Arnoldo Rocha Facanha

A bstract: Phenotypic, genetic and metabolic tumour heterogeneity is a major factor contributing to cancer progression, metastasis, and resistance to therapy. Distinctive thermal and proton flux patterns can occur in cancer cells as a function of variations in the metabolic rate of the tumour mass, tumour margins, and normal tissues, which can be detected by video thermometry (VTM) as well as by Scanning Ion-selective Electrode Techniques (SIET). This study presents distinct thermal patterns associated with tumour heterogeneity observed in canine mammary cancer using VTM and investigate the correlation between these thermometric signatures with metabolic changes related to V-ATPase activity by comparing real-time VTM data with that from concanamycin-sensitive ATP hydrolysis and cell proton flux measurements. The results demonstrate that integrating SIET and VTM data sets can reveal metabolic signatures to assist the diagnosis, surgery and therapeutic monitoring. Considering the breast cancer hallmarks conservation in human, canines and other mammals, this study provides a first bioenergetic proof-of-concept for the potential of the integration of the VTM technology with enzymatic and electrophysiological analyses sensitive to concanamycin for the development of more effective diagnostic, prognostic and therapeutic approaches in veterinary as well as in preclinical medical studies.

Climate change has intensified the proliferation of disease vectors, such as Aedes aegypti, the primary transmitter of dengue, chikungunya, and zika viruses. Although the two recently licensed dengue vaccines represent a significant advancement, vector management remains the primary strategy for preventing these urban arboviruses. In this context, the development of pesticides that offer safer alternatives for the environment and human health has become urgent. In this study, a chitosan-based nanocomposite was developed as a delivery system for rotenoids isolated from Clitoria fairchildiana seeds, leveraging their larvicidal activity against third-instar larvae of Ae. aegypti. The nanocomposite was synthesized using a controlled ionic gelation method incorporating the TPP-β-CD inclusion complex, which resulted in nanoparticles with smaller size, improved polydispersity index, and enhanced stability, evidenced by a higher zeta potential. FTIR analysis confirmed rotenoid incorporation into the nanocomposite and suggested hydrogen bonding or potential covalent interaction with chitosan functional groups. Bioassays demonstrated that the nanocomposite achieved an LC₅₀ of 91.7 ppm, representing a 23.6% increase in larvicidal efficacy compared to the rotenoids in their natural form. The nanocomposite also induced dose-dependent morphological and physiological alterations in the larvae, including damage to the peritrophic matrix, evidenced by abnormal anal excretion, and tissue melanization and formation of melanotic pseudotumors. These responses may be associated with increased production of reactive oxygen species in the larval midgut, consistent with previous findings for the nonencapsulated rotenoids. Importantly, empty nanoparticles exhibited no adverse effects on larval survival, which is attributed to the biocompatibility and nontoxic nature of chitosan, a biodegradable polysaccharide structurally related to the insect exoskeleton and widely recognized for its environmental safety. Additionally, neither rotenoids nor the CS/TPP–β-CD–rot nanocomposite exerted cytotoxic effects, confirming their favorable safety profile. These findings highlight the potential of nanotechnology to enhance the efficacy of bioactive compounds while minimizing environmental and human health risks, offering a sustainable and innovative strategy for vector control.

Comparative analysis of expression patterns of ATP6V1C1 encoding C1 subunit of V-H ⁺ -ATPase revealed that molecular alterations correlated with endometrial cancer of better prognosis were grouped with low ATP6V1C1 expression while those correlated with worse prognosis were clustered with high ATP6V1C1 expression levels. Expression patterns of C1 subunit in endometrial adenocarcinoma are associated with molecular malignancy signatures shared by histological subtypes of highest mortality rate suggesting that G3 adenocarcinoma exhibits molecular changes resembling endometrial serous carcinoma. ATP6V1C1 might serve as novel prognostic marker allowing identification of targetable pathways for high-risk endometrial cancer.

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

Attempts to improve the ascorbate (AsA) content of plants are still dealing with the limited understanding of why exists a wide variability of this powerful anti-oxidant molecule in different plant sources, species and environmental situations. In plant mitochondria, the last step of AsA synthesis is catalyzed by the enzyme L-galactone-1,4-lactone dehydrogenase (L-GalLDH). By using GalLDH-RNAi silencing plant lines, biochemical and proteomic approaches, we here discovered that, in addition to accumulate this antioxidant, mitochondria synthesize AsA to down-regulate the respiratory activity and the cellular energy provision. The work reveals that the AsA synthesis pathway within mitochondria is a branched electron transfer process that channels electrons towards the alternative oxidase, interfering with conventional electron transport. It was unexpectedly found that significant hydrogen peroxide is generated during AsA synthesis, which affects the AsA level. The induced AsA synthesis shows proteomic alterations of mitochondrial and extra-mitochondrial proteins related to oxidative and energetic metabolism. The most identified proteins were known components of plant responses to high light acclimation, programmed cell death, oxidative stress, senescence, cell expansion, iron and phosphorus starvation, different abiotic stress/pathogen attack responses and others. We propose that changing the electron flux associated with AsA synthesis might be part of a new mechanism by which the L-GalLDH enzyme would adapt plant mitochondria to fluctuating energy demands and redox status occurring under different physiological contexts.

V-ATPases are hetero-oligomeric enzymes consisting of 14 subunits and playing key roles in ion homeostasis and signaling. Differential expressions of these proton pumps have been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the V-ATPase genes expression in Esophageal Squamous Cell Carcinoma (ESCC) using gene expression microarray data and extended the analysis to other cancers the Oncomine database. Among all differentially expressed genes, those encoding the V-ATPase C isoforms exhibited striking expression patterns validated by qRT-PCR in paired ESCC samples and respective normal surrounding tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. This study reveals multi-cancer molecular signatures in the V-ATPase structure and establishes that the expression ratios of its subunits/isoforms could form a conformational code that controls the pump regulation and interactions related to tumorigenic events.