Curcumin is one of the major curcuminoids produced by the ginger family Zingiberaceae. These curcuminoids possess pharmacological properties that include anticancer activities. We have evaluated some synthetic curcumin analogues that have shown potential as anticancer drugs. These antineoplastic agents bearing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore are electrophiles which are designed to preferentially react with sulfhydryl groups present in proteins as opposed to amino and hydroxyl groups present in DNA. In previous pilot studies, three derivatives examined in this thesis showed inhibition towards human cancer cell lines such as Molt 4/C8 and CEM T-lymphocytes. In this thesis work, I determined the cytotoxicity of these derivatives and curcumin towards human colon cancer (HCT-116) cells and also normal colon epithelial (CRL-1790) cells, and examined the possible mechanism(s) involved. I hypothesized that they act via induction of reactive oxygen species (ROS) which elicit a transient surge of mitochondrial ROS generation and a phenomenon known as ROS-induced ROS release (RIRR), along with the mitochondrial permeability transition (MPT) and mitochondrion –dependent apoptosis. I asked whether these agents react with some of the key protein thiols in the mitochondria whose oxidation/alkylation results in mitochondrion - dependent apoptosis. NC-2109 and NC-2346 were found to be potent cytotoxic agents based on their GI50 values of 0.87 ± 0.38 μM and 0.90 ± 0.22 μM, respectively, and were more potent than the anticancer drug 5-fluorouracil (GI50 = 5.47 ± 0.55 μM) and curcumin (GI50 = 3.50 ± 0.36 μM). However NC-2109 was found to have a better selectivity towards cancer cells over normal cells (a selectivity index of 18.81 versus 5-FU, curcumin and NC-2346 which had selectivity indices of 1.87, 16.75 and 4.61, respectively). In the investigations of the mechanisms involved, both curcumin and curcumin analogues were able to induce mitochondrial ROS production. Moreover, curcumin and its synthetic counterparts showed a biphasic ROS profile which is most characteristic of RIRR. Treatment with these agents also led to the disruption of the mitochondrial membrane potential, suggesting oxidation of protein thiols and the opening of the mitochondrial permeability transition pore which is an important step to initiate mitochondria-directed apoptosis. This possibility was confirmed based on GSSG/GSH ratios, since curcumin, NC-2346 and NC-2109 all produced a higher GSSG/GSH ratio than the controls. In addition to their ability to depolarize the mitochondrial membrane in HCT-116 cells, that these molecules acted via the mitochondrial pathway were further authenticated based on their ability to induce mitochondrial swelling in rat liver mitochondria. In another part of this thesis I evaluated the involvement of the critical thiol protein adenine nucleotide translocase (ANT), a bifunctional protein that plays a central role in mitochondrial apoptosis. ANT has four different isoforms; ANT1 and ANT3 are proapoptotic, while ANT2 and ANT4 are antiapoptotic and are overexpressed in cancer states. A combination approach using ANT2 siRNA however did not conclusively show whether these agents acted synergistically with ANT2 knockdown to potentiate mitochondria-mediated cell death. An alternative combination approach was the use of carboxyatractyloside (CAT) which binds to and retains ANT in its ‘c’ conformation, exposing thiols and potentially driving a cell towards programmed cell death. The presence of CAT enhanced the ability of curcumin and its synthetic analogs to collapse the mitochondrial membrane potential, an important step in mitochondrial-mediated apoptosis. In conclusion, curcumin and the curcumin analogue NC-2109 were found to be cytotoxic in vitro, towards HCT-116 cells and also showed good selectivity. In addition, these two molecules were found to be ROS inducers, and coincidentally oxidized cellular thiols and caused depolarization of the mitochondrial membrane potential. The results support a mechanism of mitochondrial-mediated cell death upon MPT pore formation (mitochondrial swelling), perhaps involving ANT2. This conclusion was further supported by the potentiation of cell death in the presence of the ANT2 inhibitor, CAT.

Tender coconut water (TCW) is one of most consumed natural energy drinks. Although it is widely available in tropical countries, because of its natural health benefits it is increasingly consumed in other parts of the world. Therefore there has been an increased requirement for packaging and shipping. TCW is highly susceptible to microbial contamination during extraction and packaging, requiring pasteurization. Thermal pasteurization leads to loss of flavor and color of coconut water. The purpose of this study was to test the feasibility of a non-thermal method of pasteurization of TCW using natural antimicrobials like curcumin, eugenol, and d-limonene with curcumin. The non-thermal pasteurization method was developed in two different processes, i.e. a batch and flow-filter process. Batch cold pasteurization process was conducted by suspending liposome encapsulated antimicrobials immobilized on glassbeads in TCW at 4oC by shake flask assay. Maximum Reduction of Listeria monocytogenes and Escherichia coli W1485 in pasteurized TCW were observed to be 5 log CFU per ml of TCW within 7 days of incubation under the presence of nano-liposomes containing 50 mM of curcumin, d- limonene or eugenol. There was no change in color and aroma of TCW after 1 month of storage at 4oC. the flow filter cold pasteurization process was developed using filter medium coated with nano-liposomes encapsulated antimicrobial leading to extended release of the natural antimicrobial curcumin to the coconut water in 4oC. Listeria monocytogenes and E. coli W1485 were used as model organisms to test effectiveness of cold pasteurization. Reduction of L. monocytogenes and E. coli W1485 in pasteurized TCW was observed to be 5.2 and 4.5 log10CFU/ml of TCW respectively within 17.25 min of incubation under the presence of nano-liposomes containing 50 mM of curcumin. There was no change in color and aroma of TCW after 1 week of storage at 4oC.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-03-16T11:11:39Z No. of bitstreams: 2 Dissertação - Fernanda Almeida Andrade - 2017.pdf: 8476311 bytes, checksum: 7b482a52ab904b738a33195974b7ec65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-20T12:58:05Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda Almeida Andrade - 2017.pdf: 8476311 bytes, checksum: 7b482a52ab904b738a33195974b7ec65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-20T12:58:05Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda Almeida Andrade - 2017.pdf: 8476311 bytes, checksum: 7b482a52ab904b738a33195974b7ec65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-09 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Infections caused by yeasts of the genus Candida are called candidiasis and are considered opportunistic diseases with characteristics ranging from superficial to disseminate. Candida species may develop defense mechanisms to the major antifungal agents available for clinical use against infections, making it difficult and limiting treatment. Natural products can be a source of bioactive compounds against infectious diseases, such as the Curcuma longa Linn or saffron plant as it is popularly known that it has the compound curcumin. This vegetable is used in large scale by the population as food condiment and as natural medicine against inflammatory diseases and infectious diseases. Laboratory-synthesized compounds have been to interest in medical biotechnology, among which the hydrazone derivatives are associated with antimicrobial including antifungal, antioxidant, antiparasitic, anticonvulsive, analgesic, anti-inflammatory and antitumor activity. The objectives were to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of curcumin and n-acylhydrazone compounds, and also to verify the time-kill curve of Candida, to quantify ergosterol and to evaluate hemolysis of red blood cells after action of both compounds. Twenty isolates Candida spp. in the broth microdilution test for evaluation of the MIC and MFC of the compounds. The time-kill curve and ergosterol assay were checked after treatment of C. albicans ATCC 28367 with the compounds. To evaluate the cytotoxicity of these compounds sheep red blood cells were used. The compound curcumin showed MIC values between 16 and 256 μg/mL and MFC between 128 and values greater than 512 μg/mL. For the n-acylhydrazone derivative the MIC values ranged from 2 to 128 μg/mL and the MFC variation ranged from 256 to values greater than 512 μg/mL. The compounds showed fungistatic action on the growth kinetics of Candida over 48 hours. The determination of ergosterol after contact with the agents in MIC of 64 μg/mL of curcumin and 16 μg/mL of nacylhydrazone was reduced by 29.56 and 53.57%, respectively. The compounds showed hemolytic activity at concentrations of 512 μg/mL. The results observed in this work show that the curcumin and the n- acylhydrazone derivative have promising antifungal properties. / As infecções causadas por leveduras do gênero Candida são denominadas candidíase e são consideradas doenças oportunísticas com características superficiais e sistêmicas. Espécies de Candida podem desenvolver mecanismos de defesa aos principais antifúngicos disponíveis para uso clínico contra infecções, dificultando e limitando o tratamento. Produtos naturais podem ser fonte de compostos bioativos contra doenças infecciosas, como por exemplo a planta Curcuma longa Linn ou açafrão como é popularmente conhecida que apresenta em sua constituição o composto curcumina. Esse vegetal é utilizado em grande escala pela população como condimento alimentar e como medicamento natural contra doenças inflamatórias e doenças infecciosas. Os compostos sintetizados laboratorialmente têm sido de interesse em biotecnologia médica, entre esses, os derivados hidrazonas estão associados com atividade antimicrobiana incluindo antifúngica, antioxidante, antiparasitária, anticonvulsiva, analgésica, anti-inflamatória e antitumoral. Os objetivos deste trabalho foram determinar a concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM) dos compostos curcumina e N-acilhidrazona, e ainda verificar a curva de morte de Candida, quantificar o ergosterol e avaliar a hemólise de hemácias após ação de ambos compostos. Foram utilizados 20 isolados Candida spp. no teste de microdiluição em caldo para avaliação da CIM e CFM dos compostos. A curva de morte e doseamento de ergosterol foram verificados após tratamento de C. albicans ATCC 28367 com os compostos. Para avaliação da citotoxicidade destes compostos utilizou-se hemácias de carneiro. O composto curcumina apresentou valores de CIM entre 16 e 256 μg/mL e CFM entre 128 a valores maiores que 512 μg/mL. Para o derivado Nacilhidrazona os valores de CIM variaram de 2 a 128 μg/mL e a variação de CFM foi entre 256 a valores maiores que 512 μg/mL. Os compostos mostraram ação fungistática sobre a cinética de crescimento de Candida ao longo de 48 horas. O doseamento de ergosterol após contato com os agentes na CIM de 64 μg/mL da curcumina e 16 μg/mL de N-acilhidrazona mostrou-se reduzido em 29,56 e 53,57%, respectivamente. Os compostos apresentaram atividade hemolítica em concentrações a partir de 512 μg/mL. Os resultados observados neste trabalho evidenciam que curcumina e o derivado N-acilhidrazona apresentam propriedades antifúngicas promissoras.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-06-01T22:28:55Z No. of bitstreams: 2 Dissertação - Vivianny Aparecida Queiroz Freitas - 2017.pdf: 4680258 bytes, checksum: 0e08c83bdb454e044ddcef0daedb874a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-02T11:13:05Z (GMT) No. of bitstreams: 2 Dissertação - Vivianny Aparecida Queiroz Freitas - 2017.pdf: 4680258 bytes, checksum: 0e08c83bdb454e044ddcef0daedb874a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-02T11:13:05Z (GMT). No. of bitstreams: 2 Dissertação - Vivianny Aparecida Queiroz Freitas - 2017.pdf: 4680258 bytes, checksum: 0e08c83bdb454e044ddcef0daedb874a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cryptococcosis is a fungal disease caused by yeasts belonging to the complex Cryptococus neoformans. The antifungal arsenal available for the treatment of this disease is still restricted and is related to the high toxicity and side effects of some drugs, causing great harm to the patients. In this context, it is necessary to discover new bioactives to quell the infections and reduce the adverse effects. The plants have rich source of secondary bioactive metabolites such as tannins, terpenoids, saponins, alkaloids, flavonoids and other compounds, registered with expressive antimicrobial properties. Therefore, the research of natural compounds and derivatives of natural products has been relevant in recent years, due to their relevance in the discovery of new drugs, in addition to which the association of drugs or compounds with different mechanisms of action has been used as an alternative in conventional therapy. We evaluated the mechanisms of action and antifungal effect of natural compounds, curcumin and morin, on Cryptococcus neoformans and Cryptococcus gattii isolates. The antifungal activity was determined across the minimum inhibitory concentration (MIC) and minimum fungicide (MFC). Polyphenols showed MICs ranging from 2 to 256 μg/mL. The compounds showed a synergistic interaction in 30% (6/20) and 15% (3/20) of the isolates when dealing with morin and curcumin, respectively, associated with fluconazole. In the interaction with amphotericin B morin presented synergism in 70% (14/20) of the isolates. Both compounds did not exhibit antagonism in any of the combinations. Changes in fungal cell morphology were observed, and in contact with red blood cells, presented low toxicity. The mechanisms of action revealed that both polyphenols act on the membrane of the fungal cell, inhibiting the synthesis of ergosterol and causing damage to it. These results demonstrate the potential of the compounds as natural bioactives, with great impact on the discovery of new drugs and their efficacy to be used in the treatment of fungal infections. / A Criptococose é uma doença fúngica causada por leveduras pertencentes ao complexo Cryptococus neoformans. O arsenal antifúngico disponível para o tratamento dessa doença ainda é restrito e está relacionado à elevada toxicidade e efeitos colaterais de alguns fármacos, causando grande prejuízo aos pacientes Neste contexto, se faz necessário descobrir novos bioativos para debelar as infecções e reduzir os efeitos adversos. As plantas apresentam rica fonte de metabólitos secundários bioativos como taninos, terpenóides, saponinas, alcalóides, flavonoides e outros compostos, registrados com expressiva propriedade antimicrobiana. Portanto, a pesquisa de compostos naturais e derivados de produtos naturais tem sido pertinente nos últimos anos, devido à sua relevância na descoberta de novos medicamentos, além do que, a associação de fármacos ou compostos, com diferentes mecanismos de ação tem sido utilizada como alternativa na terapia convencional. Nós avaliamos os mecanismos de ação e o efeito antifúngico dos compostos naturais, curcumina e morina, sobre isolados de Cryptococcus neoformans e Cryptococcus gattii. A atividade antifúngica foi averiguada através da determinação da concentração inibitória mínima (CIM) e fungicida mínima (CFM). Os polifenóis, apresentaram CIMs que variaram de 2 a 256 μg/mL. Os compostos apresentaram interação sinérgica em 30% (6/20) e 15%(3/20) dos isolados se tratando da morina e curcumina, respectivamente, associada ao fluconazol. Na interação com a anfotericina B a morina apresentou sinergismo em 70% (14/20) dos isolados. Ambos os compostos não apresentaram antagonismo em nenhuma das combinações. Alterações na morfologia da célula fúngica foram observadas, e em contato com as hemácias, apresentaram baixa toxicidade. Os ensaios de mecanismos de ação revelaram que ambos os polifenóis atuam na membrana da célula fúngica inibindo a síntese de ergosterol e causando danos a mesma. Estes resultados demonstram o potencial dos compostos como bioativos naturais, apresentando grande impacto na descoberta de novos fármacos e sua eficácia para serem utilizados no tratamento de infecções fúngicas.

Le récepteur de facteur de croissance épidermique (EGFR) est une cible commune de thérapie anticancéreuse. Aujourd'hui, la recherche de nouvelles molécules inhibitrices de ce récepteur se tourne vers des substances naturelles. Un des composés naturels les plus prometteurs qui ont montré une activité anti-EGFR est la curcumine, un polyphénol présent dans les rhizomes de Curcuma longa. Il y a de nombreux rapports décrivant son effet sur l'activité kinase du récepteur, le rendement d'autophosphorylation, le niveau d'expression et les processus liés à la fonction EGFR comme la prolifération cellulaire. Néanmoins, l'ensemble des mécanismes d’intercation de la curcumine avec l'de l'EGFR n’est pas entièrement élucidée. Nous avons démontré que le mode d'action de la curcumine est double. La curcumine est capable d'inhiber partiellement, mais directement l'activité enzymatique du domaine intracellulaire de l'EGFR. Mais le travail présenté attire l'attention sur le rôle de l'environnement de la membrane de l'EGFR au niveau d'action de la curcumine. Nous avons montré que l'insertion de curcumine dans la membrane plasmique aboutit à sa rigidification et par conséquent la limitation de la diffusion du récepteur. Le suivi de particules à l'unité analyses a confirmé que le coefficient de diffusion de l'EGFR dans la membrane des cellules cancéreuses diminué de manière significative en présence de la curcumine, susceptibles d'influencer la dimérisation du récepteur et l'activation tour à tour. / Epidermal Growth Factor Receptor (EGFR) is a common target of anticancer therapy. Nowadays the search for new molecules inhibiting this receptor is turning towards natural substances. One of the most promising natural compounds that have shown an anti-EGFR activity is curcumin, the polyphenol found in the rhizomes of Curcuma longa. There are numerous reports describing its effect on the receptor kinase activity, the autophosphorylation yield, the expression level and the processes related to EGFR function like cell proliferation. Nevertheless, the entire mechanism of how curcumin interact with the EGFR is not fully elucidated. We demonstrated that the mode of action of curcumin is dual. Curcumin is able to inhibit directly but partially the enzymatic activity of the EGFR intracellular domain. But the presented work brings attention to the role of the EGFR membrane environment at the curcumin action level. We showed that the curcumin insertion in plasma membrane leads to its rigidification and as a consequence to limitation of the receptor diffusion. Single particle tracking analyses confirmed that the diffusion coefficient of EGFR in the cancer cell membrane significantly decreased in the presence of the curcumin, which might influence the receptor dimerization and in turns its activation.

O câncer é uma doença que causa grande número de mortes a cada ano e os tratamentos convencionais normalmente ocasionam efeitos colaterais indesejados além do que nem sempre são eficazes. A terapia fotodinâmica (TFD) é uma modalidade alternativa de tratamento, que se baseia na combinação de um fotossensibilizador (FS), luz e oxigênio, e tem mostrado resultados promissores em vários tipos de câncer. No entanto, existem alguns desafios para o desenvolvimento de fotossensibilizadores que cumprem os requisitos para uso clínico. Os compostos, hipericina (HY) e curcumina (CUR), estão presentes em plantas medicinais, possuem altos rendimentos quânticos e podem ser usados como FS em TFD. O objetivo deste trabalho foi comparar a eficiência fotodinâmica da HY e CUR através de suas propriedades físico-químicas e biológicas, tais como coeficiente de absortividade molar (&#949), coeficiente de partição (Log P), fotodegradação, atividade fotodinâmica de geração de oxigênio singlete e a resposta biológica usando ensaios citotóxicos e microscopia de fluorescência para avaliar o tipo de morte celular. A HY apresenta propriedade anfifílica, enquanto a CUR, hidrofóbica. A HY é mais fotoestável, enquanto a CUR fotodegrada facilmente sob irradiação em 455 nm levando à formação de fotoprodutos que possuem menor citotoxicidade que a CUR íntegra, o que sugere que a eficiência da TFD diminui conforme este corante é irradiado. Além disso, a alta eficiência de geração de oxigênio singlete de HY corrobora com a sua fototoxicidade mais elevada em comparação com a CUR. A concentração inibitória média (IC50) da HY em células de carcinoma epidermóide de laringe humana (HEp-2) é 256 vezes menor para CUR sob a mesma dose de luz. A investigação do tipo de morte celular foi realizada por microscopia de fluorescência após TFD utilizando os FSs em concentração equivalente a duas vezes o valor de IC50. HY-TFD promoveu cerca de 1,5 vezes mais apoptose nas células HEp-2 do que a CUR. Além disso, a acumulação intracelular dos FSs analisada por Microscopia Confocal de fluorescência mostrou que a CUR apresenta uma velocidade de acumulação menor que a HY. Em conclusão, os resultados obtidos sugerem que a HY é um FS mais eficiente que a CUR para TFD, porém como a CUR e a HY apresentam baixa toxicidade na ausência de luz, são compostos seguros para serem utilizados clinicamente. / Cancer is a disease that causes several deaths each year and the treatment modalities are not quite selective and usually bring about intensive side effect besides not very effective sometimes. Photodynamic therapy (PDT) is an alternative treatment, which is based on the combination of a photosensitive molecule, light activation and oxygen. Many studies have shown promising results with PDT in diverse types of cancer. However, there are some challenges for the development of photosensitizers to comply the requisites for clinical use. The compounds hypericin (HY) and curcumin (CUR), are present in medicinal plants, have high fluorescence quantum yields and can be used as PS in PDT. In this study, we compare HY and CUR through the study of theirs physic-chemical and biological properties, as well as photo degradation, determination of the partition coefficient (Log P) and the molar absorptivity coefficient (&#949), the photodynamic activity as an indirect estimation of singlet oxygen generation potential and the biological response using cytotoxicity assays as well as fluorescence microscopy. The results suggested that HY presents amphiphilic property while CUR is hydrophobic. HY is more photostable than CUR, which is easily photodegradated at 455 nm leading to the formation of photoproducts. The cytotoxicity of these photoproducts was investigated being lower than that of CUR, which suggests that the efficiency of CUR-PDT decreases while this dye is irradiated. Furthermore, the elevated oxygen singlet generation of HY corroborates with its higher phototoxicity compared to CUR. The median inhibitory concentration (IC50) of HY in human epidermoid cancer cells (HEp-2) is 256 times lower than the IC50 for CUR at equal light irradiance. The investigation on the type of cell death was carried out by fluorescence microscopy after PDT using the photosensitizers at concentration equivalent to 2x IC50 values. HY-PDT induced around more 1.5 times apoptosis cell death than CUR-PDT. Besides, the intracellular accumulation of PS analyzed by fluorescence Confocal Microscopy has demonstrated that the accumulation of CUR is slower than HY. In conclusion, our findings suggest that HY is a more efficient PS than CUR for PDT, however, as CUR and HY present low toxicity in the absence of light, they are safe compounds to be clinically used.

Autophagy is the major cellular, conservative, lysosomal catabolic process to eliminate and recycle intracellular waste and organelles through autophagosomes. Enhancing autophagy to promote the clearance of toxic proteins is developing as a promising approach to treat proteinopathy disorders like Alzheimer's disease (AD). AD is the most common aging-associated neurodegenerative disease. It is characterized by the aggregation of aberrantly hyperphosphorylated tau (p-Tau) and excessively produced Amyloid-beta (Aβ) into neurofibrillary tangles (NFTs), and amyloid plaques (AP) respectively. Reprogramming autophagy lysosomal pathway (ALP) through autophagy master controller, transcription factor EB (TFEB), is developing as an attractive strategy to treat AD. It is already proven that TFEB overexpression can promote Aβ and p-Tau lysosomal clearance, attenuate NFT and AP deposition and restore the behavioural deficits in AD mice models. Previously Song et al., 2016 have identified a small molecule curcumin derivative C1. They reported that C1 could bind to recombinant TFEB and enhance ALP both in vitro and in vivo conditions independent of mTOR inhibition. In the current study, C1 is systematically evaluated for its bioavailability, anti-AD efficacy in vitro, and in vivo AD experimental models. To validate TFEB mediated anti-AD efficacy of C1 in vitro, we tested the C1 effect on amyloid precursor protein (APP) and p-Tau degradation in vitro neuronal AD cell culture models. In N2a cells overexpressed with APP (695) and EGFP-P301L tau plasmids, C1 induced APP, CTFβ, and Tau lysosomal degradation. To demonstrate the TFEB dependent autophagic clearance effects of C1, TFEB is silenced in N2a cells with lentiviral shRNA particles. Under TFEB silenced condition, C1 induced reduction of FL-APP, CTFβ, and Tau was compromised. Overall In vitro experiments show that C1 induced lysosomal digestion of FL-APP, CTFβ, and p-Tau in a TFEB dependent manner. To further demonstrate C1 brain bioavailability, C1 and curcumin comparative pharmacokinetic studies (Pk study) in both mice (time course Pk study) and rat (single time point analysis) are conducted. In Pk studies, both C1 and curcumin are dosed at 10 mg/kg to determine their concentration in the whole brain (mice), separate brain regions (rat), CSF (rat), and plasma. The WinNonlin analysis of C1 and curcumin mice Pk study data revealed that C1 is significantly more bioavailable than curcumin in both brain and plasma, which is also corroborated by the single time point analysis in rats. To illustrate C1 anti-AD activity in vivo, C1 is screened in homozygous P301S (Tau), heterozygous 5xFAD (Aβ), and homozygous 3xTg (both Aβ and Tau) AD transgenic mice models. These mice were started to treat with C1 before the onset of AD pathology until the AD pathological phenotype is expressed to cause impairment in mice behaviour. In mice behavioural examination, C1 treatment has significantly improved mice motor function (Rotarod-P301S), restored cognitive impairment related to the cortex (contextual fear conditioning-5xFAD), hippocampus (Morris water maze-3xTg) and improved cholinergic activation (open field-3xTg). In the brain biochemical examination, C1 activated the TFEB mediated ALP pathway to degrade FL-APP, CTFα/β, Aβ, and p-Tau and reduced the amyloid plaque load, extra neuronal-NFT positive cells. Notably, C1 treatment in 5xFAD mice has significantly restored hippocampal synaptic function. In summary, the current study validates C1 as an orally bio-available potent small molecule TFEB activator which restores mice cognitive impairment, altered behaviour, and synaptic plasticity by reducing Aβ and tau levels in AD experimental models. Overall, the TFEB activator C1 can be a promising drug to treat AD.

Over the past decades, numerous studies shown curcumin, a dietary compound derived from turmeric, has a variety of health-promoting actions, such as anti-oxidant, anti-microbial, anti-inflammatory, and anti-cancer effects, making curcumin the most promising dietary compound for disease prevention. However, the underlying mechanisms by which curcumin has these health-promoting effects are not well understood. A better understanding of the molecular mechanism of curcumin could help to develop novel strategies to reduce the risks of some human diseases. Protein thiols play important roles in cell signaling, and recent studies showed that curcumin could covalently react with protein thiols, supporting that curcumin-protein interactions could contribute to the health-promoting effects of curcumin. However, the curcumin-protein interactions are under-studied. Notably, it remains unknown whether oral intake of curcumin could covalently interact with protein in vivo. In this project, we synthesized a click chemistry probe of curcumin (Di-Cur), and used this probe to characterize curcumin-protein interactions both in vitro and in vivo using a click chemistry-based imaging approach. Our results demonstrate that orally administrated curcumin could form curcumin-protein adducts in specific tissues of the mice, which may contribute to the potent biological effects and poor pharmacokinetics of curcumin.

Glycation of whey proteins results in food-grade composites with modified physicochemical properties. Here, the reaction between glucose and bovine serum albumin (BSA) is promoted under wet-heating conditions. The glycated protein is characterized in depth and compared to the native counterpart and the impact of glycation on properties like net surface charge, particle size and surface hydrophobicity are observed. Conjugation with glucose reduced the surface hydrophobicity of BSA but the interactions between albumin and curcumin became stronger, which contradicts the direct relationship between curcumin binding affinity and protein surface hydrophobicity described in the literature. Nonetheless, curcumin was still capable of quenching the intrinsic fluorescence of the protein after conjugation with glucose and leads to the conclusion that curcumin and BSA interact in a different manner upon glycation. This thesis also depicts mucin as a forthcoming model in the study of nanoparticle interactions with intestinal mucus and glycation posed no effect on such interactions.

Curcumin, a polyphenol, has been suggested to improve metabolic byproduct clearance and increase nitric oxide production in working muscle. These purported effects may delay neuromuscular fatigue. Therefore, the purpose of this study was to examine the effects of curcumin in combination with fenugreek (CUR) or fenugreek soluble fiber alone (FEN) on the neuromuscular fatigue threshold (PWCFT), time to exhaustion (Tlim) on a graded exercise test (GXT), and O2peak in untrained subjects.Forty-seven,college-aged, aerobically untrained individuals were randomly assigned to one of three supplementation groups; placebo (PLA, n=15),curcumin + fenugreek, CurQfen® (CUR, n=18), or fenugreek soluble fiber (FEN, n=14). All subjects performed a maximal GXT on a cycle ergometer to determine the PWCFT, Tlim, and O2peak before (PRE-test) and after (POST-test) 28 days of daily supplementation. Statistical analyses included 3 separate, one-way ANCOVAs to determine if there were any differences among the groups (PLA, CUR, FEN) for adjusted post-test scores for the PWCFT, O2peak, and Tlim. The respective pre-test score was used as the covariate. In addition, reliability analyses (PRE- to POST-test) for the PLA group were used to calculate the minimal difference needed to be real (MD). The adjusted POST PWCFTvalues showed no statistical differences between groups (F= 3.141p= 0.053); however pairwise LSD comparisons indicated a significant difference between the CUR and PLA groups (p= 0.016), but not between the CUR and FEN groups. Therefore, separate one-way ANCOVAs were used to examine the adjusted PWCFTmeans for the PLA vs. CUR (F = 4.906, p =0.035) and the PLA vs. FEN (F = 2.969, p = 0.097). The one- way ANCOVA for O2peak (F= 0.612 p= 0.547) and Tlim(F = 0.688, p = 0.508) values showed no statistical difference among the groups. Individual responses in each group showed ~ 20% of subjects in the CUR group, ~ 7% in the FEN group, and ~6% in the PLA group had values greater than the MD for the PWCFT, but none of the subjects in the PLA, FEN, or CUR groups exceeded the MD for O2peak or Tlim. These findings indicatedCurQfen® supplementation increased the PWCFTcompared to a placebo, but not compared to fenugreek soluble fiber alone. However, there were no effects of CurQfen® on O2 peak or Tlim. The mechanisms responsible for delaying time to neuromuscular fatigue may include increased NO production and increased blood flow to remove metabolic byproducts; however, the cellular changes which could lead to increases in Tlim andO2peak may not have been sensitive to the GXT protocol or the given dosage of curcumin supplementation. Considering individual responses, CurQfen® supplementation resulted in a real change in the PWCFTfor a small portion of the subjects (~20%). These findings suggested that CurQfen® supplementation without exercise training may help to improve time to neuromuscular fatigue in certain individuals, but the group mean analyses were not necessarily reflective of the responses for a majority of the subjects.