Differential impact of acclimation and acute water deprivation in the expression of key transcription factors in soybean roots.

2016

Modulation of cancer energy metabolism: the role of the ATPase inhibitor factor 1 (IF1) in the bioenergetics of cancer cells experiencing oxygen deprivation

IF1, the endogenous inhibitor protein of mitochondrial F1Fo-ATPase, has raised interest in cancer research due to its overexpression in solid tumours compared to normal tissues. Physiologically, IF1 protects cells from energy depletion by limiting the ATP hydrolytic activity of ATP synthase triggered by mitochondrial depolarization caused by oxygen deficiency as it occurs during ischemic episodes. Considering both the physiological function of IF1 and that cancer cells in solid tumour are frequently exposed to oxygen deprivation, we hypothesized that IF1 overexpression represents a strategy that cancer cells develop to protect themselves from energy depletion under conditions of low oxygen availability. To assess this, we assayed the bioenergetic changes in 143B and HCT116 cancer cells with different metabolic features following stable silencing of IF1. Interestingly, we found that in both cell lines exposed to oxygen deprivation conditions the presence of IF1 limits the energy dissipation due to the activation of the ATP hydrolytic activity of ATP synthase. Furthermore, the analyses of cellular growth and viability revealed that the IF1 silencing inhibited proliferation in the highly glycolytic 143B cells, while it induced more than 50% of cellular death in HCT116 OXPHOS-dependent cells, indicating that the energetic advantage conferred by IF1 is essential for cancer cell proliferation or survival depending on the energy metabolism of each cell line. Moreover, under mitochondrial depolarization conditions, both mitophagy and mitochondrial biogenesis markers were found up-regulated in IF1-expressing cells only, thus indicating a continuous renewal and preservation of the mitochondrial mass. Taken together, our results sustain the idea that IF1 overexpression supports cancer cell adaptation to hypoxic or anoxic conditions also favouring the proliferation of re-oxygenated cells by promptly providing functional mitochondria.

Analytical instrumental approaches and strategies to support the sensory assessment of virgin olive oils

At the beginning, this Ph.D. project led to an overview of the most common and emerging types of fraud and possible countermeasures in the olive oil sector. Furthermore, possible weaknesses in the current conformity check system for olive oil were highlighted. Among those, despite the organoleptic assessment is a fundamental tool for establishing the virgin olive oils (VOOs) quality grade, the scientific community has evidenced some drawbacks in it. In particular, the application of instrumental screening methods to support the panel test could reduce the work of sensory panels and the cost of this analysis (e.g. for industries, distributors, public and private control laboratories), permitting the increase in the number and the efficiency of the controls. On this basis, a research line called “Quantitative Panel Test” is one of the main expected outcomes of the OLEUM project that is also partially discussed in this doctoral dissertation. In this framework, analytical activities were carried out, within this PhD project, aimed to develop and validate analytical protocols for the study of the profiles in volatile compounds (VOCs) of the VOOs headspace. Specifically, two chromatographic approaches, one targeted and one semi-targeted, to determine VOCs were investigated in this doctoral thesis. The obtained results, will allow the possible establishment of concentration limits and ranges of selected volatile markers, as related to fruitiness and defects, with the aim to support the panel test in the commercial categorization of VOOs. In parallel, a rapid instrumental screening method based on the analysis of VOCs has been investigated to assist the panel test through a fast pre-classification of VOOs samples based on a known level of probability, thus increasing the efficiency of quality control.

Instrumental and sensory analyses for the characterization of food, phytotherapeutic or pharmaceutical hemp products

To address the request to develop rapid and easy methods for determining the cannabinoids, an HPLC-UV method (8 min) to separate and quantify the 10 main cannabinoids in hemp inflorescences was developed, and in-house validated. Moreover, the antioxidant activity of cannabidiol (CBD) in two oily matrices was investigated and compared to that of α-tocopherol, in relation to the growing market of oily solutions containing cannabidiol. Then, since no univocal legislation on the evaluation of quality and authenticity of hemp seed oil (HSO) exists, the composition and quality of cold-pressed HSOs were also explored, highlighting a great variability in terms of oxidative state minor compounds content. From the sensory point of view, a panel was trained, a specific sensory wheel and a profile sheet were developed. Due to the Covid-19 pandemic, the sensory evaluation was also performed at home. The panel showed a good performance both in the laboratory and remotely. Moreover, a focus group was used to investigate consumers’ attitudes, pointing out that a high-quality HSO has to be cold-pressed and green for them. Then, the evaluation of stability during the storage of HSOs was investigated. The results showed that photo-oxidation did not seem to significantly affect the quality of the oil during the first 3 months of storage. Finally, a study about the evolution of the volatile profile of 9 HSOs, under accelerated oxidation conditions, allowed identifying volatile markers of HSOs oxidation and freshness. This Ph.D. was developed in the context of the scholarship “Harmonized procedures of analysis of medical, herbal, food and industrial cannabis: development and validation of cannabinoids’ quality control methods, of extraction and preparation of derivatives from the plant raw material, according to the product destination” funded by Enecta S.r.l.

Sensory analysis as a tool to improve the quality of aquaculture products

Sensory analysis is a scientific discipline used to evoke, measure, analyse and interpret the responses to products that are perceived by the senses of sight, smell, taste, touch and hearing. This science is used to highlight the strengths and characteristics of a product, such as in the case of research and development products where alternative ingredients, food waste or by-products are used. It can also be used to evaluate the same characteristics over time, to highlight alterations in one of the sensory components at a given time or over time. This doctoral thesis deals with the valorisation, through characterisation, of various aquaculture fish products. In particular, the products covered by this study were analysed, depending on the objective pursued, with different sensory methods using trained judges and in one case consumers. Therefore, the sensory characterisation of the products was useful for investigating the foods considered in this doctoral research. In particular, specific research topics were taken: 1. The study of alternative ingredients, such as the outcomes of different levels of inclusion of insect larvae (Hermetia illucens) meal on the quality of sea bream (Sparus aurata) fillets. 2. The study of consumer expectations and perceptions on the use of insect meal as a feed for aquaculture products. In particular, this study was done after the characterisation by Quantitative Descriptive analysis (QDA) of the products to exclude sensory differences. 3. Development of a non-destructive and cheap device based on dielectric spectroscopy for assessing fish freshness. In particular in this study, the developed device was evaluated in correlation with a sensory method for assessing the freshness of fish product, the Quality Index Method (QIM)

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

Development And Shelf-life Determination Of Pasteurized, Microfiltered, Lactose Hydrolyzed Skim Milk.

The segment of the world population showing permanent or temporary lactose intolerance is quite significant. Because milk is a widely consumed food with an high nutritional value, technological alternatives have been sought to overcome this dilemma. Microfiltration combined with pasteurization can not only extend the shelf life of milk but can also maintain the sensory, functional, and nutritional properties of the product. This studied developed a pasteurized, microfiltered, lactose hydrolyzed (delactosed) skim milk (PMLHSM). Hydrolysis was performed using β-galactosidase at a concentration of 0.4mL/L and incubation for approximately 21h at 10±1°C. During these procedures, the degree of hydrolysis obtained (>90%) was accompanied by evaluation of freezing point depression, and the remaining quantity of lactose was confirmed by HPLC. Milk was processed using a microfiltration pilot unit equipped with uniform transmembrane pressure (UTP) ceramic membranes with a mean pore size of 1.4 μm and UTP of 60 kPa. The product was submitted to physicochemical, microbiological, and sensory evaluations, and its shelf life was estimated. Microfiltration reduced the aerobic mesophilic count by more than 4 log cycles. We were able to produce high-quality PMLHSM with a shelf life of 21 to 27d when stored at 5±1°C in terms of sensory analysis and proteolysis index and a shelf life of 50d in regard to total aerobic mesophile count and titratable acidity.

Anxiety-like Effects Of Meta-chlorophenylpiperazine In Paradoxically Sleep-deprived Mice.

Chlorophenylpiperazines (CPP) are psychotropic drugs used in nightclub parties and are frequently used in a state of sleep deprivation, a condition which can potentiate the effects of psychoactive drugs. This study aimed to investigate the effects of sleep deprivation and sleep rebound (RB) on anxiety-like measures in mCPP-treated mice using the open field test. We first optimized our procedure by performing dose-effect curves and examining different pretreatment times in naïve male Swiss mice. Subsequently, a separate cohort of mice underwent paradoxical sleep deprivation (PSD) for 24 or 48h. In the last experiment, immediately after the 24h-PSD period, mice received an injection of saline or mCPP, but their general activity was quantified in the open field only after the RB period (24 or 48h). The dose of 5mgmL(-1) of mCPP was the most effective at decreasing rearing behavior, with peak effects 15min after injection. PSD decreased locomotion and rearing behaviors, thereby inhibiting a further impairment induced by mCPP. Plasma concentrations of mCPP were significantly higher in PSD 48h animals compared to the non-PSD control group. Twenty-four hours of RB combined with mCPP administration produced a slight reduction in locomotion. Our results show that mCPP was able to significantly change the behavior of naïve, PSD, and RB mice. When combined with sleep deprivation, there was a higher availability of drug in plasma levels. Taken together, our results suggest that sleep loss can enhance the behavioral effects of the potent psychoactive drug, mCPP, even after a period of rebound sleep.

Galectin-3 Up-regulation In Hypoxic And Nutrient Deprived Microenvironments Promotes Cell Survival.

Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

High-resolution Transcript Profiling Of The Atypical Biotrophic Interaction Between Theobroma Cacao And The Fungal Pathogen Moniliophthora Perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.

Association Of Lipophilic Opioids And Hyperbaric Bupivacaine In Spinal Anesthesia For Elective Cesarean Section. Randomized Controlled Study.

To evaluate the efficacy and side-effects of fentanyl and sufentanil combined with hyperbaric spinal bupivacaine in elective cesarean section. A prospective, randomized, double-blind study with 64 term parturients, distributed into 2 groups according to the opioid combined with hyperbaric bupivacaine 0.5% (10mg): GF - fentanyl (25 µg) and GS - sufentanil (5.0 µg). The latency and maximum sensory block level; degree and duration of motor block; duration and quality of analgesia; maternal-fetal repercussions were evaluated. This was an intention-to-treat analysis with a 5% significance level. The latency period, maximum sensory block level, motor block degree and perioperative analgesia were similar in both groups. Motor block and analgesia had a longer duration in the sufentanil group. Maternal adverse effects and neonatal repercussions were similar. The incidence of hypotension was higher in the fentanyl group. In both groups, there was a predominance of patients who were awake and either calm or sleepy. The addition of fentanyl and sufentanil to hyperbaric subarachnoid bupivacaine was shown to be effective for the performance of cesarean section, and safe for the mother and fetus. Analgesia was more prolonged with sufentanil.

Comparison Of Techniques For The Isolation Of Volatiles From Cashew Apple Juice.

The aim of this study was to compare the performance of the following techniques on the isolation of volatiles of importance for the aroma/flavor of fresh cashew apple juice: dynamic headspace analysis using PorapakQ(®) as trap, solvent extraction with and without further concentration of the isolate, and solid-phase microextraction (fiber DVB/CAR/PDMS). A total of 181 compounds were identified, from which 44 were esters, 20 terpenes, 19 alcohols, 17 hydrocarbons, 15 ketones, 14 aldehydes, among others. Sensory evaluation of the gas chromatography effluents revealed esters (n = 24) and terpenes (n = 10) as the most important aroma compounds. The four techniques were efficient in isolating esters, a chemical class of high impact in the cashew aroma/flavor. However, the dynamic headspace methodology produced an isolate in which the analytes were in greater concentration, which facilitates their identification (gas chromatography-mass spectrometry) and sensory evaluation in the chromatographic effluents. Solvent extraction (dichloromethane) without further concentration of the isolate was the most efficient methodology for the isolation of terpenes. Because these two techniques also isolated in greater concentration the volatiles from other chemical classes important to the cashew aroma, such as aldehydes and alcohols, they were considered the most advantageous for the study of cashew aroma/flavor.

Preference Mapping Of Dulce De Leche Commercialized In Brazilian Markets.

Dulce de leche samples available in the Brazilian market were submitted to sensory profiling by quantitative descriptive analysis and acceptance test, as well sensory evaluation using the just-about-right scale and purchase intent. External preference mapping and the ideal sensory characteristics of dulce de leche were determined. The results were also evaluated by principal component analysis, hierarchical cluster analysis, partial least squares regression, artificial neural networks, and logistic regression. Overall, significant product acceptance was related to intermediate scores of the sensory attributes in the descriptive test, and this trend was observed even after consumer segmentation. The results obtained by sensometric techniques showed that optimizing an ideal dulce de leche from the sensory standpoint is a multidimensional process, with necessary adjustments on the appearance, aroma, taste, and texture attributes of the product for better consumer acceptance and purchase. The optimum dulce de leche was characterized by high scores for the attributes sweet taste, caramel taste, brightness, color, and caramel aroma in accordance with the preference mapping findings. In industrial terms, this means changing the parameters used in the thermal treatment and quantitative changes in the ingredients used in formulations.

Low Bone Mass In Human Immunodeficiency Virus-infected Climacteric Women Receiving Antiretroviral Therapy: Prevalence And Associated Factors.

Low bone mineral density (BMD) has been found in human immunodeficiency virus (HIV)-infected patients; however, data on associated factors remain unclear, specifically in middle-aged women. This study aims to evaluate factors associated with low BMD in HIV-positive women. In this cross-sectional study, a questionnaire was administered to 206 HIV-positive women aged 40 to 60 years who were receiving outpatient care. Clinical features, laboratory test results, and BMD were assessed. Yates and Pearson χ(2) tests and Poisson multiple regression analysis were performed. The median age of women was 47.7 years; 75% had nadir CD4 T-cell counts higher than 200, and 77.8% had viral loads below the detection limit. There was no association between low BMD at the proximal femur and lumbar spine (L1-L4) and risk factors associated with HIV infection and highly active antiretroviral therapy. Poisson multiple regression analysis showed that the only factor associated with low BMD at the proximal femur and lumbar spine was postmenopause status. Low BMD is present in more than one third of this population sample, in which most women are using highly active antiretroviral therapy and have a well-controlled disease. The main associated factor is related to estrogen deprivation. The present data support periodic BMD assessments in HIV-infected patients and highlight the need to implement comprehensive menopausal care for these women to prevent bone loss.

Eccentric Exercise Leads To Glial Activation But Not Apoptosis In Mice Spinal Cords.

The aim of this investigation was to evaluate the effects of 3 overtraining (OT) protocols on the glial activation and apoptosis in the spinal cords of mice. Rodents were divided into control (C; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR). The incremental load test, ambulation test, exhaustive test and functional behavioural assessment were used as performance evaluation parameters. 36 h after the exhaustive test, the dorsal and ventral parts of the lumbar spinal cord (L4-L6) were dissected for subsequent protein analysis by immunoblotting. The OT protocols led to similar responses of some performance parameters. The ventral glial fibrillary acidic protein (GFAP) protein levels were diminished in the OTR/up and OTR compared to CT and OTR/down groups. The ventral ionized calcium binding adaptor molecule 1 (Iba-1), and the dorsal GFAP and Iba-1 protein levels were increased in the OTR/down compared to the other groups. The ratio between the cleaved capase-3/caspase-3 and cleaved caspase-9/caspase-9 measured in the spinal cord were not sensitive to the OT protocols. In summary, the OTR/down activated the glial cells in the motor (i. e. Iba-1) and sensory (i. e. GFAP and Iba-1) neurons without leading to apoptosis.