Great advances have been made in the last 100 years in the effort to provide safe and reliable potable water. Unfortunately, organisms surviving the water treatment process still cause illnesses in the population. Acanthamoebae are ubiquitous in the environment and are resistant to commonly used disinfection methods. In addition to being pathogenic on their own, Acanthamoeba spp. are capable of acting as a host to pathogenic bacteria in potable water. The amoebae provide the bacteria with protection from chemical and physical means of disinfection. In this way many pathogens that would otherwise be killed in the water treatment process survive and are capable of infecting water customers. Most likely due to experimental limitations discussed herein, the concentration of organic carbon in solution was not found to affect the number of Acanthamoeba polyphaga surviving within reactors designed to model residential water tanks. A copper ion concentration of 1.3 mg/L was determined to be an effective disinfectant against A. polyphaga trophozoites, while free chlorine at 10 mg/L and monochloramine at 50 mg/L were deemed effective against trophozoites. Sulfate-reducing bacteria (SRB) are suspected to be causative agents in copper pitting corrosion. SRB have been found in tubercles covering pits in many homes experiencing pinhole leaks, but the mechanisms of the survival of these organisms in potable water systems are poorly understood. Nutrient studies conducted show that the absence of nitrogen in solution may encourage copper corrosion by SRB. In addition, a medium specifically designed to encourage SRB growth resulted in a large increase in copper corrosion as compared to the control water. / Master of Science

Universiẗat, Diss., 2002--Bonn.

A interação entre microrganismos endossimbiontes e mosquitos tem sido amplamente estudada. Tais estudos visam encontrar alternativas para o controle biológico de mosquitos vetores. Recentemente foi descrito que Acanthamoeba polyphaga é capaz de infectar Aedes aegypti, mosquito vetor do vírus da dengue. Visando entender melhor sobre esta interação, estudaramse aspectos comportamentais e de desenvolvimento de mosquitos alimentados com A. polyphaga, em condições de laboratório. Além disso, procurou-se localizar os sítios de infecção destas amebas no interior do corpo dos mosquitos, através de cortes histológicos. Nas condições em que os testes foram feitos, as amebas contribuíram para o aumento de produção de ovos de Ae. aegypti. Da mesma forma, não produziram patologias nos insetos, uma vez que não houve morte dos mesmos, durante o desenvolvimento dos bioensaios. Algumas amebas que serviram de alimentação às larvas, escaparam à barreira da membrana peritrófica e à resposta de defesa do mosquito, invadindo outras regiões do corpo das larvas e dos adultos. As amebas são encontradas nas formas de cistos e trofozoítos, indicando que o micro-habitat intestinal e do corpo gorduroso não parecem ser muito lesivos para as mesmas. Os resultados obtidos até a data indicam a importância da continuidade dos estudos. Também é importante estudar outros processos associados à resposta imune dos mosquitos quando as amebas conseguem escapar as barreiras de defesa e invadir outros locais do corpo. / The interaction between endosymbiont microorganisms and mosquitoes have been widely studied. Such studies have the aim to find alternatives for biological control of mosquitoes. Recently it was reported that Acanthamoeba polyphaga is able of infecting Aedes aegypti the vector mosquito of dengue viruses. In order to better understand such interaction, we studied some biological aspects and development of mosquitoes that fed on A. polyphaga, in laboratory conditions. Furthermore, we tried to clarifying the sites of infection of these amoebae inside the mosquitoes body, using histological sections. In conditions where the tests were done, the amoebas contributed to the increased of egg production of Ae. aegypti. Likewise, no pathologies were observed in the insects, since no death was observed, during the development of bioassays. Some amoebas served as food to the larvae escaped the barrier of the peritrophic membrane, as well as the defense response of the mosquito, invading other body regions of larvae and adults. Amoebas are found in the forms of cysts and trophozoites, indicating that the intestinal micro-habitat and body fat did not appear to be especially harmful to them. The results to date indicate the importance of continuing such studies. It is also important to study other processes associated with the immune response of mosquitoes when amoebae escape the defense barriers and invade other body sites.

Synthèse d’inhibiteurs potentiels non phosphorylés de la désoxyxylulose phosphate réductoisomérase et étude de la voie de biosynthèse des unités isopréniques chez Acanthamoeba polyphaga. De nombreux microorganismes pathogènes utilisent la voie du méthylérythritol phosphate (MEP) pour la synthèse des unités isopréniques (IPP et DMAPP). Absente chez l’homme, cette voie constitue une cible de choix pour lutter contre ces microorganismes. La fosmidomycine est un des meilleurs inhibiteurs connus à ce jour de la désoxyxylulose phosphate réductoisomérase (DXR), deuxième enzyme de cette voie. Afin d’améliorer d’une part la biodisponibilité de ce type d’inhibiteur et d’autre part de lutter contre le phénomène de résistance à cet antibiotique, nous avons synthétisé des analogues de la fosmidomycine où le groupement phosphonate est remplacé par un groupement tétrazole ou par un groupement squaryle. Les molécules synthétisées ont été testées sur la DXR d’ Escherichia coli et n’ont pas révélé d’activité inhibitrice significative.Par ailleurs, nous avons montré, par des expériences d’incorporation de glucose marqué au 13C, que l’amibe Acanthamoeba polyphaga, utilise la voie du mévalonate pour synthétiser les unités isopréniques nécessaires à la synthèse de ses stérols / Synthesis of potential non-phosphonate inhibitors of the deoxyxylulose phosphate reductoisomerase and study of the biosynthetic pathway for isoprenoids units synthesis using by Acanthamoeba polyphagaMany pathogenic microorganisms synthesize their isoprenoid units (IPP and DMAPP) via the methylerythritol phosphate pathway (MEP pathway). Absent in man, all enzymes of this metabolic route are potential targets for the design of new antimicrobials. This pathway is present in pathogenic bacteria, but absent in mammals. Hence, the development of small-molecule inhibitors for the MEP enzymes constitutes a novel approach for the design of new antimicrobials. Fosmidomycin is the most efficient inhibitor of the the deoxyxylulose phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. In an attempt to improve the pharmacological properties and the bioavailability of this antibiotic, we synthesized analogues of the fosmidomycin by replacing the phosphonate group by tetrazole or squaryl moieties. These synthesized compounds were tested on the DXR isolated from Escherichia coli.Otherwise, we showed by achieving incorporation experiments with 13C labeled glucose that the amoeba Acanthamoeba polyphaga utilize the mevalonate pathway to synthesize its sterols.

A interação entre microrganismos endossimbiontes e mosquitos tem sido amplamente estudada. Tais estudos visam encontrar alternativas para o controle biológico de mosquitos vetores. Recentemente foi descrito que Acanthamoeba polyphaga é capaz de infectar Aedes aegypti, mosquito vetor do vírus da dengue. Visando entender melhor sobre esta interação, estudaramse aspectos comportamentais e de desenvolvimento de mosquitos alimentados com A. polyphaga, em condições de laboratório. Além disso, procurou-se localizar os sítios de infecção destas amebas no interior do corpo dos mosquitos, através de cortes histológicos. Nas condições em que os testes foram feitos, as amebas contribuíram para o aumento de produção de ovos de Ae. aegypti. Da mesma forma, não produziram patologias nos insetos, uma vez que não houve morte dos mesmos, durante o desenvolvimento dos bioensaios. Algumas amebas que serviram de alimentação às larvas, escaparam à barreira da membrana peritrófica e à resposta de defesa do mosquito, invadindo outras regiões do corpo das larvas e dos adultos. As amebas são encontradas nas formas de cistos e trofozoítos, indicando que o micro-habitat intestinal e do corpo gorduroso não parecem ser muito lesivos para as mesmas. Os resultados obtidos até a data indicam a importância da continuidade dos estudos. Também é importante estudar outros processos associados à resposta imune dos mosquitos quando as amebas conseguem escapar as barreiras de defesa e invadir outros locais do corpo. / The interaction between endosymbiont microorganisms and mosquitoes have been widely studied. Such studies have the aim to find alternatives for biological control of mosquitoes. Recently it was reported that Acanthamoeba polyphaga is able of infecting Aedes aegypti the vector mosquito of dengue viruses. In order to better understand such interaction, we studied some biological aspects and development of mosquitoes that fed on A. polyphaga, in laboratory conditions. Furthermore, we tried to clarifying the sites of infection of these amoebae inside the mosquitoes body, using histological sections. In conditions where the tests were done, the amoebas contributed to the increased of egg production of Ae. aegypti. Likewise, no pathologies were observed in the insects, since no death was observed, during the development of bioassays. Some amoebas served as food to the larvae escaped the barrier of the peritrophic membrane, as well as the defense response of the mosquito, invading other body regions of larvae and adults. Amoebas are found in the forms of cysts and trophozoites, indicating that the intestinal micro-habitat and body fat did not appear to be especially harmful to them. The results to date indicate the importance of continuing such studies. It is also important to study other processes associated with the immune response of mosquitoes when amoebae escape the defense barriers and invade other body sites.

A interação entre microrganismos endossimbiontes e mosquitos tem sido amplamente estudada. Tais estudos visam encontrar alternativas para o controle biológico de mosquitos vetores. Recentemente foi descrito que Acanthamoeba polyphaga é capaz de infectar Aedes aegypti, mosquito vetor do vírus da dengue. Visando entender melhor sobre esta interação, estudaramse aspectos comportamentais e de desenvolvimento de mosquitos alimentados com A. polyphaga, em condições de laboratório. Além disso, procurou-se localizar os sítios de infecção destas amebas no interior do corpo dos mosquitos, através de cortes histológicos. Nas condições em que os testes foram feitos, as amebas contribuíram para o aumento de produção de ovos de Ae. aegypti. Da mesma forma, não produziram patologias nos insetos, uma vez que não houve morte dos mesmos, durante o desenvolvimento dos bioensaios. Algumas amebas que serviram de alimentação às larvas, escaparam à barreira da membrana peritrófica e à resposta de defesa do mosquito, invadindo outras regiões do corpo das larvas e dos adultos. As amebas são encontradas nas formas de cistos e trofozoítos, indicando que o micro-habitat intestinal e do corpo gorduroso não parecem ser muito lesivos para as mesmas. Os resultados obtidos até a data indicam a importância da continuidade dos estudos. Também é importante estudar outros processos associados à resposta imune dos mosquitos quando as amebas conseguem escapar as barreiras de defesa e invadir outros locais do corpo. / The interaction between endosymbiont microorganisms and mosquitoes have been widely studied. Such studies have the aim to find alternatives for biological control of mosquitoes. Recently it was reported that Acanthamoeba polyphaga is able of infecting Aedes aegypti the vector mosquito of dengue viruses. In order to better understand such interaction, we studied some biological aspects and development of mosquitoes that fed on A. polyphaga, in laboratory conditions. Furthermore, we tried to clarifying the sites of infection of these amoebae inside the mosquitoes body, using histological sections. In conditions where the tests were done, the amoebas contributed to the increased of egg production of Ae. aegypti. Likewise, no pathologies were observed in the insects, since no death was observed, during the development of bioassays. Some amoebas served as food to the larvae escaped the barrier of the peritrophic membrane, as well as the defense response of the mosquito, invading other body regions of larvae and adults. Amoebas are found in the forms of cysts and trophozoites, indicating that the intestinal micro-habitat and body fat did not appear to be especially harmful to them. The results to date indicate the importance of continuing such studies. It is also important to study other processes associated with the immune response of mosquitoes when amoebae escape the defense barriers and invade other body sites.

Consumer exposure to opportunistic pathogens in potable water systems poses a significant challenge to public health as manifested by numerous cases of pneumonia, non-tuberculosis lung disease, and keratitis eye infections. Water utilities have extensive understanding in control of heterotrophic and coliform bacteria re-growth in water distribution systems via disinfection, control of assimilable organic carbon (AOC), and biologically degradable organic carbon (BDOC). However, much little is known about the effect of AOC on the proliferation of heterotrophic bacteria and pathogens within premise plumbing. This thesis is the first systematic examination of opportunistic pathogen persistence and amplification in simulated glass water heaters (SGWH) as a function of influent organic matter concentration. The role of plumbing conditions that may internally generate AOC is critically examined as part of this evaluation. Strong correlations were often observed between influent organic matter and heterotrophic bacteria in effluent of SGWH as indicated by 16S rRNA gene abundance (average R2 value of 0.889 and 0.971 for heterotrophic organisms and 16S rRNA respectively). The correlation was strongest if water turnover was more frequent (every 48-72 hours) and decreased markedly when water changes were less frequent (stagnation up to 7 days). No simple correlations were identified between the concentration of pathogenic bacteria (L. pneumophila, M. avium, A. polyphaga, and H. vermiformis) and AOC, although correlations were observed between M. avium and TOC over a limited range (and only for a subset of experiments). Indeed, there was little evidence that Legionella and Acanthamoeba proliferated under any of the conditions tested in this work. Parallel experiments were conducted to examine the extent to which factors present in premise plumbing (e.g. sacrificial magnesium anode rods, cross-linked polyethylene, nitrifying bacteria, and iron) could influence water chemistry and influence growth of bacteria or specified pathogens. Although these factors could strongly influence pH, dissolved oxygen concentrations, and levels of organic matter (e.g. iron, magnesium, nitrifying), there was no major impact on effluent concentrations of either heterotrophic bacteria or premise plumbing pathogens under the conditions investigated. While additional research is needed to confirm these findings, at present, there is no evidence of correlations between organic matter and pathogen concentrations from SGWH under conditions tested. Substantial effort was also invested in attempting to identify SGWH and oligotrophic nutrient conditions that would consistently support L. pneumophila and A. polyphaga amplification. A review of the literature indicates no prior examples of large scale amplification of these microorganisms at nutrient levels commonly found in synthesized potable water. It is likely that a complex combination of abiotic and biotic factors (i.e. micronutrients, necrotrophic growth, ambient water temperature, disinfectant type and dose, plumbing materials, water usage patterns), which are not yet fully understood, control the amplification and viability of these pathogenic organisms in premise plumbing systems. / Master of Science

Les virus sont présents dans tous les écosystèmes, et sont les entités les plus abondantes dans le milieu marin. Bien que nous associons systématiquement virus aux maladies, la plupart d'entre eux coexistent cependant en équilibre avec leur hôte. Les virus sont associés à tous les règnes de la vie, même les virus qui affectent d'autres virus(virophages). La définition aujourd'hui d'un virus chez les virologues, c'est qu'un virus est un parasite génétique qui utilise des systèmes cellulaires pour sa propre réplication. Les hôtes les plus couramment utilisés par les virus que nous avons étudiés sont principalement des protozoaires. Ainsi, les Amoebozoa font l'objet de nombreuses études et sont utilisés pour isoler de nouvelles espèces intracellulaires( virus, bactéries). Ces espèces ont évolué de manière à résister aux effets consécutifs à la phagocytose ou à l'ingestion dans des vacuoles, et restent viable dans le cytoplasme de l'amibe, et ont le potentiel de se multiplier dans les parasites. Dans cette étude, nous avons dans un premier temps étudier les diverses interactions existantes entre virus Acanthamoeba polypaghaga Mimivirus(APMV) et des bactéries au sein de l'amibe. Pour cela, nous avons choisi un système original basé sur la co-culture de l'APMV, soit seul ou en combinaison avec deux autres microorganismes isolés individuellement à partir de l'amibe. Il s'agit d'une bactérie intracellulaire stricte(BABL1) et le virophage de APMV (Sputnik). Cela nous a permis de mettre en évidence, d'une part la capacité du virophage à moduler la virulence d'APMV tout en révélant, d'autre part, la bataille qui a eu lieu entre eux au cours de l'infection de l'hôte. dans un deuxième temps, nous avons examiné l'activité virucide des biocides couramment utilisés en pratique clinique pour la désinfection des équipements hospitaliers. APMV et Marseillevirus montrent une grande résitance aux biocides chimiques, en particulier l'alcool. Seule la température de 75°C et le glutaraldéhyde ont réussi à réduire les titres d'APMV et Marseillevirus à des niveaux indétectables. Après dessiccation ou exposition aux rayonnements ultraviolets, APMV et marseillevirus ont démontré leur stabilité durable. Précédent le pré-traitement des échantillons de l'environnement par l'éthanol à 70°C, a permis la disparition des contaminants bactériens sans réduire la charge virale, permettant leur isolement sur amibe, sans avoir besoin d'utiliser des antibiotiques, qui peuvent avoir un effet délétère su les amibes. / In this study, we first examined the various interactions taking place between the virus Acanthamoeba polyphaga Mimivirus (APMV) and bacteria within the amoeba. We chose an original system based on a co-culture of APMV either alone or in combination with two other organisms isolated from amoeba, i.e a strict intracellular bacterium (BABL1) and the virophage of APMV (Sputnik). This allowed us to highlight, on the one hand, the possibility to modulate the virulence of APMV while revealing, on the other hand, the battle which occurs between them during the infection of the host. We then examined the virucidal activity of biocides commonly used in clinical practice for the disinfection of hospital equipment. APMV and Marseillevirus show high resistance to chemical biocides, especially to alcohol. Only a temperature of 75°C or glutaraldehyde were able to reduce APMV and Marseillevirus titres to undetectable levels. Whether dried or under ultraviolet, APMV and Marseillevirus demonstrated their lasting stability. Previous pre-treatment of environmental samples by ethanol 70° allowed disappearance of bacterial contaminating bacteria without reducing giant virus load allowing their isolation on amoeba without need the use of antibiotic that may have a deleterious effect on amoebae.

Cette thèse présente nouveaux organismes trouvés dans d'échantillons d'eaux usées proviennent la zone sud de Jeddah, en Arabie Saoudite. Legionella saoudiensis, Kaumoebavirus, moumouvirus saoudien (SDMV), Yasminevirus et Bunga messiliensis qui sont isolés par une méthode de co-culture amibienne d'infection par Dictyostelium discoideum ATCC 44841, Vermamoeba vermiformis CDC-19, Acanthamoeba polyphaga Linc AP-1 , et Acanthamoeba griffinii, respectivement. Legionella saoudiensis, une souche bactérienne Gram-négative, en forme de bacille, LS-1T appartient au genre Legionella de la famille des Legionellaceae, basée sur des séquences de gène 16S rRNA et d'autres 4 gènes (mip, rpoB, rnpB et 23S-5S). D'une part, le KAUmoebavirus a des capsides icosaédriques de ~ 250 nm-large, un génome d'ADN de 350 731 pb, et une densité de codage de 86%, correspondant à 465 gènes. La plupart de ces gènes (59%) sont étroitement liés aux gènes de Faustoviruses (43%) et Asfarviruses (23%). D'autre part, le moumouvirus saoudien est un nouveau virus géant appartenant à la lignée Mimivirus B, de l'hôpital universitaire King Abdulaziz à Djeddah, et a présenté des particules icosaédriques de 500 nm avec un génome de 1 046 087 pb, plus grand que les génomes moumouvirus qui ont été décrites dans le passé. Il a été prédit que son génome code pour 868 ORF, dont la taille varie de 54 à 2 914 acides aminés. En outre, il code pour 40 nouveaux gènes (ORFans) sans similitude avec d'autres séquences. Ces résultats montrent que la dispositiond’une carte élargie des protistes conduit à découvrir de nouveaux virus géants. / This thesis displays novel organisms that are found in sewage water samples from southern area of Jeddah, Saudi Arabia. These organisms are Legionella saoudiensis, Kaumoebavirus, Saudi moumouvirus (SDMV), Yasminevirus, and Bung messiliensis that are isolated by amoebal co-culture method of infection with Dictyostelium discoideum ATCC 44841, Vermamoeba vermiformis CDC-19, Acanthamoeba polyphaga Linc AP-1, and Acanthamoeba griffinii, respectively. Legionella saoudiensis, a Gram-negative, bacilli shaped bacterial strain, LS-1T belongs to the genus Legionella in the family Legionellaceae based on 16S rRNA gene sequences and other 4 genes (mip, rpoB, rnpB, and 23S-5S). On one hand, KAUmoebavirus has ~250-nm-large icosahedral capsids, a 350,731 bp DNA genome, and a coding density of 86%, corresponding to 465 genes. Most of these genes (59%) are closely related to genes from Faustoviruses (43%) and Asfarviruses (23%). On the other hand, Saudi moumouvirus is a new giant virus belonging to Mimivirus lineage B, from the King Abdulaziz University hospital in Jeddah, and presented 500 nm icosahedral particles with a 1,046,087 bp genome, which is larger than moumouvirus-like genomes which have been described in the past. Its genome was predicted to encode 868 ORFs, ranging in size from 54 to 2,914 amino acids. Furthermore, this genome was predicted to encode 40 new genes (ORFans) without similarity with other sequences. These findings show that the widen chart of protists apply lead to discover new giant viruses.

Made available in DSpace on 2016-08-17T18:39:49Z (GMT). No. of bitstreams: 1 5518.pdf: 1765927 bytes, checksum: 354bfee295ad1873956c15264ad51854 (MD5) Previous issue date: 2013-08-29 / Financiadora de Estudos e Projetos / The photodynamic inactivation (PDI), used for biological control of microorganisms, involves the action of a photosensitizer (PS), activated by visible light, in order to oxidize organic substrates, resulting in cytotoxic effect. The control of free-living amoebae is important both for its pathogenicity and its microorganisms harboring. Additionally, some species may develop serious infections in humans. The present study evaluated the in vitro effectiveness of PDI in Acanthamoeba polyphaga. Salt of curcuminoids, curcumin, methylene blue and Photogem® were used as FS. Besides, this study intended to observe morphological changes caused by the salt of curcuminoids in the microorganism. The samples were grown at 37 ºC in PYG medium for a period of 48 to 72 hours. Curcumin was solubilized in 1 mL DMSO and further diluted in distilled water to obtain final concentrations. The other PS s were directly solubilized in distilled water. The irradiation light-emitting diodes were used at wavelengths 460 and 630 nm at doses of 30 and 50 J/cm2. After treatments, resazurin was added to evaluate the respiratory activity of A. polyphaga and the samples were incubated at 37 °C for 4 hours. The fluorescence intensity was measured in a fluorescence spectrophotometer. In PDI with salt of curcuminoids at concentrations 500, 1000 and 1500 µg/mL, there was a reduction of 27.7%, 61.4% and 82.5% at 30 J/cm2 and 75.2%, 85.0% and 95.9% at 50 J/cm2, respectively. In PDI with curcumin at concentrations 35, 70 and 140 µg/mL, there was a reduction of 16.2%, 24.0% and 25.7% at 30 J/cm2, and 25.4%, 28.3 % and 30.5% at 50 J/cm2, respectively. In PDI with methylene blue at concentrations 24 and 32 µg/mL, there was a reduction of 14.1% and 28.3% at 30 J/cm2, with no reduction in the concentration of 16 µg/mL and 18.7%, 36.9% and 23.9% at 50 J/cm2, respectively. Finally, in PDI with Photogem® at concentrations 50, 100 and 200 µg/mL, there was a reduction of 20.1%, 37.6% and 53.5% at 30 J/cm2, and 17.1%, 38.9% and 57.3% at 50 J/cm2, respectively. The removal of the PS before irradiation showed that the salt of curcuminoids probably didn t stay inside of amoebas, because the reduction obtained previously was not observed in this condition. The PS was toxic to the amoebae in the absence of light, at the concentrations tested, and the isolated use of light showed no phototoxic effect, except the dose of 50 J/cm2 at 460 nm wavelength. The phototoxicity by doses of light together the PS contributed to the death of the amoebae, being more efficient with salt of curcuminoids. The analysis of confocal microscopy images showed that the salt of curcuminoids caused damage in amoebae, confirming its toxicity in the dark. Therefore, it is concluded that contact with only the PS is already able to induce morphological changes in A. polyphaga, leading some of them to death. / A inativação fotodinâmica (IFD), utilizada no controle biológico de microrganismos, envolve a ação de um fotossensibilizador (FS), ativado por luz visível, no intuito de oxidar substratos biológicos, resultando em efeito citotóxico. O controle de amebas de vida livre é importante, tanto pela sua patogenicidade quanto pelo fato de abrigarem microrganismos. Além disso, algumas espécies podem desenvolver sérias infecções em humanos. O presente estudo propôs analisar a efetividade in vitro da IFD em Acanthamoeba polyphaga utilizando sal de curcuminóides, curcumina, azul de metileno e Photogem® como FS, além de observar alterações morfológicas causadas pelo sal de curcuminóides neste microrganismo. As amostras foram cultivadas em meio PYG, incubadas a 37°C por 48 a 72 horas. A curcumina foi solubilizada em 1 mL de DMSO e posteriormente diluída em água destilada para obtenção das concentrações finais. Os demais FS foram solubilizados diretamente em água destilada. Para a irradiação, diodos emissores de luz foram utilizados nos comprimentos de onda 460 e 630 nm, nas doses de 30 e 50 J/cm2. Após os tratamentos, resazurina foi adicionada para avaliar a viabilidade de A. polyphaga, sendo as amostras incubadas a 37°C por 4 horas. A intensidade de fluorescência foi medida em espectrofotômetro de fluorescência. Na IFD com sal de curcuminóides nas concentrações 500, 1000 e 1500 µg/mL, houve redução de 27,7%, 61,4% e 82,5% com 30 J/cm2, e de 75,2%, 85,0% e 95,9% com 50 J/cm2, respectivamente. Já na IFD com curcumina nas concentrações 35, 70 e 140 µg/mL, houve redução de 16,2%, 24,0% e 25,7% com 30 J/cm2, e de 25,4%, 28,3% e 30,5% com 50 J/cm2, respectivamente. Na IFD com azul de metileno nas concentrações 24 e 32µg/mL, houve redução de 14,1% e 28,3% com 30 J/cm2, não havendo redução na concentração de 16 µg/mL e de 18,7%, 36,9% e 23,9% com 50 J/cm2, respectivamente. Por fim, na IFD com Photogem® nas concentrações 50, 100 e 200 µg/mL, houve redução de 20,1%, 37,6% e 53,5% com 30 J/cm2, e de 17,1%, 38,9% e 57,3% com 50 J/cm2, respectivamente. A retirada do FS antes da irradiação mostrou que o sal de curcuminóides provavelmente não permaneceu no interior das amebas, pois a redução obtida anteriormente não foi observada nesta condição. Os FS apresentaram toxicidade para as amebas, na ausência de luz, nas concentrações testadas e o uso isolado da luz não apresentou efeito fototóxico, exceto a dose 50 J/cm2 no comprimento de onda 460 nm. A fototoxicidade proporcionada pelas doses de luz junto aos FS utilizados contribuiu para o efeito de morte das amebas, sendo a IFD eficiente com o sal de curcuminóides. A análise das imagens obtidas pela microscopia confocal mostrou que o sal de curcuminóides causou danos em amebas, confirmando a toxicidade apresentada no escuro. Portanto, conclui-se que apenas o contato com o FS já é capaz de induzir mudanças morfológicas em A. polyphaga, levando algumas células à morte.