Navegando por Palavras-chave "Caenorhabditis Elegans"
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- ItemAcesso aberto (Open Access)Avaliação de resistência aos antifúngicos e virulência em isolados de Candida tropicalis provenientes de infecções de corrente sanguínea(Universidade Federal de São Paulo (UNIFESP), 2019-12-18) Favarello, Larissa Molina [UNIFESP]; Melo, Analy Salles De Azevedo [UNIFESP]; http://lattes.cnpq.br/8922840487452492; http://lattes.cnpq.br/0360899625657312; Universidade Federal de São Paulo (UNIFESP)Objective: To evaluate isolates of Candida tropicalis from bloodstream infections from Brazilian medical centers included in surveillance studies (2007-2018) to evaluate susceptibility to azoles and to characterize potential virulence in vivo model with the nematode Caenorhabditis elegans. Material and Methods: Clinical isolates stored in the LEMI Microorganism Bank previously identified by phenotypic methods such as Candida tropicalis were selected. The isolates were thawed and seeded on Sabouraud dextrose agar and CHROMagar® Candida media for reactivation and purity evaluation; Species were confirmed by the Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass (MALDI-TOF MS, Bruker®) method using 25% formic acid in a protein extraction protocol; In vitro susceptibility was performed with the antifungals: amphotericin B, fluconazole, voriconazole and anidulafungin using the broth microdilution method recommended by the Clinical & Laboratory Standards Institute (CLSI), document M27ED4. Concomitantly, the prevalence of trailing with low-high phenotype was observed for azole antifungals; virulence was evaluated using the survival curve in C. elegans specimens infected with C. tropicalis, C. albicans, C. auris reference strains and clinical isolates of C. tropicalis according to azole resistance profiles. Results: 200 sequential isolates were recovered from hospitalized patients with positive blood cultures, which were previously identified by phenotypic methods as C. tropicalis over the period of 2007 to 2018. The sequencing of 21 clinical isolates (10.5%) and the reference strain C. tropicalis ATCC 750 was performed to improve the in-house protein database. The other 179 C. tropicalis isolates (89.5%) were properly identified using the in-house database (MALDI-TOF MS). Regarding the susceptibility profile, all isolates were susceptible to amphotericin B, one isolate was resistant to anidulafungin. It was found that 3.5% of the isolates were non-susceptible to azoles. To facilitate the analysis, we divided the isolates into two periods: P1 (2007-2012) and P2 (2013-2018). In P1 (2007-2012) the resistance rate was 1.9% for fluconazole and 0% for voriconazole, being found for P2 (2013-2018) resistance rate of 3.2% for fluconazole and 1% for voriconazole, no statistical difference was observed (p = 0.42). The virulence assay with C. elegans was observed on the second day after infection, the survival rate for C. auris was 90%; 50% for C. albicans; and 0%\20% for the clinical isolate (1534/17) and reference strains (ATCC 750) of C. tropicalis respectively. Conclusions: (i) It was possible to accurately identify by the MALDI-TOF MS method the isolates of C. tropicalis, after enriching the in house database; (ii) Five isolates of C. tropicalis with resistance profile to azole antifungals were found; (iii) Comparing the periods P1 and P2, there was an increase tendency in the rate of azoles resistance, but there was no statistical difference; (iv) One anidulafungin resistant C. tropicalis isolate was found; (v) Survival curves with C. elegans suggested that C. tropicalis, regardless of susceptibility profile, was more virulent than C. auris (P <0.05).
- ItemSomente MetadadadosCaracterização fenotípica do mutante Scav-1 de caenorhabditis elegans(Universidade Federal de São Paulo (UNIFESP), 2020-07-30) Silva, Rafaella Cecilia Da [UNIFESP]; Cunha, Fernanda Marques Da [UNIFESP]; Universidade Federal de São PauloCD36 is a scavenger receptor involved in several homeostatic processes of mammals such as, for example, control of angiogenesis, the innate immune response and lipid metabolism. Although the functions of CD36 and the molecular pathways involved in angiogenesis and the immune response are well characterized, there is still much to be defined about, for example, the role of CD36 in lipid metabolism. CD36 has been implicated in the deregulated metabolism of fatty acids under pathophysiological conditions. Data gathered so far indicate that CD36 has a wide participation in lipid metabolism, being involved in aspects ranging from the absorption of lipids from the diet to the composition of the cellular lipidome. However, the molecular aspects of CD36 function are not clear. Historically, the use of organisms simpler than rodents such as the nematode C. elegans has been of great value in elucidating complex biological processes at the molecular level. Thus, the present study aimed to characterize basic aspects of the biology of C. elegans mutants for scav-1, a possible CD36 orthologue in the worm. For this, analyzes of fertility, pharynx pumping rate, mobility, life span, development time and biometric measurements were carried out. In addition, proteins and carbohydrates of these animals were quantified. The data obtained show that the absence of SCAV-1 delays the development of the animal and impacts on reproduction, decreasing the number of eggs laid by hermaphrodites. Health parameters such as pharynx pumping and body movement were affected by the absence of SCAV-1, especially during aging, suggesting that the absence of SCAV-1 leads to premature aging. Corroborating this hypothesis, longevity data indicate that scav-1 mutants have a significantly reduced mean lifespan when compared to wild type worms. In addition, it was observed that the mutation leads to reduced body protein content, but does not alter the level of carbohydrates. Together, the results show that SCAV-1 is fundamental for several aspects of the biology of C. elegans
- ItemSomente MetadadadosO impacto da mutação no gene LIPL-5 sobre a expressão da enzima Lipase-like 4 e resposta ao estresse em Caenorhabditis elegans(Universidade Federal de São Paulo (UNIFESP), 2021) Assis, Carolina Gomes De [UNIFESP]; Cunha, Fernanda Marques Da [UNIFESP]; Universidade Federal de São PauloIntroduction: Mitochondria are important organelles for the organism, as they are involved in several physiological processes crucial to the maintenance of homeostasis. In a study conducted previously by our group, it was observed that C. elegans with a loss-of-function mutation for the enzyme Lipase-Like 5 (LIPL-5) presented lipid profile alterations including increased amounts of ceramides and mitochondrial signature lipids. It was also observed that the mitochondria of these mutants had improved oxidative capacity when compared to mitochondria from wild type animals. However, the mechanisms by which LIPL-5 deficiency modulates the mitochondrial phenotype are unknown. Objective: To investigate the impact of the lipl-5 gene mutation on the expression of the enzyme Lipase-Like 4 (LIPL-4) and on the unfolded protein response (UPR) in C. elegans. Methods: To analyze the cytoplasmic UPR, a reporter strain mutant for lipl-5 was generated by crossing. For mitochondrial UPR and endoplasmic reticulum UPR, the lipl-5 gene was silenced by RNAi in the respective reporter strains. The activation of stress responses was assessed by fluorescence microscopy followed by quantification. Silencing was confirmed by qPCR, a method that was also used to assess the impact of LIPL-5 deficiency on lipl-4 relative expression, under conditions of bacterial deprivation and ad libitum. Results: lipl-5 silencing in the reporter animals for mitochondrial UPR resulted in a significant increase in the mean fluorescence intensity, indicating activation of UPRmt. lipl-5 silencing in the reporter animals for endoplasmic reticulum UPR, by itself, did not cause UPRer activation in basal conditions, but it impaired its activation in response to tunicamycin (TM) treatment. lipl-5 gene mutation did not provoke a stress response in the cytoplasm under baseline conditions nor significantly affected the response of the animals when challenged with heat shock. Regarding the impact of LIPL-5 deficiency on lipl-4 expression, when evaluating the expression of lipl-4 and lipl-5 in wild type animals (WT), it was seen that both are expressed in response to bacterial deprivation, but with different expression profiles. When investigating lipl-4 expression in lipl-5 mutants, it was possible to observe that, after 6 hours of bacterial deprivation, relative levels of lipl- 4 in the mutant were considerably higher than in WT, but without significant difference after 24 h of starvation. There was also no difference in lipl-4 expression levels in lipl-5 animals in ad libitum condition compared to WT. Conclusions: LIPL- 5 deficiency per se activates mitochondrial UPR, and impairs the response to endoplasmic reticulum UPR induced by TM. The effect of the absence of LIPL-5 is specific since the cytoplasmic UPR is not affected by its deficiency. The lack of LIPL- 5 does not affect lipl-4 expression in control conditions, but induces an early and intense increase in the lipl-4 expression in response to bacterial deprivation. The effects of LIPL-5 deficiency on UPRmt, UPRer and lipl-4 expression may be involved in mitochondrial phenotype alterations previously reported in lipl-5 animals.