A informação relativa aos orientadores da tese foi retirada da página do SIFEUP / Tese de doutoramento. Engenharia Química e Biológica. Faculdade de Engenharia. Universidade do Porto. 2009

Alzheimer’s disease (AD) is a deleterious neurodegenerative disease caused in major part by the aberrant processing and accumulation of amyloid beta peptides. In this dissertation, we systematically investigated the role of N-terminal region (NTR) residues of amyloid (1-40) (Aβ40) peptide in amyloidogenesis, lipid bilayer membrane interaction and damage, as well as neurotoxicity. Herein, we investigated the role of NTR residues on the aggregation and amyloid fibril formation process, to gain understanding on the electrostatic and hydrophobic constituents of the mechanism. This was achieved by substituting specific charged residues located in the NTR of Aβ40 and investigating their effects through a variety of techniques. We also investigated the role of NTR charged residues in their interaction with supported phospholipid bilayer membranes through the use of Quartz Crystal Microbalance with Dissipation (QCM-D) monitoring to gain insight on the mechanistic details of the interaction. To further understand the implications of substituting charged NTR residues on membrane interaction, pore formation and damage, we utilized a carboxyfluorescein dye leakage assay to quantify the membrane damage caused by Aβ40 and the NTR mutants. We also performed neurotoxicity assay with SH-SY5Y neuroblastoma cells to shed light on the effects of NTR substitutions on cellular toxicity. Finally, we synthesized a polymer, trimethyl chitosan (TMC), and utilized it as a polyelectrolyte monitor of electrostatic interactions occurring between TMC and the NTR of Aβ40. Our results demonstrate that the NTR charged residues of Aβ40 contribute significantly to the aggregation process, amyloidogenesis, and phospholipid membrane interaction and perturbation by means of electrostatic, thermodynamic and hydrophobic forces. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Epidemiologische Studien zeigen, dass eine obst-, gemüse- und ballststoffreiche Ernährung mit einer verminderten Inzidenz des kolorektalen Karzinoms verbunden ist. Von den vielen Komponenten einer solchen Ernährung wurde insbesondere für die Gruppe der Carotinoide wie beta-Carotin und Lycopin eine tumorprotektive Wirkung beschrieben. Es gibt eine Vielzahl von möglichen Mechanismen für diese Schutzwirkung: Carotinoide sind potente Antioxidantien, und oxidativer Stress ist ein etablierter Faktor in der Karzinogenese. Auch können viele Carotinoide zu Retinoiden umgewandelt werden, die bekannte anti-kanzerogene Eigenschaften besitzen. Um zu untersuchen, ob Carotinoide tumorprotektive Eigenschaften durch eine Beeinflussung der Zellzyklusprogression ausüben, wurden in dieser Arbeit die möglichen Effekte von beta-Carotin und Lycopin auf die Proliferation von humanen Kolonkarzinomzelllinien und die Expression der an der Zellzyklusregulation beteiligten Proteine cdk2, p53 und PCNA sowie der Cdk-Inhibitoren p21waf1/cip1 und p27kip1 untersucht. Hierzu dienten die humanen Kolonkarzinomzelllinien HT-29 und SW-620 als in vitro-Modell. Im Western Blot wurde anschließend die Expression der entsprechenden Proteine bestimmt. Die Inkubation der Zellen mit Lycopin und beta-Carotin führte zu verminderter Proliferation der untersuchten Zelllinien. Dies steht in Einklang zu anderen Untersuchungen, die anti-proliferative Effekte von beta-Carotin und Lycopin zeigten. Es ließen sich jedoch keine Effekte auf die Expression der untersuchten, an der Zellzykluskontrolle beteiligten Proteine nachweisen. Es ergibt sich somit aus dieser Arbeit kein Anhalt dafür, dass die proliferationshemmenden Effekte dieser Carotinoide durch eine Modulation der Progression im Zellzyklus vermittelt werden. Vielmehr scheinen andere Mechanismen eine Rolle zu spielen. Weitere Untersuchungen wären nötig, z.B. mittels Transcriptom- und Proteom-Analytik, um die hier ursächlichen genetischen Veränderungen zu identifizieren. Ein weiterer beschriebener Mechanismus der Proliferationshemmung in Kolonkarzinomzellen ist die Aktivierung von PPAR-gamma. PPAR-gamma (peroxisome proliferator-activated receptor gamma) ist ein nukleärer Hormonrezeptor, der durch Liganden wie Prostaglandin D2, mehrfach ungesättigte Fettsäuren sowie Zyklooxygenase-Inhibitoren und Thiazolidindione aktiviert wird. Butyrat, das bei der bakteriellen Fermentation unverdauter Kohlenhydrate im Kolon entsteht, ist ein weiterer Ernährungsfaktor, der positiv mit einer erniedrigten kolorektalen Tumorinzidenz korreliert. In dieser Arbeit wurde untersucht, ob die anti-proliferativen in vitro-Wirkungen der kurzkettigen Fettsäure Butyrat auf Kolonkarzinomzellen mit einer Modulation der Expression von PPAR-gamma einhergehen. Butyrat führte in dieser Arbeit zu einer Proliferationshemmung der Kolonkarzinomzelllinien HT-29, SW-480 und SW-620. Darüber hinaus induzierte es in allen drei untersuchten Zelllinien die Expression von PPAR-gamma-Protein, welches in unbehandelten Zellen nur minimal nachweisbar war. Sollten Wirkungen von Butyrat zumindest teilweise über PPAR-gamma vermittelt werden, so müsste auch dessen Heterodimerisierungspartner RXR-alpha(retinoid X receptor alpha) in den Zellen vorhanden sein. Es zeigte sich, dass alle drei Zelllinien unbehandelt RXR-alpha exprimieren. Unter Butyrat-Inkubation verringerte sich interessanterweise die Expression von RXR-alpha-Protein. Diese Ergebnisse lassen sich möglicherweise als negativer Feedback-Mechanismus erklären, um einen überschießenden Effekt von PPAR-gamma oder seiner Liganden zu verhindern. Die untersuchten Carotinoide hatten dagegen keinen Effekt auf die Expression von PPAR-gamma. Zur Untersuchung des molekularen Wirkmechanismus von Butyrat diente Trichostatin A (TSA), welches die Histondeacetylase (HDAC) hemmt, die DNA so zugänglicher für Transkriptionsfaktoren macht und dadurch die Expression verschiedener Proteine moduliert. TSA inhibierte in gleicher Weise wie Butyrat die Proliferation der Zellen, induzierte die Expression von PPAR-gamma und supprimierte die Expression von RXR-alpha. Es ist also eine HDAC-Inhibition als Wirkweg der Butyrat-Wirkung auf die Zellen am wahrscheinlichsten, wodurch vermehrt PPAR-gamma gebildet wird. Denkbar ist, dass durch die so erhöhten intrazellulären PPAR-gamma-Spiegel die tumorprotektive Wirkung physiologischer oder synthetischer Liganden verstärkt wird. Eine Kombinationstherapie aus Butyrat und PPAR-gamma-Liganden zur Tumorprotektion ist so prinzipiell denkbar. Zukünftige Untersuchungen müssen das Augenmerk insbesondere auch auf die Klärung der umgebungsabhängigen PPAR-gamma-Wirkung richten, um die teils diskrepanten Befunde an Tumorzellkulturen und Tiermodellen einerseits, im natürlichen Epithelzellverband andererseits zu klären. / Epidemiological studies show that diets rich in fruit, vegetable and fiber are associated with a reduced incidence of colorectal cancer. Of special interest is for example the group of carotenoids, such as beta-carotene and lycopene. There are a number of possible tumor protective effects of carotenoids. They are potent antioxidants, and oxidative stress is a well-established factor in carcinogenesis. Also, many carotenoids are converted to retinoids, which have well-known anti-carcinogenic properties. We investigated whether carotenoids exert their tumor protective effects by influencing the cell cycle progression, possible effects of beta-carotene and lycopene on proliferation of human colon carcinoma cell lines (HT-29, SW-620) and expression of proteins involved in cell cycle regulation (cdk2, p53, PCNA, p21Waf1/Cip1 and p27kip1). Incubation of cells with lycopene and beta-carotene resulted in decreased proliferation of the cell lines examined. However, no effects on the expression of the examined proteins involved in cell cycle regulation could be observed. Another well known mechanism of the inhibition of proliferation in colon cancer cells is the activation of PPAR-gamma. PPAR-gamma (peroxisome proliferator-activated receptor gamma) is a nuclear hormone receptor, which is activated by ligands such as prostaglandin D2, polyunsaturated fatty acids, cyclooxygenase inhibitors, and thiazolidinediones. Butyrate which is produced during the bacterial fermentation of undigested carbohydrates in the colon is another dietary factor which is positively correlated with decreased colorectal cancer incidence. We investigated if antiproliferative effects of the short-chain fatty acid butyrate on colon cancer cells is exerted by modulating the expression of PPAR-gamma. Butyrate resulted in inhibition of proliferation of colon carcinoma cell lines HT-29, SW-480 and SW-620. Moreover, Butyrate induced the expression of PPAR-gamma protein in the three cell lines. The necessary heterodimerisation partner of PPAR-gamma is RXR-alpha (retinoid X receptor alpha). It was found that all three untreated cell lines express RXR-alpha. Under incubation with butyrate, the expression of RXR-alpha protein decreased. These results can be explained as a negative feedback mechanism to prevent excessive effect of PPAR-gamma and its ligands. The examined carotenoids, however, had no effect on the expression of PPAR-gamma. To study the molecular mechanism of action of butyrate, we used trichostatin A (TSA). TSA inhibits histone deacetylase (HDAC), which makes DNA accessible for transcription factors, and thereby modulates the expression of various proteins. TSA, in the same way as butyrate, inhibited proliferation of the cells, induced expression of PPAR-gamma and suppressed expression of RXR-alpha. Thus, inhibition of HDAC is likely to be at least one way in which butyrate effects cell by forming increased PPAR-gamma. Tumor protective effects of physiological or synthetic ligands may be enhanced by increased intracellular PPAR-gamma. Combined treatment with butyrate and PPAR-gamma ligands might be an effective anti-tumor strategy.

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Bladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-?? and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ??1, in the TGF- ?? pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ??1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.

在眾多產生貝他分配的方法中，我們研究Kennedy的演算法。在本文中，我們探討在小樣本下，不同參數組合(k,p,q,r) 產生同一貝他分配的情形。 / There are mAny methods for generating a beta distribution. In this study, we focus on the method proposed by Kennedy (1988). Let [A₁,B₁]=[0,1] And [A_n,B_n] be rAndom subinterval of [0,1] defined recursively as follows. Take C , D to be the minimum And maximum of k i.i.d rAndom points uniformly distributed on [A_n,B_n]; And choose [A_n+1,B_n+1] to be [C_n,B_n], [A_n,D_n] or [C_n,D_n] with probabilities p, q, r respectively such that p+q+r=1. Kennedy showed that the limiting distribution of [A_n,B_n] has a beta distribution on [0,1] with parameters k(p+r) And k(q+r). 　　Based upon the known asymptotic result, we study the small-sample behaviors among those combinations of k, p, q, r that have the same Beta(m, n) distribution, where m = k(p+r), n = k(q+r), through simulations. We conclude that smaller k's basically have better performAnces.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

<p>Muscle fatigue has always been of vital importance in most sports. A few possible factors have been reported to be the cause of muscular fatigue during high intensity exercise; depletion of glycogen, oxidative stress, disruption of contractile mechanisms and accumulation of metabolites. One of the theories of the cause of muscular fatigue, both in endurance and intermittent sports, is decreased pH levels due to increased concentration of H+ ions dissociated from lactic acid in muscle. Carnosine, a fairly unnoticed ergogenic aid, taken in the form of β-alanine has shown to potentially delay the onset of fatigue. Supplementation of β-alanine, would increase carnosine levels in muscle and may counteract the decrease in pH since carnosine functions as a H+ buffer. The purpose of the present study was to examine the effects of 8 weeks supplementation of β-alanine in distance runners and Swedish division four soccer players on aerobic capacity, intermittent recovery and muscular fatigue. The runners (n = 15) were tested in lactate profiling tests and the soccer players (n = 22) were tested in the Yo-Yo intermittent endurance test pre and post the 8-week test-period. The yo-yo test did not result in significant difference between the soccer players’ β-group and control-group (p = 0,29). Neither did the lactate test result in significant differences between the distance runners’ β-group and control-group in any of the five variables measured. However, a trend in difference was seen between groups in both velocity at lactate threshold (VLT) (p = 0,11) and recovery blood lactate (RBL) (p = 0,14) where the β-group had increased slightly from 16,8 ± 1,6 km/h to 17,0 ± 1,2 km/h in VLT and decreased from 4,5 ± 1,6 mmol∙L-1 to 3,1 ± 1,0 mmol∙L-1 in RBL. The results suggested that β-alanine may delay the onset of fatigue and improve performance in endurance sports such as running by increasing the removal of lactate acid from muscle.</p>