Philosophiae Doctor - PhD / Wastewater is normally composed of a mixture of pollutants. The type and composition of pollutants in a particular wastewater depend on the source of origin. The source and characteristics of a particular wastewater determine the ideal method of sewage treatment. Specific treatment techniques are effective in the removal of certain types of pollutants and may have no impact on the levels of other types of pollutants. Therefore, a combination of treatments and assessment of the quality of effluent before release into the environment is normally recommended. The assessment of effluent can be achieved by various techniques including chemical analysis and biological assays. Chemical analyses are commonly employed; however, they often pose detection problems and are considered to be uneconomical.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-05-24T17:29:59Z No. of bitstreams: 1 elainecarlosscherrerramos.pdf: 912925 bytes, checksum: 8f525d975ce8281bb5d0fd17c5f85f70 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-09-03T12:11:20Z (GMT) No. of bitstreams: 1 elainecarlosscherrerramos.pdf: 912925 bytes, checksum: 8f525d975ce8281bb5d0fd17c5f85f70 (MD5) / Made available in DSpace on 2018-09-03T12:11:20Z (GMT). No. of bitstreams: 1 elainecarlosscherrerramos.pdf: 912925 bytes, checksum: 8f525d975ce8281bb5d0fd17c5f85f70 (MD5) Previous issue date: 2018-03-01 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O ácido ursólico (ácido 3β-hidroxi-urs-12-en-28-oico) é um triterpeno pentacíclico, composto de 30 átomos de carbono, usualmente obtido a partir da extração e da purificação de diversas espécies vegetais. Estudos demostram efeitos benéficos do ácido ursólico (AU), incluindo sua capacidade imunomoduladora, anti-inflamatória, antitumoral e inibidor da metástase. Estudos sugerem que modificações estruturais do AU podem ampliar os efeitos biológicos benéficos deste composto. Desta forma, o objetivo deste trabalho foi avaliar in vitro o efeito citotóxico e imunomodulador de derivados do ácido ursólico. Dezoito derivados semissintéticos foram preparados e cedidos para a verificação de atividades biológicas. Inicialmente, foram realizados testes em células de adenocarcinoma alveolar humano (A549), avaliando o efeito citotóxico e pró-apoptótico dos compostos. O efeito dos derivados do ácido ursólico foi também avaliado em macrófagos RAW264.7, analisando a viabilidade celular, a produção de óxido nítrico e a produção de fator de necrose tumoral. Em ambas as linhagens de células foi verificada a influências dos derivados na expressão do fator de transcrição NF-κB. As células A549 e RAW264.7 foram tratadas com os derivados nas concentrações de (7,5, 15, 30, 60 e 90µM), por 3, 24 e 48 horas. Os compostos AUD1, AUD5, AUD6, AUD8, AUD11, AUD12, AUD13 e AUD16 foram capazes de reduzir a viabilidade e a expressão de NF-κB nas células A549, em uma ou mais concentrações testadas. O composto AUD1 foi capaz de reduzir a viabilidade celular nas concentrações 30, 60 e 90μM, sendo significativamente mais eficaz que o AU nestas concentrações. Além disso, AUD1, AUD5, AUD6 e AUD8 induziram apoptose, sendo os derivados AUD1 e AUD8 significativamente mais eficaz que AU na indução da apoptose. Em relação aos macrófagos RAW264.7, somente os compostos AUD12, AUD13, AUD15 e AUD16 não promoveram redução da produção de NO. Todos os demais compostos reduziram a produção de NO em pelo menos uma das concentrações testadas, sem afetar a viabilidade celular. AUD2 promoveu a melhor produção de NO na menor concentração (7,5 μM) com IC50>90µM. Os compostos AUD1, AUD3, AUD4, AUD5, AUD6, AUD7, AUD8, AUD11, AUD12, AUD13, AUD14, AUD15 e AUD18 foram capazes de reduzir a expressão de NF-κB em todas as concentrações testadas. Em relação ao TNF, todos os compostos avaliados foram capazes de inibir a produção desta citocina pelas células RAW264.7 nas concentrações de 60 e 90μM. Ao realizar o estudo das modificação estruturais em relação as atividade biológicas é possível sugerir que a esterificação na posição C-28 ou ainda a manutenção do grupamento hidroxila no C-3 parecem importantes para o aumento da citotoxicidade dos compostos quando testadas em células A549. Além disso, quando avaliado o efeito imunomodulador na resposta inflamatória, a esterificação (C-3 e/ou C-28) potencializa a ação dos derivados do ácido ursólico em células RAW264.7. / The ursolic acid (3β-hydroxy-urs- 12-en- 28-oic acid) is a pentacyclic triterpene constituted by 30 carbon atoms that is usually obtained through the extraction and purification in different plants. The anti-inflammatory, anti-tumoral, immunomodulatory and anti-metastatic activity of ursolic acid (UA) has already been shown in different studies. Structural modifications in important portions of UA can amplify the beneficial biological effects of this compound. With this, it was synthesized 18 semisynthetic compound derivatives of ursolic acid (UAD1UAD18), which can be promising molecules to treat diseases that need new therapeutic options. The aim of this study was to evaluate in vitro the cytotoxic and immunomodulatory effects of these ursolic acid derivatives. To verify the biological activities was realized tests in the adenocarcinoma human alveolar basal epithelial cells (A549) evaluating the cytotoxic and the proapoptotic effects of these compounds. The murine macrophage cells (RAW264.7) were used to evaluate the effects of the UADs on cellular viability, nitric oxide production and tumoral necrosis factor production. In both cells was evaluated the expression of NF-κB. A549 and RAW264.7 were treated with UA or UADs in the concentrations of 7.5µM, 15µM, 30µM, 60µM and 90µM during 3, 24 or 48 hours. The compounds UAD1, UAD5, UAD6, UAD8, UAD11, UAD12, UAD13 and UAD16 were able to reduce the viability and the expression of NF-κB in A549 cells, in at least one of the concentrations. UAD1 was able to reduce the cellular viability in the concentrations of 30µM, 60µM and 90µM, being more effective than UA. Moreover, UAD1, UAD5, UAD6 and UAD8 induced the apoptosis of A549, being, UAD1 and UAD8, significantly more effective than UA. In RAW264.7, only the compounds AUD12, AUD13, AUD15 and AUD16 were not capable of promoting the reduction of NO production. The other compounds reduced the NO production in at least one of the concentrations, without cytotoxicity (IC50). AUD2 showed the better reduction of NO in the lower concentration, with IC50>90µM. The compounds UAD1, UAD3, UAD4, UAD5, UAD6, UAD7, UAD8, UAD11, UAD12, UAD13, UAD14, UAD15 and UAD18 were able to reduce the NF-κB expression in all tested concentrations, however, all the compounds reduced the production of TNF in the RAW264.7 cells in the concentrations of 60µM and 90µM. In conclusion, when was related the structure to the biological activity of the compounds, can be suggested that the esterification at C-28 or the conservation of the hydroxyl at C-3 seems to be important for the cytotoxicity of the compounds against A549 cells. Furthermore, when evaluated the immunomodulatory effect on the inflammatory response, the esterification (C-3 and/or C-28) enhances the action of the ursolic acid derivatives on RAW264.7 cells.

Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH). The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types. Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results. In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.

Chapter 1: Introductionn Staphylococci are gram positive rotund bacteria that grow in clusters; and hence get their name. The genus of Staphylococcus comprises of over 30 species of which S. epidermidis and S.aureus are significant in their interaction with humans and are known to cause diseases. S.aureus invades various soft tissues and causes a vast multitude of diseases spanning from simple boils and abscesses to osteomyelitis and endocarditis, which can become fatal upon the onset of bacteremia and toxic shock. S. aureus has also been established as one of the leading causes of nosocomial infections especially because of their multi-drug resistant traits and their ability to colonize prosthetic devices and catheters. The increasing incidence of the multi-drug resistant strains and the rising prevalence of community acquired S. aureus infections mandates a comprehensive understanding of the pathogen and its biology, its intracellular fate and the defense mechanisms in the host. Towards this end, we have attempted to delineate some aspects of the pathogen’s virulence and the host responses to them. S. aureus normally inhabits the skin and mucosal surfaces as a commensal. Upon the onset of permissive circumstances it turns into an opportunistic pathogen. Immuno-compromised conditions or breach of skin can serve as the portals of entry for the pathogen. Upon entry, the bacteria encounter macrophages as the first line of defense in the host. Macrophages appear at the site of infection and phagocytose the bacteria, subjecting the pathogen to phagolysosomal degradation which facilitates antigen presentation and pathogen clearance. As part of their immune evasion mechanism, various pathogens are known to adopt a multitude of strategies to subvert this fate and survive in the host cells. This dissertation work aims at gaining insight into the staphylococcus-macrophage interaction in the ongoing host-pathogen duel, to gain better understanding about the pathophysiology and etiology of the disease. Chapter 2: Intracellular Trafficking of Staphylococcus aureus in Macrophages Successful targeting of the pathogen necessitates a comprehensive understanding of its biology and physiology in its interactions with the host. With this objective we undertook a study to uncover the intracellular niche of S. aureus in RAW264.7 murine macrophage-like cells. Any invading pathogen once internalized by the macrophage is contained in a phagosome, which undergoes progressive acidification and maturation from the early endosome to late endosome and ultimately fuses with the phagolysosome, where where the invading pathogen is subject to degradation. Through exhaustive electron microscopy of the infected macrophages, we show that S. aureus is present as a single bacterium per vacuole through the entire period of infection. We have further monitored the intracellular trafficking of the bacteria in the macrophage through confocal studies with endosomal markers which serve as indicators of vesicle maturation. Soon after the onset of the infection, the bacteria were found to be present in the early endosome (EEA-1 positive vesicles) which gradually matured into LAMP1 positive, late endosomal vesicles. However, only a small fraction of the bacteria containing vesicles were found to fuse with the lysosomes, suggesting that the bacteria prevented phagolysosomal fusion. We further observed that the bacteria did not prevent the acidification of the vesicles they resided in, but only limited their fusion with the lysosome. Taken together, our studies delineating the intracellular niche of S. aureus in RAW macrophages revealed that the pathogen has successfully evolved immune evasion mechanisms to overcome its phagolysosomal relegation. Chapter 3: Staphylococcus aureus Succumbs to the Hepcidin in Murine Macrophage We have further attempted to study the intracellular fate of the bacteria in macrophages towards gaining greater insight into its biology. Our studies on the intracellular fate of S. aureus in RAW264.7 cells revealed a distinct biphasic fate of the bacteria. The pathogen was found to replicate initially and this proliferative phase was subsequently followed by a gradual fall in its numbers. Interestingly however, the pathogen is never found to be cleared from the system suggesting the presence of a residual infective pool in the macrophages. We thus explored the possible mechanisms which could attribute to this biphasic intracellular fate of the bacteria. Macrophages come armed with a rich repertoire of defense mechanisms to incapacitate the invading pathogens. They have in their arsenal, reactive oxygen (ROS) and nitrogen species (RNS) and many potent anti-microbial peptides, apart from the lysosomal machinery, to degrade the invading pathogen. Upon investigation, we find that the RAW macrophages do not mount a ROS/RNS response when infected with S. aureus. Induction of these responses in the macrophage by alternate means further reveals that the pathogen is recalcitrant to death by these oxidative/nitrosative bursts. Of the antimicrobial peptides (AMPs) harbored by macrophages, we find that Hepcidin is up-regulated upon infection with S. aureus. Hepcidin is a peptide which is known to have a key regulatory role in iron homeostasis in addition to its potent antimicrobial functions. Since Hepcidin is known to be induced upon increased iron availability; we pre-treated the host cells with iron and monitored the effect of the same on bacterial fate. As expected, we observed that Hepcidin induction by pre-treatment with iron equips the macrophage to counter the pathogen better and thus leads to hastened and heightened clearance of the bacteria. This induction of hepcidin is significant at the mRNA and protein levels and is also corroborated by increased co-localisation of the bacteria with the anti-microbial peptide. Our studies thus identify hepcidin as a key line of the host defense towards countering the bacterial infection thus explaining the near complete bacterial clearance observed. Chapter 4: Global gene expression studies offering insight into potential immune evasion strategies of S.aureus in countering host offences. The interactions between host and the pathogen are multi-layered with the involvement of numerous players and many signaling cascades. In this light, we have attempted to get a holistic view of the host-pathogen interplay through microarray studies. These global profiling studies were aimed at identifying the important players in bacterial virulence and the macrophage response factors involved in countering the same in the context of S. aureus infection. The array was uniquely designed to incorporate both bacterial and host probes so as to facilitate parallel analysis of the host and pathogen gene expression profiles in the same sample. The expression profiling studies were carried out at three time points which represent the key phases of the bacterial infection viz. internalization, replication and clearance. A comprehensive analysis of the bacterial and host gene expression profiles under these phases provided insights into bacterial virulence and the host’s strategies to counter the same. We observe a large scale metabolic shut down in S. aureus subsequent to its internalization. We find the distinct up-regulation of a small subset of genes, majority of which are as yet uncharacterized. Amongst these were a few well-characterized virulence genes which remained active, representing the bacterial strategies to subvert the host immune response. The large scale down-regulation of gene expression can be possibly explained as the adaptation of the bacteria to the available metabolites and its submission to a quiescent phase of existence in the macrophage. In parallel, the host system exhibits the induction of TNF-α and up regulation of TLR2 and Nod2, which are typically triggered by a gram-positive infection. But simultaneously, we also observed a marked increase in the expression of anti-apoptotic and anti-inflammatory responses. This was re-iterated by a significant down-regulation in some of the pro-inflammatory, pro-apoptotic and antigen presentation involved genes and processes. We further find that the time course of the infection did not largely influence the gene expression kinetics. The macrophages were influenced and committed to a fate conducive for the bacteria fairly early in the infection regime. Thus, our studies of the expression profiles of the pathogen and the host under the different phases of the infection provide us with a comprehensive understanding the strategies of bacterial offense and host defenses thereby offering a window into this fascinating world of host-pathogen interactions. Chapter 5: Conclusion To summarize, we have attempted to study the intracellular fate of the S. aureus pathogen in macrophages. Our studies suggest that the bacterium attempts to evade clearance by the host immune system by actively preventing fusion with the lysosomal vesicles. We also find that despite these defenses, the pathogen appears to succumb to the host immune system as it is targeted by Hepcidin, an anti-microbial peptide. The lack of complete bacterial clearance under these conditions is however suggestive of an underlying strategy by the pathogen, possibly to maintain a chronic infective state in the host system. The microarray studies, in addition, shed light on the other possible immune evasion strategies that S.aureus might be employing to escape the host offences. The results are indicative of the bacteria influencing anti-apoptotic, anti-inflammatory and antigen presentation responses and thereby prolonging its survival in the macrophage. In conclusion, given the fact that the macrophages are itinerant cells with a long life span, the light thrown by our findings of the various immune evasion strategies that S.aureus is adopting; it suggests that the macrophages could serve as potential carriers which could account for the dissemination of the infection to new sites, which has perpetually been a major concern for any Staphylococcal infection.