ~500 nm was tested to determine the effects of structural influences on the yields of supercoiled DNA photo-converted to nicked or linear forms. Possible mechanisms and optimal parameters for near- quantitative DNA photocleavage with a symmetrical quinoline pentamethine cyanine dye are discussed.

M.Sc. (Zoology) / Please refer to full text to view abstract

The fundamental challenge to prevent species from going extinct is difficult but of grave importance. Halting species from going extinct minimizes the loss of biodiversity. One way of researching biodiversity is by studying species on a genetic level. This creates a dilemma as studying species genetically often requires using destructive sampling and is not desirable or even allowed when studying threatened species. Thus, there is a necessity for alternate sampling methods. In this study both non-lethal and lethal methods were used to gather tissues from the butterfly species Vanessa cardui. The DNA extractions turned out to give varying amounts of DNA, but it was successfully extracted from all the different tissue types. Amplifiable DNA was successfully gained using PCR and confirmed using gel electrophoresis. Existing and newly designed primers for multi-copy genes were used and several of them gave amplifiable DNA. Even if amplifiable DNA has been obtained in other studies, using various tissues, it turned out to only work with a live butterfly’s body in this study.

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