Orientador: Leticia Helena Theodoro / Coorientador Valdir Gouveia Garcia / Banca: Sandra Helena Penha de Oliveira / Banca: Rossana Abud Cabrera Rosa / Resumo: Introdução: A presença de bolsas residuais representa um fator de risco preditor de progressão da doença periodontal. A presença de Diabetes Mellitus (DM) aumenta a prevalência de periodontite e influencia negativamente na capacidade de reparo tecidual. O objetivo do estudo foi avaliar a eficácia clínica da terapia fotodinâmica antimicrobiana (aPDT) com curcumina e LED, como terapia coadjuvante à raspagem e alisamento radicular (RAR), no tratamento de bolsas residuais de pacientes com DM tipo 2. Métodos: Para este estudo clínico controlado randomizado de boca dividida, vinte e cinco pacientes foram selecionados. Em cada paciente, todas as bolsas residuais com profundidade de sondagem (PS) ≥5 mm e sangramento à sondagem (SS), por quadrante, foram aleatoriamente alocados para receber: 1) RAR (grupo RAR); 2) RAR e irrigação com solução de curcumina (grupo CUR); 3) RAR e irradiação com LED (grupo LED); 4) RAR e terapia fotodinâmica antimicrobiana (grupo aPDT). Para a aPDT, utilizou-se solução de curcumina (100 mg/L) seguida de irradiação com LED (InGaN, 465 - 485 nm, 100 mW/cm², 60 segundos). Os parâmetros clínicos de PS, recessão gengival (RG), nível de inserção clínica (NIC), SS e índice de placa visível (IP) foram avaliados no início (baseline), 3 e 6 meses após os tratamentos. Resultados: Na comparação intergrupo, não houve diferença estatisticamente significante nos valores médios dos parâmetros clínicos avaliados (PS, RG, NIC, SS e IP) no início do estudo (baseline), aos 3 ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Residual pockets represent a risk factor for periodontal disease progression, which is exacerbated by Diabetes Mellitus (DM) by increasing the prevalence of periodontal disease and negatively influencing healing capacity. The present study aimed to evaluate the clinical efficacy of antimicrobial photodynamic therapy (aPDT) with curcumin and LED, as an adjunctive therapy to scaling and root planing (SRP), in the treatment of residual pockets in patients with type 2 DM. Methods: A randomized and controlled split-mouth clinical trial was conducted with twenty-five patients. In each patient, all residual pockets with probing depth (PD) ≥5 mm and bleeding on probing (BOP), per quadrant, were randomly allocated to receive: 1) SRP (SRP group); 2) SRP and irrigation with curcumin solution (CUR group); 3) SRP and LED irradiation (LED group); 4) SRP and aPDT (aPDT group). The aPDT was performed with curcumin solution (100 mg/L) followed by LED irradiation (InGaN, 465 - 485 nm, 100 mW/cm², 60 seconds). Clinical parameters of PD, gingival recession (GR), clinical attachment level (CAL), BOP and visible plaque index (PI) were evaluated at baseline, 3 and 6 months post-therapies. Results: In an intergroup comparison, the mean values for PD, GR, CAL, BOP and PI were not different at baseline, 3 and 6 months (p > 0.05). Similarly, the mean difference in the reduction of PD and CAL gain between baseline and 3 or 6 months were not statistically different (p > 0.05). The intragrou... (Complete abstract click electronic access below) / Mestre

Orientador: Cristiane Duque / Coorientador: AiméeMaria Guiotti / Banca: Marcelle Danelon / Banca: Aline Rogéria Freire de Castilho / Resumo: Embora o tratamento endodôntico convencional reduza significativamente a microbiota presente no interior dos canais radiculares, a permanência de microrganismos devido à complexidade anatômica do sistema de canais radiculares e a resistência destes ao tratamento químico-mecânico pode ocasionar infecções persistentes ou secundárias. Muitos estudos têm explorado o uso de fitoquímicos, buscando obter novos compostos que apresentem propriedades farmacológicas. A curcumina, pigmento amarelo isolado dos rizomas da Curcuma longa (Zingiberaceae), e o cinamaldeido, substância volátil responsável pelo odor e sabor das cascas dos caules de plantas do gênero Cinnamomum (Lauraceae) são possíveis substâncias promissoras. O objetivo do estudo foi avaliar o efeito citotóxico e ação antimicrobiana/antibiofilme de compostos híbridos de curcumina e cinamaldeído sobre microrganismos de interesse endodôntico. Foram realizados ensaios para determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) do cinamaldeído, da curcumina e dos 23 híbridos sobre Enterococcus faecalis, Streptococcus mutans, Lactobacillus casei, Actinomyces israelii e Fusobacterium nucleatum. Os melhores compostos foram avaliados em ensaios de biofilme simples (cada cepa bacteriana isoladamente) e dual-espécies (E. faecalis + L. casei, E. faecalis + S. mutans, E. faecalis + A. israelii, E. faecalis + F. nucleatum) em placas de poliestireno objetivando-se determinar o efeito sobre o metabolismo ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although conventional endodontic treatment significantly reduces the microbiota present inside the root canals, the permanence of microorganisms due to the anatomical complexity of the root canal system and their resistance to chemical-mechanical treatment can lead to persistent or secondary infections. Many studies have explored the use of phytochemicals, seeking to obtain new compounds that present pharmacological properties. Curcumin, a yellow pigment isolated from Curcuma longa rhizomes (Zingiberaceae), and cinnamaldehyde, the volatile substance responsible for the odor and taste of plant stems of the genus Cinnamomum (Lauraceae) are possible promising substances. The objective of the study was to evaluate the cytotoxic effect and antimicrobial action / antibiofilm of hybrid compounds of curcumin and cinnamaldehyde on microorganisms of endodontic interest. The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBM) of cinnamaldehyde, curcumin and 23 hybrids on Enterococcus faecalis, Streptococcus mutans, Lactobacillus casei, Actinomyces israelii and Fusobacterium nucleatum were determined. The best compounds were evaluated on single biofilms (single bacterial strain) and dual-species biofilms (E. faecalis + L. casei, E. faecalis + S. mutans, E. faecalis + A. israelii, E. faecalis + F. nucleatum) in polystyrene plates to determine the effect on bacterial metabolism using the XTT assay and its viability by counting Colony Forming Units (CFUs) after ... (Complete abstract click electronic access below) / Mestre

Les cancers hormono-dépendants : le cancer du sein et de la prostate, sont les plusfréquents respectivement chez la femme et chez l’homme. Ces cancers représentent unproblème majeur de santé publique. Malgré les améliorations du dépistage et des traitementscuratifs, ces cancers peuvent devenirs métastatiques et le traitement devient alors palliatif. Ace stade, les thérapeutiques sont déjà très avancées avec une prise en charge qui repousse deplus en plus la fin de vie du patient tout en conservant au mieux sa qualité de vie. Au stademétastatique, différentes stratégies peuvent être envisagées pour tenter de rallonger la surviedes patients : une médecine personnalisée en ciblant des voies de signalisation particulière ;une autre voie d’approche peut être une intervention nutritionnelle. En effet, les bienfaits decertains composants nutritionnels « anti-cancer » sont de plus en plus connus et étudiés.L’objectif de ce travail a été d’étudier ces deux approches dans la prise en chargethérapeutique de ces 2 cancers hormono dépendants en prenant le cas de l’évérolimus commethérapie ciblée et le cas de la curcumine comme composé nutritionnel.Une base de données rétrospective a été réalisée chez des patientes atteintes d’uncancer du sein métastatique (n = 63) et recevant de l’évérolimus en combinaison avecl’hormonothérapie (EHT). L’objectif de ce travail était d’étudier la réponse à cette nouvellecombinaison associant une thérapie ciblée et une hormonothérapie et de comparer cesdonnées à une ancienne base de données (PMID 21852136) qui cherchait à savoir l’intérêt detraiter les patientes avec plusieurs lignes de chimiothérapie. Il a été montré une améliorationde la survie chez les patientes traitées par EHT comparativement à une chimiothérapie et cettedifférence est significative lorsque les patientes sont traitées avec moins de 2 lignes dechimiothérapies antérieures.La seconde approche était d’évaluer la combinaison de l’association Taxotère® pluscurcumine dans le cancer de la prostate résistant à la castration et dans le cancer du seinmétastatique.Un essai pilote de phase II a évalué l’association Taxotère® plus curcumine enpremière ligne de traitement des cancers de la prostate métastatiques hormono-résistants sur30 patients. Cette association a permis d’obtenir un taux de réponse du PSA de 59% et untaux de réponse objective de 40% pour les patients présentant des cibles mesurables. Letraitement a été très bien toléré avec une dose de curcumine de 6 grammes par jour pendant 7jours.Deux études de phase II randomisés sont en cours :- CURRYTAX, un essai multicentrique randomisé de phase II comparant le Taxotère® enmonothérapie à l’association Taxotère® plus curcumine en première ou deuxième ligne detraitement des cancers du sein inopérables, en rechute ou métastatiques, Her2 négatifs.- CURTAXEL, un essai multicentrique randomisé de phase II en double aveuglecomparant l’association Taxotère® plus curcumine versus Taxotère® plus placebo enpremière ligne de traitement des cancers de la prostate métastatiques résistants à la castration.Des résultats préliminaires vont être présentés dans ce manuscrit, avant l’analyseintermédiaire prévue après l’inclusion et le suivi des 50 premiers patients. L’étude de lapopulation, de la compliance, de la réponse objective, de la survie, du temps jusqu’àprogression, de la réponse du PSA, du suivi des marqueurs (CEA, CA15-3) vont êtreprésentés dans ce manuscrit sur 30 patientes dans l’étude CURRYTAX et sur 33 patients dansl’étude CURTAXEL.Ces travaux montrent l’intérêt de telles approches et ouvrent des pistes à exploiter en cequi concerne une intervention biologique dans le cancer du sein et celui de la prostate que cesoit par une intervention nutritionnelle telle que la curcumine ou bien via un mécanisme surune voie de signalisation. / Hormone dependent cancers like breast cancer and prostate cancers are the two mostcommon cancers in woman and man and is a major health concern. Despite the improvementof early detection methods and curative treatments, cancers become metastatic and treatmentbecome palliative. At metastatic stage, different treatments can be tested to control the growthlonger and relieve symptoms caused by it to help prolong life and preserve quality of life.Metastatic cancer may be treated with targeted therapy depending on specific characteristic,nutrition therapy can also help as the benefit of nutritional science against cancer knowledgehas increased and more research are being done on this method to show its effectiveness.The purpose of this work was to compare 2 different therapies using the Everolimus plusHormonotherapy (EHT) treatment as targeted therapy and Taxotere plus Curcumin asnutrition therapy.A retrospective database was realised on 63 metastatic breast cancer patients who receivedEverolimus plus Hormonotherapy (EHT) as targeted therapy. This aim of this database was toevaluate the efficacy of this combined treatment and compare data with an existing database(PMID21852136) which looked at the benefits of treating patients with numerous lines ofchemotherapy.The survival was improved for patients who received EHT compared to patients whoreceived chemotherapy. The study shows that EHT resulted in a longer overall survival ratefor patient pre-treated with less than 2 CT lines.In the second part of this work, we analysed the efficacy of a combination Taxotere pluscurcumin in castration resistant prostate cancer and metastatic breast cancer patients.A phase II study evaluated the combination of Taxotere® and curcumin in first linetreatment of metastatic castration resistant prostate cancer on 30 patients. The PSA responserate was 59% and the objective tumor response rate was 40% for patient with measurabletargets. This treatment was well tolerated with a daily dose of 6g for 7days.Two phase II randomized studies are currently recruiting:-CURRYTAX, a randomized multicenter phase II trial assessing Taxotere as amonotherapy versus Taxotere plus curcumin in first and second lines cancer treatments forinoperable, recurring or metastatic HER2- Negative breast cancer patients.-CURTAXEL, a randomized double blind multicenter phase II trial comparing Taxotereplus curcumin versus Taxotere plus placebo in first line cancer treatment for metastaticcastration resistant prostate cancer patients.Preliminary results will be included in this manuscript before the planned intermediateanalysis, after enrollment of 50 patients. Population, compliance, objective response, overallsurvival, time to progression, PSA response, markers assessment (CEA, CA15-3) will also bepresented in this manuscript on 30 and 33 patients in CURRYTAX and CURTAXEL trials,respectively.These clinical results show the interest of such approaches and permit to explore furtherbiological pathway in breast and prostate cancer, either through nutritional therapy usingcurcumin or through a signalling pathway.

The molecular mechanisms of putative anti-inflammatory nutrients, i.e., fish oil, curcumin and limonin, were investgated with respect to CD4+ T cell function. Initially, using a DO11.10 mouse model which exhibits a transgenic T cell receptor specific to OVA 323-339 peptide, we demonstrated that dietary fish oil suppresses antigen-specific Th1 clonal expansion in vivo. Following immunization, the accumulation of adoptively transferred transgenic cells in wild type recipient mouse lymph nodes was suppressed. In addition, cell division analysis by carboxyfluorescein succinimidyl ester (CFSE) revealed that both total cell number in lymph nodes as well as cell division were decreased by fish oil. Since n-3 polyunsaturated fatty acids (PUFA), active long chain fatty acids in fish oil, elicit favorable effects on a variety of cell types, e.g., anti-tumor effect on colonocytes, amelioration of coronary heart disease and anti-inflammatory effects involving T cells, B cells, dendritic cells and macrophages, we postulated that a fundamental mechanism of action may explain the multiple effects observed. In a series of experiments described herein, we demonstrated that n-3 PUFA alters the formation/location of membrane subdomains, referenced to as lipid rafts. Specifically, lipid raft formation at the immunological synapse (IS) in CD4+ T cells was suppressed following membrane enrichment with n-3 PUFA. The alteration of lipid rafts down-regulated the localization of select signaling proteins, including F-actin, PKC and PLC-1, and phosphorylation of PLC-1 at the IS. Consequently, CD4+ T cell proliferation was suppressed as assessed by CFSE analysis and radioactive thymidine incorporation. Phytochemicals have been used for chemopreventive and chemotherapeutic purposes. We examined the putative anti-inflammatory effects of curcumin (1%) and limonin (0.02%) with respect to CD4+ T cell function. Dietary curcumin and limonin suppressed NF-B activation in CD4+ T cells. In addition, CD4+ T cell proliferation was modulated by 2% curcumin. We further investigated the combined therapeutic potential of phytochemicals and fish oil, containing n-3 PUFA. Interestingly, fish oil and limonin together significantly (P<0.05) suppressed T cell proliferation, whereas feeding either fish oil or limonin alone showed little effect. In summary, our data indicated that dietary fish oil alters proximal signaling of T cells by perturbing lipid raft formation. Curcumin and limoin are capable of suppressing NF-B in T cells, thereby exhibiting a synergistic effect when combined with fish oil. Further studies are required to elucidate the relationship of dietary dose of active compoments with respect to mechanism of actions.

Methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) is a synthetic triterpenoid that inhibits growth of Panc1 and Panc28 pancreatic cancer cell lines and activates peroxisome proliferator-activated receptor B (PPARB)-dependent transactivation in these cells. CDODA-Me has also induced p21 and p27 protein expression and downregulated cyclin D1; however, these responses were receptor-independent. CDODA-Me induced apoptosis, which was accompanied by receptor-independent induction of the proapoptotic proteins early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), and activating transcription factor-3 (ATF3). Induction of NAG-1 in Panc28 cells was p38-mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K)-dependent, but Egr-1-independent, whereas induction in Panc1 cells was associated with activation of p38-MAPK, PI3-K and p42-MAPK and was only partially Egr-1-dependent. Specificity protein (Sp) transcription factors Sp1, Sp3 & Sp4 are overexpressed in multiple tumor types and negative prognostic factors for survival. Since Sp proteins regulate genes associated with survival (survivin), angiogenesis [vascular endothelial growth factor and its receptors] and growth [cyclin D1, epidermal growth factor receptor], research in this laboratory has focused on development of anticancer drugs that decrease Sp protein expression. Arsenic trioxide, curcumin, 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid (CDDO), CDDO-Me, and celastrol exhibit antiproliferative, antiangiogenic and proapoptotic activity in many cancer cells and tumors. Treatment of cancer cells derived from urologic and gastrointestinal tumors with arsenic trioxide decreased Sp1, Sp3 and Sp4 transcription factors and cotreatment with the proteosome inhibitor MG132 did not inhibit downregulation of Sp proteins in these cancer cells. Mechanistic studies suggested that compound-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide, demonstrating that the mitochondriotoxic effects of these compounds are important for their anticancer activities. Moreover, repression of Sp and Sp-dependent genes by CDDO-Me and celastrol was due to downregulation of microRNA-27a and induction of ZBTB10, an Sp repressor, and these responses were also reversed by antioxidants. Thus, the anticancer activity of CDDO-Me and celastrol is due, in part, to activation of ROS which in turn targets the microRNA-27a:ZBTB10?Sp transcription factor axis to decrease growth inhibitory, pro-apoptotic and antiangiogenic genes and responses.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Curcumin (CURC) was incorporated in liposomes as free drug or after formation of hydropropyl-β- or hydroxypropyl-γ-cyclodextrin (HPβCD or HPγCD) complexes prepared by co-precipitation and characterized by X-ray diffractometry. Liposomes encapsulating CURC as free drug or CD-complexes (hybrid formulations) were prepared by the dehydration–rehydration vesicle (DRV) method followed by extrusion, and characterized for size, zeta-potential and CURC loading. CURC stability (at 0.01 and 0.05 mg/mL) in 80% (v/v) fetal bovine serum (FBS) was evaluated at 37 oC. Results demonstrate that HPβCD stabilizes CURC more than HPCD, but liposome encapsulation provides substantially more protection, than CDs. CURC stabilization is similar, when encapsulated as free compound or CD-complex. However, the last method increases CURC loading by 23 times (depending on the lipid composition of liposomes and the CD used), resulting in higher solubility. The stability profile of CURC in serum depends on the composition of liposomes and CURC concentration, since at lower concentrations larger CURC fractions are protected due to protein binding. Compared to the corresponding CD complexes, hybrid formulations provide intermediate CURC solubility (comparable to HPβCD) but profoundly higher stabilization. After identifying that CURC formulations are active against B16 melanoma cells (in vitro), the optimal concentrations of CURC and doxorubicin (DOX) combinations (which provided highest anticancer activity [in vitro]) were identified. In a second step, combination hybrid-liposomal formulations of CURC and DOX were prepared, as a technique to deliver the specific combination of the two drugs to cancer cells following in vivo administration (since the two drugs have different pharmacokinetics and would not reach cancer cells at the same ratio if administered as free drugs). Additionally the effect of ceramide incorporation in the liposomal membrane on the anticancer activity of the later combination-formulations was investigated. The liposomal CURC-DOX combination formulations demonstrated significantly enhanced anticancer activity, compared to liposomes with DOX only. As confirmed by FACS analysis and uptake studies, this enhanced effect was due to increased uptake of DOX by the cells, in the presence of CURC (which was not seen when free compounds were used). Although a positive effect of ceramide addition in liposomal-DOX anticancer activity was demonstrated, in agreement with previous studies, ceramide addition did not result in further increase of the anticancer activity of the CURC-DOX combination formulations, when tested by MTT assay. However, mechanistic studies revealed that the CURC-DOX formulations resulted in higher percentage of early apoptotic cells, compared to the ceramide-DOX formulations which resulted mainly in late-apoptotic cells. When all CURC-DOX and Ceramide were combined in the same formulation, the percentages of early and late apoptotic cells were additive. / Η Κουρκουμίνη (CURC) ενσωματώθηκε σε λιποσώματα ως ελεύθερο μόριο ή ως σύμπλοκο εγκλεισμού με υδροξυπροπυλο-β-κυκλοδεξτρίνη ή υδροξυπροπυλο-γ-κυκλοδεξτρίνη (HPβCDorHPCD); Τα σύμπλοκα παρασκευάστηκαν με την τεχνική της συγκαταβύθισης και χαρακτηρίστηκαν με ακτίνες-Χ. Συμβατικά λιποσώματα (που ενσωματώνουν την CURC ωε ελεύθερο μόριο) και υβριδικά, που την εγκλωβίζουν ως σύμπλοκο-εγκλεισμού, παρασκευάστηκαν με την τεχνική DRV και εξώθηση διαμέσου μεμβρανών, και αφού χαρακτηρίστηκαν φυσικοχημικά (φόρτωση CURC, μέση διάμετρος, πολυδιασπορά και ζ-δυναμικό), μελετήθηκε η σταθερότητα της CURC (σε συγκέντρωση 0.01 and 0.05 mg/mL), όταν τα παρασκευάσματα επωάστηκαν σε ρυθμιστικό διάλυμα και ορρό (80% ο/ο FCS) στους 37 oC. Τα αποτελέσματα έδειξαν ότι η HPβCD σταθεροποιεί την CURC περισσότερο από ότι η HPCD, αλλά η ενσωμάτωση σε λιποσώματα προσφέρει σημαντικά μεγαλύτερη σταθεροποίηση από ότι οι CDs (είτε όταν εγκλωβίζετε ως ελεύθερο μόριο ή ως CD-σύμπλοκο). Όμως η τελευταία μέθοδος πετυχαίνει αύξηση της φόρτωσης έως και κατά 23 φορές (ανάλογα με τη λιπιδική σύσταση και το σύμπλοκο που χρησιμοποιείται), με αποτέλεσμα να αυξάνει σημαντικά η διαλυτότητα της CURC. Η σταθερότητα της CURC παρουσία ορρού συνδέεται με την λιπιδική σύσταση των λιποσωμάτων και τη συγκέντρωση της CURC, αφού σε χαμηλότερες συγκεντρώσεις μεγαλύτερο κλάσμα της CURCfractions προστατεύεται λόγο σύνδεσης στις πρωτεΐνες. Σε σύγκριση με τα σύμπλοκα εγκλεισμού, οι υβριδικές λιποσωμικές μορφές προσφέρουν ενδιάμεση διαλυτότητα στην CURC (συγκρίσιμη με αυτήν της HPβCD) αλλά σημαντικά αυξημένη σταθερότητα. Μετά την απόδειξη ότι οι μορφές της CURC είναι δραστικές κατά κυττάρων μελανώματος B16 (invitro), εντοπίστηκαν οι βέλτιστες συγκεντρώσεις συνδυασμών CURC και δοξορουβικίνης (DOX) που προσφέρουν βέλτιστη αντικαρκινική δράση [invitro]. Σε δεύτερο βήμα, παρασκευάστηκαν υβριδικές λιποσωμικές μορφές συνδυασμών CURC -DOX, ως τεχνική χορήγησης συνδυασμών που θα φθάσουν στα καρκινικά κύτταρα στις αρχικές συγκεντρώσεις (κάτι που δεν θα συμβεί εάν χορηγηθούν ως ξεχωριστά φάρμακα λόγο της διαφορετικής κινητικής τους). Επιπρόσθετα μελετήθηκε η επίδραση της ενσωμάτωσης ceramideς στην λιποσωμική μεμβράνη στην αντικαρκινική δράση των μορφών. Οι μορφές συνδυασμού CURC-DOX έδειξαν αυξημένη αντικαρκινική δράση σε σύγκριση με λιποσώματα που είχαν μόνο DOX (κάτι που δεν παρατηρήθηκε όταν χρησιμοποιήθηκαν ελεύθερα τα δύο μόρια). Όπως επιβεβαιώθηκε με FACSanalysis και με πειράματα κυτταρικής πρόσληψης, αυτή η αυξημένη δράση των μορφών συνδυασμού οφείλετε σε αύξηση της πρόσληψης DOX από τα κύτταρα, παρουσία CURC (πάλι δεν συνέβηκε όταν χρησιμοποιήθηκε ελεύθερη CURC). Εάν και παρατηρήθηκε αυξημένη δραστικότητα λιποσωμικής -DOX όταν τα λιποσώματα είχαν ceramides στη σύσταση τους, σε συμφωνία με προηγούμενες μελέτες, η προσθήκη, ceramideς δεν είχε θετικό αποτέλεσμα στη δραστικότητα των μορφών συνδυασμού CURC-DOX όταν μετρήθηκε με την τεχνική MTT. Εντούτοις, μηχανιστικές μελέτες έδειξαν ότι οι μορφές συνδυασμού CURC-DOX έδωσαν αυξημένο ποσοστό κυττάρων που είναι σε αρχικά στάδια απόπτωσης, σε σχέση με τα λιποσωματα DOX με ceramides που έδωσαν μεγαλύτερα ποσοστά κυττάρων σε τελικά στάδια απόπτωσης. Στις μορφές που συνδύαζαν τα πάντα, CURC-DOX και Ceramides τα ποσοστά κυττάρων σε πρώιμη και προχωρημένη απόπτωση ήταν αθροιστικά.

Foodborne diseases are one among the diseases with high morbidity and mortality rate. The concern is raised with the emergence of pathogenic strains that are resistant to the available set of antibiotics. Conventional regimens fail to treat the infections caused by these pathogens prolonging the sickness leading to increased morbidity and mortality. The situation can get further complicated with the dietary intake of the host. Of late it has been understood that the dietary flavonoids play an important role in regulating the immune system. Curcumin, a pigment from turmeric, is one among such bioflavonoid with an immunomodulatory potential. Curcumin has been a front-line topic of mainstream scientific research for a variety of diseases from cancer to Alzheimer’s to infectious diseases. Curcumin being considered as a spicy panacea is not a remedy for all diseases. Its ability to act differentially as an antioxidant or pro-oxidant can be either beneficial or harmful for the host. It exhibits antioxidant properties at concentrations achievable in the body; this can make the host vulnerable to infections due to the suppression of innate immune responses. Curcumin also suppresses the type 1 immune response, which might lead to alleviation of type 1 immune response disorders. However, the inhibition of type 1 immune response might invite infections with opportunistic pathogens. We have chosen curcumin to assess the effect of diet on the regulation of pathogenesis of Salmonella along with few medically important pathogens like Yersinia enterocolitica, Staphylococcus aureus, Shigella flexneri and Listeria monocytogenes. The thesis is divided into five chapters. As the main focus of the thesis is on Salmonella, in Chapter 1 we introduce diverse aspects of curcumin and the basic biology of Salmonella. Initially the properties of curcumin, the molecule of interest are introduced followed by brief overview to Salmonella biology and pathogenesis. Various activities of curcumin dealing with the variety of diseases are discussed. Further, the introduction to the intricate underlying mechanisms and the functional determinants of curcumin is given. The subsequent sections give an overview of different phases of Salmonella pathogenesis and the molecular mechanisms of Salmonella virulence and host defense. Towards the end of the chapter we discuss the strength, limitations and the distinctive characteristics of the murine model of typhoid fever. Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Its anti-bacterial effect has been demonstrated in bacteria, like B. subtilis, H. pylori and E. coli. Contrary to this, the results of the Chapter 2 reveals that curcumin at a nontoxic concentration to both host and pathogen, regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in reticuloendothelial organs when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. It up-regulated the genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that the iron chelation property of curcumin has a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ two-component regulatory system (TCS). Curcumin downregulates SPI-1 genes required for entry into epithelial cells and upregulates SPI-2 genes required for intracellular survival, through PhoPQ TCS. Thus, this common regulator (PhoPQ) could explain curcumin's mode of action. Another important factor for the pathogen’s success is its ability to counteract the action of antibiotics. Almost all the bactericidal antibiotics act via production of reactive oxygen species in the bacteria. Curcumin has anti-oxidant property that might interfere with the action of antibiotics. Ciprofloxacin is a commonly used anti-typhoidal drug. It kills the bacteria by inhibiting DNA replication and increasing reactive oxygen species in bacterial cell. In Chapter 3 we present the results obtained after the investigation of the interference of curcumin with the anti-bacterial action of ciprofloxacin against Salmonella. We found that curcumin indeed increased the proliferation of Salmonella Typhi and Salmonella Typhimurium in ciprofloxacin treated macrophages by reducing the ciprofloxacin-induced reactive oxygen species. It also inhibited ciprofloxacin mediated DNA damage and the resultant SOS response and filamentation. However, curcumin was unable to rescue the ciprofloxacin induced gyrase inhibition. The reduced antibiotic (ciprofloxacin) efficacy against Salmonella by curcumin might aggravate the disease. Thus, the results of chapter 1 and 2 urge us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks. Bacteria modulate its virulence determinants in response to the environmental cues. Salmonella being a foodborne pathogen has a very likely chance of getting exposed to turmeric and hence curcumin. In Chapter 4 we have assessed the modulation of motility of S. Typhimurium, an important virulence determinant, by curcumin. We show that curcumin reduced the motility of the S. Typhimurium by decreasing the flagellar density around it. Surprisingly, this was achieved without affecting the expression of the flagellin gene and protein. Curcumin physically adhered to the flagella making it fragile and breaking it into fragments. This can hinder bacterial motility, chemotaxis, adherence and invasion into the host cells. However, aflagellate bacteria are hypervirulent as is the case with our experimental results with curcumin treated bacteria. Curcumin regulates myriad of bacterial (Salmonella) activities increasing its pathogenicity. Curcumin is known to regulate the host defenses in response to the disease. In Chapter 5 we have sought to address the effect of curcumin treatment of host cells on the outcome of infection by different pathogens. Pathogens have evolved different strategies to evade the host innate immune system, one of them being avoiding lysosome mediated degradation. Pathogens like Salmonella, Yersinia, Mycobacterium and Staphylococcus have acquired molecular machinery to inhibit the fusion of the pathogen containing vacuole with lysosomes and multiply within the vacuole whereas other pathogens like Shigella, Listeria and Rickettsia escape into and multiply in the cytosol. In our study we show that pretreatment of macrophage with curcumin increased the fold proliferation of S. Typhimurium, S. aureus and Y. enterocolitica whereas decreased that of S. flexneri and L. monocytogenes. From the results obtained, we can state that curcumin differentially regulates the pathogenesis of vacuolar and cytosolic pathogen. We hypothesized that curcumin pretreatment stabilizes the membrane of pathogen containing vacuole retarding the lysis of the phagolysosome harboring the cytosolic pathogen and hence facilitating its clearance. We indeed observed that the membrane stabilizing effect of curcumin led to increased fusion of cytosolic pathogen with the lysosome, decreasing its proliferation in the cells. As the vacuolar pathogens have an inherent ability to inhibit this fusion, they proliferate better in curcumin treated cells. In a nutshell curcumin can have multiple and sometimes unexpected effects not only on a pathogen’s potential to successfully cause infection but also on the host’s ability to counter it. A brief summary of the study that does not directly deal with the modulation of bacterial pathogenesis by curcumin is included in the Appendix. In this study a novel, simple, sensitive and efficient PCR based assay was devised to detect Salmonella contamination in milk, fruit juice and ice-cream without any pre-enrichment.

Cryptosporidium parvum is an obligatory intracellular parasitic protist that belongs to the phylum Apicomplexa. Cryptosporidiosis is an infection for which no satisfactory efficient curative treatment is known, especially in immunocompromised individuals. Furthermore, the parasite oocysts show considerable tenacity in the environment. Therefore, new potent drugs along with a simple and reliable experimental model for evaluation of anticryptosporidial measures are urgently needed. The present studies were undertaken to establish a combined cell culture and quantitative PCR assay (cc-qPCR) to assess efficacy of pharmacological compounds against C. parvum. Human ileocecal adenocarcinoma cells (HCT-8) were selected for culture of C. parvum. Oocysts were excysted directly on confluent monolayers for infection. After 3 h of incubation the non invasive parasite remains were removed by washing. At the end of the incubation period the cells were harvested and subjected to DNA extraction. Real time PCR was performed to quantify the target parasite DNA (fragments of 70 kDa heat shock protein gene) copy numbers. Each reaction was run in triplicate. A standard curve calculated on the basis of serial dilutions of plasmid DNA or infected control culture DNA was run in each experiment. A series of oocyst suspensions were applied to cell cultures to determine the sensitivity of the cc-qPCR assay and also to generate a calibration curve to calculate the infectivity of oocysts. A dilution series of heat inactivated oocysts (70°C for 1 h) were used to determine the size of the oocyst inoculum at which complete elimination of extracellular parasite material by washing is reliably achieved. The results obtained by the assays were reproducible and the method sensitive with a detection limit of infection with 10 oocysts 48 h post infection (p.i.) and with 100 oocysts 24 h p.i. Percent effects of drugs and disinfectants were enumerated by comparing DNA copies between treated and non treated samples. The suitability of cc-qPCR for screening of pharmacological compounds was validated by confirming the in vitro efficacy of monensin (98.15% ± 1.09 at 0.144 µM) and halofuginone (98.05% ± 0.59 at 25 µM) over the entire incubation period with a dose dependent reduction of parasite multiplication demonstrated 27 h p.i. The inhibition of parasite proliferation by 0.144 µM monensin in the period from 3 h p.i (time defined to represent the initial level of parasite development before drug application) to 27 h p.i. or 45 h p.i. was 97 and 99% respectively, and by 25 µM halofuginone 99% (27 h p.i.). Hexadecylphosphocholine (miltefosine), a new anti-leishmanial compound, was tested against cryptosporidia and provided a maximum of 98% reduction of parasite multiplication at 45 h p.i. The potential activity of curcumin (extract from the herb Curcuma longa) against C. parvum was also evaluated by cc-qPCR. Curcumin appeared to be sensitive to degradation after prolonged incubation and the observed inhibition of multiplication of C. parvum was significantly increased when medium was replaced by fresh medicated medium after 12 h of exposure. The effects on parasite multiplication (>95% inhibition with IC50 value of 13 µM) and on sporozoite invasion (assessed 3 h p.i.; 65% inhibition at 200 µM) suggest that further exploration of anticryptosporidial efficacy of curcumin may be rewarding. The cc-qPCR was further optimized to analyse inactivation measures directed against oocysts of C. parvum. The suitability of the assay for assessment of inactivation measures was confirmed by the reproducible demonstration of effectiveness of cresolic disinfectants at the recommended concentration of 4% and incubation period of 2 h (Neopredisan® 135-1, Menno Chemie, Norderstedt, Germany: 99.91% ± 0.08; Aldecoc® TGE, EWABO Chemikalien GmbH & Co. KG, Wietmarschen, Germany: 99.91± 0.05) and by using thermally inactivated oocysts (complete inactivation by 56°C and 70°C for 20 min). Based on the in vitro results and previously obtained data from the chicken infection model 99.5% inactivation is proposed as a suitable threshold value that needs to be consistently exceeded by a product to be considered efficient. Application of Neopredisan® 135- 1 and Aldecoc® TGE (4% for 2h) consistently inactivated more than 99.5% of oocysts while other disinfectants that are not certified as anticoccidial products like Aldecoc® XD (EWABO Chemikalien GmbH & Co. KG, Wietmarschen, Germany) and IGAVET® FF spezial (COS OHLSEN Chemie & Gerätevertrieb GmbH, Geltorf-Esprehm, Germany) and bleach (sodium hypochlorite) did not. It can be concluded that the cc-qPCR method is suited to easily and reliably assess anticryptosporidials in vitro. The method demonstrated that miltefosine and curcumin display anticryptosporidial efficacy under the applied conditions. The cc-qPCR is a highly standardized method supposedly appropriate to replace the chicken infection model for Eimeria tenella as currently practised for certification of anticoccidial disinfectants according to the guidelines of DVG (German Veterinary Society).

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 168-181.