The anti-cancer agent 4, 4-dihydroxybenzophenone-2, 4- dinitrophenylhydrazone (A007) forms complexes with pdelocalized lymphangitic dyes that allow its penetration through the skin effectively delivering it to a meta-stable type cancerous site. Previous in vitro studies, combined with gas phase mass spectrometry studies, have shown that a stronger binding affinity equates to a greater efficacy of the drug. For the determination of drug:dye complex binding strength coefficients in solution, two methods have been developed by affinity capillary electrophoresis (ACE), and cation exchange liquid chromatography (CELC). The methods demonstrated that A007 non-covalent binding strength was greatest for methylene green, followed by methylene blue, and lastly toluidine blue. Bond dissociation energies and apparent reaction enthalpies for the fragmentation pathways of lithiated acylglycerols were experimentally determined by collision activation in a triple quadrupole mass spectrometer. A developed novel derived effective path length approach for predicting bond dissociation energies (BDE) for electrostatic complex's alkali metal adducts (Li+), and halide adducts (Cl-) of acylglycerols was applied to the major fragmentation product ions of a lithiated mono-acylglycerol, a 1, 2-diacylglycerol, and a 1, 3-diacylglycerol, to predict the covalent bond dissociation energies involved in fragmentation pathways. The model's calculated apparent reaction enthalpies are used in conjunction with the energy-resolved mass spectrometry method of breakdown graphs to give a more complete quantitative aspect to the interpretation of the fragmentation processes. The dry eye condition affects millions of individuals world wide. The symptoms can be a result of simple irritation to the eye or a serious disease state. A dry eye model was developed using rabbits in order to study the compositional makeup of the tear components in hopes of identifying an underlying cause, or expressed effect of the dry eye condition. The major non-polar lipids of the tear were identified by mass spectrometry as mono and diacylglycerols, with a smaller extent of triacylglycerols. The major polar phosphorylated lipids were identified in the tear extract revealing that sphingomyelin based species were being expressed in the dry eye condition. The major proteins were determined to be the lower molecular weight lipid binding proteins where two specific species were found to increase in expression for the dry eye condition.

Previous studies showed that memantine inhibits tau hyperphosphorylation in vitro. In this study, phosphorylated tau (P-tau) and total tau (T-tau) were measured before and after 6 month treatment with memantine in 12 subjects ranging from normal cognition with subjective memory complaints, through mild cognitive impairment to mild Alzheimer's disease. Thirteen non-treated individuals served as controls. Treatment was associated with a reduction of P-tau in subjects with normal cognition. No treatment effects were seen among impaired individuals, suggesting that longer treatment time may be necessary to achieve biomarker effect in this group.

Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-α (PFT-α), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 μM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-α followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-α also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-α interferes with multiple steps of Dox-induced apoptosis.

Zirconia is one of the most promising column packing materials for High Performance Liquid Chromatography (HPLC). The perfect HPLC support material should be energetically homogenous, have a high surface area on which different chemical species can reversibly attach and be physically and chemically stable over a wide range of pH, temperature and solvent conditions. Most existing supports do not have all of these properties. This project is also focused on a proteomics study. Zirconia, hafnium oxide and titanium oxide which are some of the more promising materials currently available, can be used for the separation and analysis of phosphorylated proteins. Adenosine triphosphate, Adenosine diphosphate and Adenosine monophosphate were used as prototypes for phosphorylated proteins. Separation, absorption, fluorescence and SEM studies were performed to determine the adsorption of Adenosine phosphates species at a particular pH on Zirconia. Zirconia was also used for the purification of Fibrinogen Growth Factor (FGF) protein, which are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique was used to analyze the off-column purification and separation of this protein. This research suggests that, at acidic conditions, adenosine monophosphate has more favorable absorption on the Zirconia surface. On the other hand, the separation study suggests that basic conditions are more favorable for the absorption of ATP, ADP and AMP when mixed together on Zirconia 500. Furthermore, it was found that Zirconia is a very promising material for the purification of FGF protein.

Οι σπειραματονεφρίτιδες (ΣΝ) αποτελούν μια ομάδα νοσημάτων του σπειράματος ανοσολογικής αιτιολογίας που σε πολλές περιπτώσεις οδηγούν στην πλήρη καταστροφή του νεφρικού παρεγχύματος με αποτέλεσμα τη νεφρική ίνωση και την ανάγκη υποκατάστασης της νεφρικής λειτουργίας με μεθόδους εξωνεφικής κάθαρσης. Τα τελευταία χρόνια έχει σημειωθεί σημαντική πρόοδος στην ανίχνευση των μοριακών μηχανισμών που οδηγούν στη νεφρική ίνωση. Κεντρικό ρόλο στη διαδικασία αυτή φαίνεται να διαδραματίζει το Smad σηματοδοτικό μονοπάτι που διαμεσολαβεί τα σήματα του TGF-β στα νεφρικά κύτταρα. Ο ρόλος του TGF-β στη νεφρική βλάβη είναι πλέον καλά τεκμηριωμένος. Αντίθετα ελάχιστα είναι γνωστά για την έκφραση και τη λειτουργία του Smad σηματοδοτικού μονοπατιού στις ανθρώπινες ΣΝ. Ο σκοπός της παρούσας μελέτης ήταν η διερεύνηση της έκφρασης των παραγόντων pSmad2/3, p300, Sp1, Smad7 και Ski (μορίων που συμμετέχουν στο Smad σηματοδοτικό μονοπάτι) σε νεφρικές βιοψίες ασθενών με ΣΝ και η ανίχνευση της δράσης τους όσον αφορά την παθογένεση και την εξέλιξη της νεφρικής βλάβης. Χρησιμοποιήθηκαν 157 βιοψίες ασθενών με ΣΝ και 15 φυσιολογικά νεφρικά δείγματα από ασθενείς που είχαν υποβληθεί σε νεφρεκτομή για καρκίνο του νεφρού. Οι σπειραματονεφρίτιδες κατηγοριοποιήθηκαν σε πρωτοπαθείς (n=91) και δευτεροπαθείς (n=66) ή υπερπλαστικές (n=86) και μη υπερπλαστικές (n=71). Η μελέτη της έκφρασης των pSmad2/3, p300, Sp1, Smad7 και Ski έγινε με τη χρήση ανοσοϊστοχημικής μεθόδου. Έγιναν συσχετίσεις με κλινικά δεδομένα των ασθενών (κρεατινίνη ορού και λεύκωμα ούρων 24ώρου), καθώς και δείκτες ιστολογικής βλάβης (σπειραματοσκλήρυνση, διάμεση ίνωση, σωληναριακή ατροφία και διάμεση φλεγμονή). Παρατηρήθηκε πολύ μεγάλη αύξηση της έκφρασης των pSmad2/3, Sp1 και p300 σε όλα τα σπειραματικά κύτταρα των ΣΝ σε σχέση με την ομάδα ελέγχου. Επίσης, αυξήθηκε η έκφραση του πυρηνικού Smad7 στις υπερπλαστικές κυρίως ΣΝ με ταυτόχρονη εξάλειψη της κυτταροπλασματικής έκφρασής του σε όλες τις ΣΝ και παράλληλη μείωση της έκφρασης του Ski. Όσον αφορά τα σωληνάρια, στα εγγύς παρατηρήθηκε αύξηση της έκφρασης των pSmad2/3, Sp1 και p300 σε όλες τις ΣΝ και αύξηση του πυρηνικού Smad7 στις υπερπλαστικές, ενώ στα άπω και τα αθροιστικά παρατηρήθηκε αύξηση της έκφρασης του pSmad2/3 και του πυρηνικού Smad7 με ταυτόχρονη μείωση του κυτταροπλασματικού Smad7. Το Ski παρουσίασε σημαντική μείωση της έκφρασής του στις ΣΝ σε όλους τους τύπους σωληναρίων. Η σπειραματική έκφραση των pSmad2/3, p300 και του πυρηνικού Smad7 ήταν αυξημένη στις υπερπλαστικές ΣΝ και κυρίως τις δευτεροπαθείς. Η σωληναριακή έκφραση των pSmad2/3 και Smad7 και η εγγύς σωληναριακή των p300, Sp1 και του κυτταροπλασματικού Smad7 ήταν επίσης αυξημένες στις υπερπλαστικές ΣΝ. Αντίθετα, το Ski δεν παρουσίασε διαφορές στην έκφραση μεταξύ των υπερπλαστικών και μη υπερπλαστικών ΣΝ. Ακόμη, ανιχνεύθηκαν σημαντικές συσχετίσεις μεταξύ μεταξύ της έκφρσης των pSmad2/3, p300, Sp1 και Smad7 στο σπείραμα και τα εγγύς σωληνάρια. Η σπειραματική και εγγύς σωληναριακή έκφραση των pSmad2/3, Sp1 και η σπειραματική του πυρηνικού Smad7 έδειξαν να συσχετίζονται θετικά με τα επίπεδα της κρεατινίνης του ορού, ενώ η έκφραση αυτών και του p300 σε διαφόρους τύπους σωληναρίων εμφάνισαν θετική στσχέτιση με δείκτες ιστολογικής βλάβης. Τέλος η έκφραση όλων των μορίων (εκτός του Ski) ήταν συχνότατη στις στοιχειώδεις σπειραματικές βλάβες. Συμπερασματικά, το Smad σηματοδοτικό μονοπάτι φαίνεται να ενεργοποιείται στις ανθρώπινες ΣΝ. Μάλιστα, η ενεργοποίησή του αυτή πιθανώς συμβάλλει στην παθογένεση αλλά και την εξέλιξη της νεφρικής βλάβης. Μελλοντικές θεραπευτικές στρατηγικές που θα στοχεύουν στην αναστολή του μονοπατιού αυτού ίσως συμβάλλουν στην παρεμπόδιση της εξέλιξης και τη θεραπεία των ανθρωπίνων ΣΝ. / -

The aim of this thesis was to investigate the prognostic role of the proliferation markers cyclin B1 and Phosphorylated Histone 3 (PPH3) in breast cancer (BC). In paper I we used an experimental study design, we compared women dying early from their BC with women free from relapse more than eight years after initial diagnosis. All women had stage I, node-negative and hormone receptor positive disease. None had received adjuvant chemotherapy. We found that low-risk node negative patients with high expression of cyclin B1 had a significantly worse outcome than patients with low expression of cyclin B1. In paper II a population-based case control study was performed to further investigate the prognostic value of cyclin B1. One hundred and ninety women who died from BC were defined as cases and 190 women alive at the time for the corresponding case’s death were defined as controls. Inclusion criteria were tumor size 50 mm, no lymph node metastases, and no adjuvant chemotherapy. Two investigators evaluated the stainings independently. Cyclin B1 was found to be a prognostic factor for BC death that could identify high-risk patients with a good to very good reproducibility. Paper III aimed to investigate the role of proliferation in male breast cancer (MBC). One hundred and ninety-seven MBC tumors were stained for cyclin A, B1, D1 and Ki67. Overexpression of cyclin A and B1 and elevated mitotic count were predictive of breast cancer death. Ki67 was re-evaluated and different cut-offs were used, but no prognostic value could be demonstrated. On the other hand high levels of cyclin D1 were associated with better outcome in MBC. In paper IV we applied the immunohistochemichal panel suggested from international guidelines to the same patient material as in paper II, to discriminate luminal A from luminal B BC. We wanted to evaluate if different cut-off values of Ki67, cyclin A or B1 could more clearly separate luminal A from B. Cyclin A, B1 and Ki67 (cut-off 20%) could detect difference in outcome between these subtypes with cyclin A showing greater prognostic value. The aim of paper V was to examine the prognostic role of PPH3 compared to the proliferation markers Ki67, cyclin A and cyclin B1 with focus on ER positive disease. PPH3 was found to be a prognostic factor for breast cancer death but in the multivariate analysis including all proliferation markers, only cyclin A remained a prognostic factor. Finally, we conclude that both cyclin B1 and PPH3 are prognostic factors for breast cancer death, but are outperformed by cyclin A in ER positive patients. In male breast cancer prognostic factors need to be further studied.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os objetivos deste trabalho foram: modificar quimicamente o amido de inhame (Dioscorea esculenta L.) por oxidaÃÃo e fosforizaÃÃo, elaborar blendas com quitosana (CH) e poli (alcool vinilico) (PVA) e avaliar suas caracterÃsticas fÃsico-quÃmicas e funcionais. O amido oxidado (AOX) obtido com um alto grau de substituiÃÃo (GS= 1,86) promoveu grandes modificaÃÃes na estrutura do granulo do amido, que apresentou caracterÃsticas de um material amorfo, enquanto a fosforizaÃÃo foi bem sutil, (GS=0,002) conservando a estrutura e padrÃo cristalino tipo B do amido de inhame nÃo modificado. O amido fosforilado (AF) apresentou aumento na estabilidade de pasta e baixa tendÃncia a retrogradarÃo, ao contrario do amido oxidado que nÃo apresentou perfil de pasta devido a despolimerizaÃÃo decorrente da reaÃÃo. As blendas de AF/CH/PVA apresentaram boas caracterÃsticas de permeabilidade ao vapor dâÃgua, baixas solubilidades em tampÃo fosfato e Ãgua destilada em comparaÃÃo com o amido nÃo modificado. As blendas AF mostraram significativo intumescimento em tampÃo devido a reticularÃo entre os grupos fosfatos da soluÃÃo e do filme, o que e desejÃvel para uma provÃvel aplicaÃÃo desses filmes como curativos. As modificaÃÃes nas intensidades e formas das bandas nos espectros de infravermelho, assim como variaÃÃes nas temperaturas de gelatinizarÃo confirmaram a interaÃÃo do PVA e quitosana com as cadeias de amido nos filmes. A adiÃÃo de PVA melhorou as propriedades mecÃnicas dos filmes proporcionando maior flexibilidade. As blendas AF nÃo apresentaram atividade na inibiÃÃo de S. aureus e P. aeruginosa, porem as blendas AOX, apesar de nÃo serem efetivas na inibiÃÃo de P. aeruginosa, apresentaram atividade contra cepas de S. aureus, principalmente as blendas AOX 50 e AOX70, o que poderà favorecer sua utilizaÃÃo como inibidores da proliferaÃÃo desse microorganismo em infecÃÃes. / The aim of this study were to modify chemically the yam starch (Dioscorea esculenta L.) by oxidation and phosphorylation reactions, produce blends with chitosan (CH) and poly (vinyl alcohol) (PVA) and evaluate their physico-chemical and functional properties. The oxidized starch (AOX) had a high degree of substitution (DS=1.86) which promoted major changes in the granular structure showing characteristics of an amorphous material, whereas the phosphorylation was very subtle (GS=0.002), remaining its granular structure and crystalline pattern B-type of native yam starch. The phosphorylated starch (AF) showed improvement in paste stability and low tendency to retrogradation, unlike the AOX which showed no viscosity profile due to depolymerization caused by the reaction. The AF/CH/PVA blends showed good characteristics of water vapor permeability, low solubility in phosphate buffer and distilled water as compared to native starch. The blends AF showed significant swelling in buffer due to crosslinking between the phosphate groups of the solution and the film, which is desirable for a possible application of such films as dressings. The changes in intensities and shapes of the FTIR bands as well as variations in temperatures of gelatinization confirmed the interaction of PVA and chitosan with the starch chains in the films. The addition of PVA improved the characteristics of the films offering high flexibility. The AF blends showed no activity in the inhibition of S. aureus and P. aeruginosa, but the AOX blends, although not showing effectiveness in inhibition of P. aeruginosa, showed activity against strains of S. aureus, especially AOX50 and AOX70 blends, which may favor their use as inhibitors against proliferation of these microorganisms in infections.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22357号 / 医博第4598号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 伊佐 正, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les travaux de cette thèse ont porté sur le développement de nouvelles méthodes de synthèse autour de la chimie des réactions multicomposants et des réactions de Tsuji-Trost des dérivés nitrés et phosphorylés. Dans le chapitre I, nous avons développé une nouvelle voie d’accès aux indoles propargyliques impliquant des adduits A3 et une réaction d’élimination d’amine en présence du 1,2-dibromoethane. Dans le chapitre II, nous avons démontré la possibilité d’exploiter des réactions de Tsuji-Trost par la formation de produits O-allylés issus des α-hydroxyphosphonates. La méthatèse cyclisante appliquée sur une gamme d’α-hydroxyphosphonates O-allylés a permis de synthétiser des phosphono-oxahétérocycles. Par ailleurs nous avons aussi démontré l’intérêt des α-hydroxyphosphonates du cinnamaldehyde pour des tandems Tsuji-Trost/ Claisen par la formation de dérivés d’acides α allylés. Dans le chapitre III, nous avons décrit un couplage entre les acides boroniques et les nitronates silylés pour aboutir à des oximes α, β insaturés. Par la suite nous avons convertis les oximes α,β-insaturés obtenus en isoxazoles par une cyclisation oxydante . Finalement dans le chapitre IV, nous avons poursuivi sur les réactions de Tsuji-Trost en mettant au point une synthèse de diènes à partir de dérivés nitrés allyliques. Cette réaction a été élargie à la synthèse des naphatlènes substitués en position 1. / The work of this thesis focused on the development of new synthesis methods around the chemistry of multicomponent reactions and Tsuji-Trost reactions of nitro and phosphorylated derivatives. In Chapter I, we developed a new pathway to propargylic indoles involving A3 adducts and an amine removal reaction in the presence of 1,2-dibromoethane. In Chapter II, we demonstrated the possibility of exploiting Tsuji-Trost reactions by the formation of O-allyl products from α-hydroxyphosphonates. The ring closing metathesis applied to a range of O-allylated α-hydroxyphosphonates allowed phosphono-oxaheterocycles to be synthesized. Furthermore, we also demonstrated the interest of α-hydroxyphosphonates from cinnamaldehyde for Tsuji-Trost / Claisen tandems by the formation of α-allylated acid derivatives. In Chapter III, we described a coupling between boronic acids and silylated nitronates to produce α, β-unsaturated oximes. Subsequently we converted the α, β-unsaturated oximes obtained in isoxazoles by an oxidizing cyclization. Finally, in Chapter IV, we continued the reactions of Tsuji-trost by developing a synthesis of dienes from allylic nitro derivatives. This reaction has been extended to the synthesis of naphatlene substituted at the 1-position.

BACKGROUND: Prostate cancer is by far the most common cancer among Swedish men. Some patients have an aggressive lethal disease, but the majority of affected men have long expected survival. Unfortunately, the diagnostic tools available are insufficient in predicting disease aggressiveness. Novel prognostic markers are therefore urgently needed. Furthermore, metastatic prostate cancer is generally treated with castration, but the long-term effects are insufficient. Additional studies are therefore needed to explore how the effects of this therapy can be enhanced. Prostate growth and regression is beside testosterone controlled by locally produced regulators. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) are two of the major regulators in the normal prostate and in prostate tumours. MATERIALS AND METHODS: VEGF and EGFR were explored in the prostate, by treating rats with either anti-VEGF or anti-EGFR treatment during castration and testosterone-stimulated prostate growth. Rats with implanted androgen-independent prostate tumours were treated with an inhibitor of both VEGF receptor-2 (VEGFR-2) and EGFR. Stereological techniques, immunohistochemistry, western blotting and quantitative real-time PCR were used to evaluate these experiments. Furthermore, prostate tissue from untreated prostate cancer patients was used to retrospectively explore the expression of phosphorylated-EGFR (pEGFR) in relation to outcome. RESULTS: Anti-VEGF treatment during testosterone-stimulated prostate growth, inhibited vascular and prostate growth. Anti-EGFR treatment during castration and testosterone-stimulated prostate growth resulted in enhanced castration effects and inhibited prostate growth. Anti-vascular treatment of androgen-independent prostate cancer with an inhibitor of VEGFR-2 and EGFR, that targets the normal and tumour vasculature, enhanced the effects of castration. Low immunoreactivity for pEGFR in prostate epithelial cells, both in the tumour and also in the surrounding non-malignant tissue, was associated with good prognosis. CONCLUSIONS: Anti-vascular treatment, with an inhibitor of VEGFR-2 and EGFR, in combination with castration could be an effective way to treat androgen-insensitive prostate tumours. VEGF and EGFR signalling are necessary components in testosterone-stimulated prostate growth. Phosphorylation of EGFR could be a useful prognostic marker for prostate cancer patients. Tumours may affect the surrounding non-malignant tissue and pEGFR immunoreactivity in the morphologically normal prostate tissue can be used to retrieve prognostic information.