Search results for: Granados Gómez

Authors: Tomás Sobrino, Lara García-Varela, Marta Aramburu-Núñez, Mónica Castro, Noemí Gómez-Lado, Mariña Rodríguez-Arrizabalaga, Antía Custodia, Juan Manuel Pías-Peleteiro, José Manuel Aldrey, Daniel Romaus-Sanjurjo, Ángeles Almeida, Pablo Aguiar, Alberto Ouro

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Introduction: Alzheimer’s disease (AD) and other tauopathies are primary causes of dementia, causing progressive cognitive deterioration that entails serious repercussions for the patients' performance of daily tasks. Currently, there is no effective approach for the early diagnosis and treatment of AD and tauopathies. This study suggests a theragnostic approach based on the importance of phosphorylated tau protein (p-Tau) in the early pathophysiological processes of AD. We have developed a novel theragnostic monoclonal antibody (mAb) to provide both diagnostic and therapeutic effects. Methods/Results: We have developed a p-Tau mAb, which was doped with deferoxamine for radiolabeling with Zirconium-89 (89Zr) for PET imaging, as well as fluorescence dies for immunofluorescence assays. The p-Tau mAb was evaluated in vitro for toxicity by MTT assay, LDH activity, propidium iodide/Annexin V assay, caspase-3, and mitochondrial membrane potential (MMP) assay in both mouse endothelial cell line (bEnd.3) and cortical primary neurons cell cultures. Importantly, non-toxic effects (up to concentrations of p-Tau mAb greater than 100 ug/mL) were detected. In vivo experiments in the tauopathy model mice (PS19) show that the 89Zr-pTau-mAb and 89Zr-Fragments-pTau-mAb are stable in circulation for up to 10 days without toxic effects. However, only less than 0.2% reached the brain, so further strategies have to be designed for crossing the Brain-Blood-Barrier (BBB). Moreover, an intraparenchymal treatment strategy was carried out. The PS19 mice were operated to implement osmotic pumps (Alzet 1004) at two different times, at 4 and 7 months, to stimulate the controlled release for one month each of the B6 antibody or the IgG1 control antibody. We demonstrated that B6-treated mice maintained their motor and memory abilities significantly compared with IgG1 treatment. In addition, we observed a significant reduction in p-Tau deposits in the brain. Conclusions /Discussion: A theragnostic pTau-mAb was developed. Moreover, we demonstrated that our p-Tau mAb recognizes very-early pathology forms of p-Tau by non-invasive techniques, such as PET. In addition, p-Tau mAb has non-toxic effects, both in vitro and in vivo. Although the p-Tau mAb is stable in circulation, only 0.2% achieve the brain. However, direct intraventricular treatment significantly reduces cognitive impairment in Alzheimer's animal models, as well as the accumulation of toxic p-Tau species.

Keywords: alzheimer's disease, theragnosis, tau, PET, immunotherapy, tauopathies

Procedia PDF Downloads 39

Authors: Gómez S. Jennifer, Méndez V. Camila, Moncayo M. Diana, Vega M. Lizeth

Abstract:

The cape gooseberry is a climacteric fruit; Colombia is one of the principal exporters in the world. The environmental condition of temperature and relative moisture decreases the titratable acidity and pH. These conditions and fruit maturation result in the fungal proliferation of Botrytis cinerea disease. Plastic packaging for fresh cape gooseberries was used for mechanical damage protection but created a suitable atmosphere for fungal growth. Beta-cyclodextrins are currently implemented as coatings for the encapsulation of hydrophobic compounds, for example, with bioactive compounds from essential oils such as cinnamaldehyde, which has a high antimicrobial capacity. However, it is a volatile substance. In this article, the casting method was used to obtain a polylactic acid (PLA) polymer film containing the beta-cyclodextrin-cinnamaldehyde inclusion complex, generating an insert that allowed the controlled release of the antifungal substance in packed cape gooseberries to decrease contamination by Botrytis cinerea in a latent state during storage. For the encapsulation technique, three ratios for the cinnamaldehyde: beta-cyclodextrin inclusion complex were proposed: (25:75), (40:60), and (50:50). Spectrophotometry, colorimetry in L*a*b* coordinate space and scanning electron microscopy (SEM) were made for the complex characterization. Subsequently, two ratios of tween and water (40:60) and (50:50) were used to obtain the polylactic acid (PLA) film. To determine mechanical and physical parameters of colourimetry in L*a*b* coordinate space, atomic force microscopy and stereoscopy were done to determine the transparency and flexibility of the film; for both cases, Statgraphics software was used to determine the best ratio in each of the proposed phases, where for encapsulation it was (50:50) with an encapsulation efficiency of 65,92%, and for casting the ratio (40:60) obtained greater transparency and flexibility that permitted its incorporation into the polymeric packaging. A liberation assay was also developed under ambient temperature conditions to evaluate the concentration of cinnamaldehyde inside the packaging through gas chromatography for three weeks. It was found that the insert had a controlled release. Nevertheless, a higher cinnamaldehyde concentration is needed to obtain the minimum inhibitory concentration for the fungus Botrytis cinerea (0.2g/L). The homogeneity of the cinnamaldehyde gas phase inside the packaging can be improved by considering other insert configurations. This development aims to impact emerging food preservation technologies with the controlled release of antifungals to reduce the affectation of the physico-chemical and sensory properties of the fruit as a result of contamination by microorganisms in the postharvest stage.

Keywords: antifungal, casting, encapsulation, postharvest

Procedia PDF Downloads 45

Authors: Ana-María Gómez-Carvajal, Hernando Santamaría-García, Mateo Bernal, Mario Valderrama, Daniela Lizarazo, Juliana Restrepo, María Fernanda Barreto, Angélica Parra, Paula Torres, Diana Matallana, Jaime Silva, José Santamaría-García, Sandra Baez

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Higher levels of oxytocin are associated with better performance on social cognition tasks. However, higher levels of oxytocin have also been associated with increased levels of envy and schadenfreude. Considering these antecedents, this study aims to explore social emotions (i.e., envy and schadenfreude) and other components of social cognition (i.e. ToM and empathy), in women in the puerperal period and their respective partners, compared to a control group of men and women without children or partners. Control women should be in the luteal phase of the menstrual cycle or taking oral contraceptives as they allow oxytocin levels to remain stable. We selected this population since increased levels of oxytocin are present in both mothers and fathers of newborn babies. Both groups were matched by age, sex, and education level. Twenty-two parents of newborns (11 women, 11 men) and 15 controls (8 women, 7 men) performed an experimental task designed to trigger schadenfreude and envy. In this task, each participant was shown a real-life photograph and a description of two target characters matched in age and gender with the participant. The task comprised two experimental blocks. In the first block, participants read 15 sentences describing fortunate events involving either character. After reading each sentence, participants rated the event in terms of how much envy they felt for the character (1=no envy, 9=extreme envy). In the second block, participants read and reported the intensity of their pleasure (schadenfreude, 1=no pleasure, 9=extreme pleasure) in response to 15 unfortunate events happening to the characters. Five neutral events were included in each block. Moreover, participants were assessed with ToM and empathy tests. Potential confounding variables such as general cognitive functioning, stress levels, hours of sleep and depression symptoms were also measured. Results showed that parents of newborns showed increased levels of envy and schadenfreude. These effects are not explained by any confounding factor. Moreover, no significant differences were found in ToM or empathy tests. Our results offer unprecedented evidence of specific differences in envy and schadenfreude levels in parents of newborns. Our findings support previous studies showing a negative relationship between oxytocin levels and negative social emotions. Further studies should assess the direct relationship between oxytocin levels in parents of newborns and the performance in social emotions tasks.

Keywords: envy, empathy, oxytocin, schadenfreude, social emotions, theory of mind

Procedia PDF Downloads 287

Authors: Kathrina Aseanne Acapulco-Gomez, Shayne Julieane Morales, Tzar Francis Verame

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Mean platelet volume (MPV) is the most accurate measure of the size of platelets and is routinely measured by most automated hematological analyzers. Several studies have shown associations between MPV and cardiovascular risks and outcomes. Although its measurement may provide useful data, MPV remains to be a diagnostic tool that is yet to be included in routine clinical decision making. The aim of this systematic review and meta-analysis is to determine summary estimates of the diagnostic accuracy of mean platelet volume for the diagnosis of myocardial infarction among adult patients with angina and/or its equivalents in terms of sensitivity, specificity, diagnostic odds ratio, and likelihood ratios, and to determine the difference of the mean MPV values between those with MI and those in the non-MI controls. The primary search was done through search in electronic databases PubMed, Cochrane Review CENTRAL, HERDIN (Health Research and Development Information Network), Google Scholar, Philippine Journal of Pathology, and Philippine College of Physicians Philippine Journal of Internal Medicine. The reference list of original reports was also searched. Cross-sectional, cohort, and case-control articles studying the diagnostic performance of mean platelet volume in the diagnosis of acute myocardial infarction in adult patients were included in the study. Studies were included if: (1) CBC was taken upon presentation to the ER or upon admission (within 24 hours of symptom onset); (2) myocardial infarction was diagnosed with serum markers, ECG, or according to accepted guidelines by the Cardiology societies (American Heart Association (AHA), American College of Cardiology (ACC), European Society of Cardiology (ESC); and, (3) if outcomes were measured as significant difference AND/OR sensitivity and specificity. The authors independently screened for inclusion of all the identified potential studies as a result of the search. Eligible studies were appraised using well-defined criteria. Any disagreement between the reviewers was resolved through discussion and consensus. The overall mean MPV value of those with MI (9.702 fl; 95% CI 9.07 – 10.33) was higher than in those of the non-MI control group (8.85 fl; 95% CI 8.23 – 9.46). Interpretation of the calculated t-value of 2.0827 showed that there was a significant difference in the mean MPV values of those with MI and those of the non-MI controls. The summary sensitivity (Se) and specificity (Sp) for MPV were 0.66 (95% CI; 0.59 - 0.73) and 0.60 (95% CI; 0.43 – 0.75), respectively. The pooled diagnostic odds ratio (DOR) was 2.92 (95% CI; 1.90 – 4.50). The positive likelihood ratio of MPV in the diagnosis of myocardial infarction was 1.65 (95% CI; 1.20 – 22.27), and the negative likelihood ratio was 0.56 (95% CI; 0.50 – 0.64). The intended role for MPV in the diagnostic pathway of myocardial infarction would perhaps be best as a triage tool. With a DOR of 2.92, MPV values can discriminate between those who have MI and those without. For a patient with angina presenting with elevated MPV values, it is 1.65 times more likely that he has MI. Thus, it is implied that the decision to treat a patient with angina or its equivalents as a case of MI could be supported by an elevated MPV value.

Keywords: mean platelet volume, MPV, myocardial infarction, angina, chest pain

Procedia PDF Downloads 48

Authors: Liliana Gomez Cardona, Echo Parent-Racine, Joy Outerbridge, Arlene Laliberté, Outi Linnaranta

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Suicide rates among Inuit in Nunavik are six to eleven times larger than the Canadian average. The colonization, religious missions, residential schools as well as economic and political marginalization are factors that have challenged the well-being and mental health of these populations. In psychiatry, screening for mental illness is often done using questionnaires with which the patient is expected to respond how often he/she has certain symptoms. However, the Indigenous view of mental wellbeing may not fit well with this approach. Moreover, biomedical treatments do not always meet the needs of Indigenous peoples because they do not understand the culture and traditional healing methods that persist in many communities. Assess whether the questionnaires used to measure symptoms, commonly used in psychiatry are appropriate and culturally safe for the Inuit in Quebec. Identify the most appropriate tool to assess and promote wellbeing and follow the process necessary to improve its cultural sensitivity and safety for the Inuit population. Qualitative, collaborative, and participatory action research project which respects First Nations and Inuit protocols and the principles of ownership, control, access, and possession (OCAP). Data collection based on five focus groups with stakeholders working with these populations and members of Indigenous communities. Thematic analysis of the data collected and emerging through an advisory group that led a revision of the content, use, and cultural and conceptual relevance of the instruments. The questionnaires measuring psychiatric symptoms face significant limitations in the local indigenous context. We present the factors that make these tools not relevant among Inuit. Although the scale called Growth and Empowerment Measure (GEM) was originally developed among Indigenous in Australia, the Inuit in Quebec found that this tool comprehends critical aspects of their mental health and wellbeing more respectfully and accurately than questionnaires focused on measuring symptoms. We document the process of cultural adaptation of this tool which was supported by community members to create a culturally safe tool that helps in resilience and empowerment. The cultural adaptation of the GEM provides valuable information about the factors affecting wellbeing and contributes to mental health promotion. This process improves mental health services by giving health care providers useful information about the Inuit population and their clients. We believe that integrating this tool in interventions can help create a bridge to improve communication between the Indigenous cultural perspective of the patient and the biomedical view of health care providers. Further work is needed to confirm the clinical utility of this tool in psychological and psychiatric intervention along with social and community services.

Keywords: cultural adaptation, cultural safety, empowerment, Inuit, mental health, Nunavik, resiliency

Procedia PDF Downloads 81

Authors: Malek Miladi, Miguel Vegara, Maria Perez-Infantes, Khaled Mohamed Ramadan, Antonio Ruiz-Canales, Damaris Nunez-Gomez

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Coffee is the second consumed commodity worldwide, yet it also generates colossal waste. Proper management of coffee waste is proposed by converting them into products with higher added value to achieve sustainability of the economic and ecological footprint and protect the environment. Based on this, a study looking at the recovery of coffee waste is becoming more relevant in recent decades. Spent coffee grounds (SCG's) resulted from brewing coffee represents the major waste produced among all coffee industry. The fact that SCGs has no economic value be abundant in nature and industry, do not compete with agriculture and especially its high oil content (between 7-15% from its total dry matter weight depending on the coffee varieties, Arabica or Robusta), encourages its use as a sustainable feedstock for bio-oil production. The bio-oil extraction is a crucial step towards biodiesel production by the transesterification process. However, conventional methods used for oil extraction are not recommended due to their high consumption of energy, time, and generation of toxic volatile organic solvents. Thus, finding a sustainable, economical, and efficient extraction technique is crucial to scale up the process and to ensure more environment-friendly production. Under this perspective, the aim of this work was the statistical study to know an efficient strategy for oil extraction by n-hexane using indirect sonication. The coffee waste mixed Arabica and Robusta, which was used in this work. The temperature effect, sonication time, and solvent-to-solid ratio on the oil yield were statistically investigated as dependent variables by Central Composite Rotatable Design (CCRD) 23. The results were analyzed using STATISTICA 7 StatSoft software. The CCRD showed the significance of all the variables tested (P < 0.05) on the process output. The validation of the model by analysis of variance (ANOVA) showed good adjustment for the results obtained for a 95% confidence interval, and also, the predicted values graph vs. experimental values confirmed the satisfactory correlation between the model results. Besides, the identification of the optimum experimental conditions was based on the study of the surface response graphs (2-D and 3-D) and the critical statistical values. Based on the CCDR results, 29 ºC, 56.6 min, and solvent-to-solid ratio 16 were the better experimental conditions defined statistically for coffee waste oil extraction using n-hexane as solvent. In these conditions, the oil yield was >9% in all cases. The results confirmed the efficiency of using an ultrasound bath in extracting oil as a more economical, green, and efficient way when compared to the Soxhlet method.

Keywords: coffee waste, optimization, oil yield, statistical planning

Procedia PDF Downloads 89

Authors: Federico Herrera, Valle Gomez García de Soria, Itxaso Portero Sainz, Carlos Fernández Arandojo, Mercedes Royg, Ana Marcos Jimenez, Anna Kreutzman, Cecilia MuñozCalleja

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Introduction: Graft-versus-host disease (GVHD) still remains the major complication associated with allogeneic stem cell transplantation (SCT). The pathogenesis involves migration of donor naïve T-cells into recipient secondary lymphoid organs. Two molecules are important in this process: CD62L and CCR7, which are characteristically expressed in naïve/central memory T-cells. With this background, we aimed to study the influence of CCR7 and CD62L on donor lymphocytes in the development and severity of GVHD. Material and methods: This single center study included 98 donor-recipient pairs. Samples were collected prospectively from the apheresis product and phenotyped by flow cytometry. CCR7 and CD62L expression in CD4+ and CD8+ T-cells were compared between patients who developed acute (n=40) or chronic GVHD (n=33) and those who did not (n=38). Results: The patients who developed acute GVHD were transplanted with a higher percentage of CCR7+CD4+ T-cells (p = 0.05) compared to the no GVHD group. These results were confirmed when these patients were divided in degrees according to the severity of the disease; the more severe disease, the higher percentage of CCR7+CD4+ T-cells. Conversely, chronic GVHD patients received a higher percentage of CCR7+CD8+ T-cells (p=0.02) in comparison to those who did not develop the complication. These data were also confirmed when patients were subdivided in degrees of the disease severity. A multivariable analysis confirmed that percentage of CCR7+CD4+ T-cells is a predictive factor of acute GVHD whereas the percentage of CCR7+CD8+ T-cells is a predictive factor of chronic GVHD. In vitro functional assays (migration and activation assays) supported the idea of CCR7+ T-cells were involved in the development of GVHD. As low levels of CD62L expression were detected in all apheresis products, we tested the hypothesis that CD62L was shed during apheresis procedure. Comparing CD62L surface levels in T-cells from the same donor immediately before collecting the apheresis product, and the final apheresis product we found that this process down-regulated CD62L in both CD4+ and CD8+ T cells (p=0.008). Interestingly, when CD62L levels were analysed in days 30 or 60 after engraftment, they recovered to baseline (p=0.008). However, to investigate the relation between CD62L expression and the development of GVHD in the recipient samples after the engraftment, no differences were observed comparing patients with GVHD to those who did not develop the disease. Discussion: Our prospective study indicates that the CCR7+ T-cells from the donor, which include naïve and central memory T-cells, contain the alloreactive cells with a high ability to mediate GVHD (in the case of both migration and activation). Therefore we suggest that the proportion and functional properties of CCR7+CD4+ and CCR7+CD8+ T-cells in the apheresis could act as a predictive biomarker to both acute and chronic GVHD respectively. Importantly, our study precludes that CD62L is lost in the apheresis and therefore it is not a reliable biomarker for the development of GVHD.

Keywords: CCR7, CD62L, GVHD, SCT

Procedia PDF Downloads 258

Authors: E. Akdur Öztürk, F. İrvasa Bilgiç, A. Ludovisi , O. Gülbahar, D. Dirim Erdoğan, M. Korkmaz, M. Á. Gómez Morales

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Anisakidosis is a zoonotic human fish-borne parasitic disease caused by accidental ingestion of anisakid third-stage larvae (L3) of members of the Anisakidae family present in infected marine fish or cephalopods. Infection with anisakid larvae can lead to gastric, intestinal, extra-gastrointestinal and gastroallergic forms of the disease. Anisakid parasites have been reported in almost all seas, particularly in the Mediterranean Sea. There is a remarkably high level of risk exposure to these zoonotic parasites as they are present in economically and ecologically important fish of Europe. Anisakid L3 larvae have been also detected in several fish species from the Aegean Sea. Turkey is a peninsular country surrounded by Black, Aegean and the Mediterranean Sea. In this country, fishing habit and fishery product consumption are highly common. In recent years, there was also an increase in the consumption of raw fish due to the increasing interest in the cuisine of the Far East countries. In different regions of Turkey, A. simplex (inMerluccius Merluccius Scomber japonicus, Trachurus mediterraneus, Sardina pilchardus, Engraulis encrasicolus, etc.), Anisakis spp., Contraceucum spp., Pseudoterronova spp. and, C. aduncum were identified as well. Although it is accepted both the presence of anisakid parasites in fish and fishery products in Turkey and the presence of Turkish people with allergic manifestations after fish consumption, there are no reports of human anisakiasis in this country. Given the high prevalence of anisakid parasites in the country, the absence of reports is likely not due to the absence of clinical cases rather to the unavailability of diagnostic tools and the low awareness of the presence of this infection. The aim of the study was to set up an IgE-Western Blot (WB) based test to detect the anisakidosis sensitization among Turkish people with a history of allergic manifestation related to fish consumption. To this end, crude worm antigens (CWA) and allergen enriched fraction (50-66% ) were prepared from L3 of A. simplex (s.l.) collected from Lepidopus caudatus fished in the Mediterranean Sea. These proteins were electrophoretically separated and transferred into the nitrocellulose membranes. By WB, specific proteins recognized by positive control serum samples from sensitized patients were visualized on nitrocellulose membranes by a colorimetric reaction. The CWA and 50–66% fraction showed specific bands, mainly due to Ani s 1 (20-22 kD) and Ani s 4 (9-10 kD). So far, a total of 7 serum samples from people with allergic manifestation and positive skin prick test (SPT) after fish consumption, have been tested and all of them resulted negative by WB, indicating the lack of sensitization to anisakids. This preliminary study allowed to set up a specific test and evidence the lack of correlation between both tests, SPT and WB. However, the sample size should be increased to estimate the anisakidosis burden in Turkish people.

Keywords: anisakidosis, fish parasite, serodiagnosis, Turkey

Procedia PDF Downloads 101

Authors: Liliana Gomez Cardona, Mary McComber, Kristyn Brown, Arlene Laliberté, Outi Linnaranta

Abstract:

The history of colonialism and more contemporary political issues have resulted in the exposure of Kanien'kehá:ka: non (Kanien'kehá:ka of Kahnawake) to challenging and even traumatic experiences. Colonization, religious missions, residential schools as well as economic and political marginalization are the factors that have challenged the wellbeing and mental health of these populations. In psychiatry, screening for mental illness is often done using questionnaires with which the patient is expected to respond to how often he/she has certain symptoms. However, the Indigenous view of mental wellbeing may not fit well with this approach. Moreover, biomedical treatments do not always meet the needs of Indigenous people because they do not understand the culture and traditional healing methods that persist in many communities. Assess whether the questionnaires used to measure symptoms, commonly used in psychiatry are appropriate and culturally safe for the Mohawk in Quebec. Identify the most appropriate tool to assess and promote wellbeing and follow the process necessary to improve its cultural sensitivity and safety for the Mohawk population. Qualitative, collaborative, and participatory action research project which respects First Nations protocols and the principles of ownership, control, access, and possession (OCAP). Data collection based on five focus groups with stakeholders working with these populations and members of Indigenous communities. Thematic analysis of the data collected and emerging through an advisory group that led a revision of the content, use, and cultural and conceptual relevance of the instruments. The questionnaires measuring psychiatric symptoms face significant limitations in the local indigenous context. We present the factors that make these tools not relevant among Mohawks. Although the scale called Growth and Empowerment Measure (GEM) was originally developed among Indigenous in Australia, the Mohawk in Quebec found that this tool comprehends critical aspects of their mental health and wellbeing more respectfully and accurately than questionnaires focused on measuring symptoms. We document the process of cultural adaptation of this tool which was supported by community members to create a culturally safe tool that helps in growth and empowerment. The cultural adaptation of the GEM provides valuable information about the factors affecting wellbeing and contributes to mental health promotion. This process improves mental health services by giving health care providers useful information about the Mohawk population and their clients. We believe that integrating this tool in interventions can help create a bridge to improve communication between the Indigenous cultural perspective of the patient and the biomedical view of health care providers. Further work is needed to confirm the clinical utility of this tool in psychological and psychiatric intervention along with social and community services.

Keywords: cultural adaptation, cultural safety, empowerment, Mohawks, mental health, Quebec

Procedia PDF Downloads 104

Authors: Juan C. Pérez-Mesa, Emilio Galdeano-Gómez, Jerónimo De Burgos-Jiménez, José F. Bienvenido-Bárcena, José F. Jiménez-Guerrero

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Environmental cost and transport congestion on roads resulting from product distribution in Europe have to lead to the creation of various programs and studies seeking to reduce these negative impacts. In this regard, apart from other institutions, the European Commission (EC) has designed plans in recent years promoting a more sustainable transportation model in an attempt to ultimately shift traffic from the road to the sea by using intermodality to achieve a model rebalancing. This issue proves especially relevant in supply chains from peripheral areas of the continent, where the supply of certain agrifood products is high. In such cases, the most difficult challenge is managing perishable goods. This study focuses on new approaches that strengthen the modal shift, as well as the reduction of externalities. This problem is analyzed by attempting to promote intermodal system (truck and short sea shipping) for transport, taking as point of reference highly perishable products (vegetables) exported from southeast Spain, which is the leading supplier to Europe. Methodologically, this paper seeks to contribute to the literature by proposing a different and complementary approach to establish a comparison between intermodal and the “only road” alternative. For this purpose, the multicriteria decision is utilized in a p-median model (P-M) adapted to the transport of perishables and to a means of shipping selection problem, which must consider different variables: transit cost, including externalities, time, and frequency (including agile response time). This scheme avoids bias in decision-making processes. By observing the results, it can be seen that the influence of the externalities as drivers of the modal shift is reduced when transit time is introduced as a decision variable. These findings confirm that the general strategies, those of the EC, based on environmental benefits lose their capacity for implementation when they are applied to complex circumstances. In general, the different estimations reveal that, in the case of perishables, intermodality would be a secondary and viable option only for very specific destinations (for example, Hamburg and nearby locations, the area of influence of London, Paris, and the Netherlands). Based on this framework, the general outlook on this subject should be modified. Perhaps the government should promote specific business strategies based on new trends in the supply chain, not only on the reduction of externalities, and find new approaches that strengthen the modal shift. A possible option is to redefine ports, conceptualizing them as digitalized redistribution and coordination centers and not only as areas of cargo exchange.

Keywords: environmental externalities, intermodal transport, perishable food, transit time

Procedia PDF Downloads 67

Authors: Arinze Ezieke, Antonio Serrano, William Clarke, Denys Villa-Gomez

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Berry cultivation results in discharge of high organic strength putrescible solid waste which potentially contributes to environmental degradation, making it imperative to assess options for its complete management. Anaerobic digestion (AD) could be an ideal option when the target is energy generation; however, due to berry fruit characteristics high carbohydrate composition, the technology could be limited by its high alkalinity requirement which suggests dosing of additives such as buffers and trace elements supplement. Overcoming this limitation in an economically viable way could entail replacement of synthetic additives with recycled by-product waste. Consequently, ash from co-smouldering of high COD characteristic AD digestate and coco-coir could be a promising material to be used to enhance the AD of berry fruit waste, given its characteristic high pH, alkalinity and metal concentrations which is typical of synthetic additives. Therefore, the aim of the research was to evaluate the stability and process performance from the AD of BFW when ash from co-smoldered digestate and coir are supplemented as alkalinity and trace elements (TEs) source. Series of batch experiments were performed to ascertain the necessity for alkalinity addition and to see whether the alkalinity and metals in the co-smouldered digestate ash can provide the necessary buffer and TEs for AD of berry fruit waste. Triplicate assays were performed in batch systems following I/S of 2 (in VS), using serum bottles (160 mL) sealed and placed in a heated room (35±0.5 °C), after creating anaerobic conditions. Control experiment contained inoculum and substrates only, and inoculum, substrate and NaHCO3 for optimal total alkalinity concentration and TEs assays, respectively. Total alkalinity concentration refers to alkalinity of inoculum and the additives. The alkalinity and TE potential of the ash were evaluated by supplementing ash (22.574 g/kg) of equivalent total alkalinity concentration to that of the pre-determined optimal from NaHCO3, and by dosing ash (0.012 – 7.574 g/kg) of varying concentrations of specific essential TEs (Co, Fe, Ni, Se), respectively. The result showed a stable process at all examined conditions. Supplementation of 745 mg/L CaCO3 NaHCO3 resulted to an optimum TAC of 2000 mg/L CaCO3. Equivalent ash supplementation of 22.574 g/kg allowed the achievement of this pre-determined optimum total alkalinity concentration, resulting to a stable process with a 92% increase in the methane production rate (323 versus 168 mL CH4/ (gVS.d)), but a 36% reduction in the cumulative methane production (103 versus 161 mL CH4/gVS). Addition of ashes at incremental dosage as TEs source resulted to a reduction in the Cumulative methane production, with the highest dosage of 7.574 g/kg having the highest effect of -23.5%; however, the seemingly immediate bioavailability of TE at this high dosage allowed for a +15% increase in the methane production rate. With an increased methane production rate, the results demonstrated that the ash at high dosages could be an effective supplementary material for either a buffered or none buffered berry fruit waste AD system.

Keywords: anaerobic digestion, alkalinity, co-smoldered digestate ash, trace elements

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Authors: M. Amo, A. Alvaro, A. Astudillo, R. Mc Culloch, J. C. del Castillo, M. Gómez, J. M. Martín

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Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) of course comprise a range of highly toxic compounds that may exist as particulates within the air or accumulate within water supplies, soil, or vegetation. They may be created either ubiquitously or naturally within the environment as a product of forest fires or volcanic eruptions. It is only since the industrial revolution, however, that it has become necessary to closely monitor their generation as a byproduct of manufacturing/combustion processes, in an effort to mitigate widespread contamination events. Of course, the environmental concentrations of these toxins are expected to be extremely low, therefore highly sensitive and accurate methods are required for their determination. Since ionization of non-polar compounds through electrospray and APCI is difficult and inefficient, we evaluate the performance of a novel low-flow Atmospheric Pressure Photoionization (APPI) source for the trace detection of various dioxins and furans using rapid Mass Spectrometry workflows. Air, soil and biota (vegetable matter) samples were collected monthly during one year from various locations within the vicinity of an industrial incinerator in Spain. Analytes were extracted and concentrated using soxhlet extraction in toluene and concentrated by rotavapor and nitrogen flow. Various ionization methods as electrospray (ES) and atmospheric pressure chemical ionization (APCI) were evaluated, however, only the low-flow APPI source was capable of providing the necessary performance, in terms of sensitivity, required for detecting all targeted analytes. In total, 10 analytes including 2,3,7,8-tetrachlorodibenzodioxin (TCDD) were detected and characterized using the APPI-MS method. Both PCDDs and PCFDs were detected most efficiently in negative ionization mode. The most abundant ion always corresponded to the loss of a chlorine and addition of an oxygen, yielding [M-Cl+O]- ions. MRM methods were created in order to provide selectivity for each analyte. No chromatographic separation was employed; however, matrix effects were determined to have a negligible impact on analyte signals. Triple Quadrupole Mass Spectrometry was chosen because of its unique potential for high sensitivity and selectivity. The mass spectrometer used was a Sciex´s Qtrap3200 working in negative Multi Reacting Monitoring Mode (MRM). Typically mass detection limits were determined to be near the 1-pg level. The APPI-MS2 technology applied to the detection of PCDD/Fs allows fast and reliable atmospheric analysis, minimizing considerably operational times and costs, with respect other technologies available. In addition, the limit of detection can be easily improved using a more sensitive mass spectrometer since the background in the analysis channel is very low. The APPI developed by SEADM allows polar and non-polar compounds ionization with high efficiency and repeatability.

Keywords: atmospheric pressure photoionization-mass spectrometry (APPI-MS), dioxin, furan, incinerator

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Authors: Katherine Del Carmen Camacho Zorogastua, Victor Hugo Gallo Ramos, Jhon Walter Gomez Lora

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Marine hydrocarbon exploration (oil and natural gas) activities are developed using 2D, 3D and 4D seismic prospecting techniques where sound waves are directed from a seismic vessel emitted every few seconds depending on the variety of air compressors, which cross the layers of rock at the bottom of the sea and are reflected to the surface of the water. Hydrophones receive and record the reflected energy signals for cross-sectional mapping of the lithological profile in order to identify possible areas where hydrocarbon deposits can be formed. However, they produce several significant negative environmental impacts on the marine ecosystem and in the social and economic sectors. Therefore, the objective of the research is to publicize the negative impacts and environmental measures that must be carried out during the development of these activities to prevent and mitigate water quality, the population involved (fishermen) and the marine biota (e.g., Cetaceans, fish) that are the most vulnerable. The research contains technical environmental aspects based on bibliographic sources of environmental studies approved by the Peruvian authority, research articles, undergraduate and postgraduate theses, books, guides, and manuals from Spain, Australia, Canada, Brazil, and Mexico. It describes the negative impacts on the environment and population (fishing sector), environmental prevention, mitigation, recovery and compensation measures that must be properly implemented and the cases of global sea species stranding, for which international experiences from Spain, Madagascar, Mexico, Ecuador, Uruguay, and Peru were referenced. Negative impacts on marine fauna, seawater quality, and the socioeconomic sector (fishermen) were identified. Omission or inadequate biological monitoring in mammals could alter their ability to communicate, feed, and displacement resulting in their stranding and death. In fish, they cause deadly damage to physical-physiological type and in their behavior. Inadequate wastewater treatment and waste management could increase the organic load and oily waste on seawater quality in violation of marine flora and fauna. The possible estrangement of marine resources (fish) affects the economic sector as they carry out their fishing activity for consumption or sale. Finally, it is concluded from the experiences gathered from Spain, Madagascar, Mexico, Ecuador, Uruguay, and Peru that there is a cause and effect relationship between the inadequate development of seismic exploration activities (cause) and marine species strandings (effect) since over the years, stranded or dead marine mammals have been detected on the shores of the sea in areas of seismic acquisition of hydrocarbons. In this regard, it is recommended to establish technical procedures, guidelines, and protocols for the monitoring of marine species in order to contribute to the conservation of hydrobiological resources.

Keywords: 3D seismic prospecting, cetaceans, significant environmental impacts, prevention, mitigation, recovery, environmental compensation

Procedia PDF Downloads 146

Authors: Wendy De Gomez

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The University of Waterloo is situated in central Canada in the Province of Ontario- one hour from the metropolitan city of Toronto. For over 30 years, it has held Canada’s top spot as the most innovative university; and has been consistently ranked in the top 25 computer science and top 50 engineering schools in the world. Waterloo benefits from the federal government’s over 100 domestic innovation policies which have assisted in the country’s 15th place global ranking in the World Intellectual Property Organization’s (WIPO) 2022 Global Innovation Index. Yet undoubtedly, the University of Waterloo’s unique characteristics are what propels its innovative creativeness forward. This paper will provide a contextual definition of innovation in higher education and then demonstrate the five operational attributes that contribute to the University of Waterloo’s innovative reputation. The methodology is based on statistical analyses obtained from ranking bodies such as the QS World University Rankings, a secondary literature review related to higher education innovation in Canada, and case studies that exhibit the operationalization of the attributes outlined below. The first attribute is geography. Specifically, the paper investigates the network structure effect of the Toronto-Waterloo high-tech corridor and the resultant industrial relationships built there. The second attribute is University Policy 73-Intellectal Property Rights. This creator-owned policy grants all ownership to the creator/inventor regardless of the use of the University of Waterloo property or funding. Essentially, through the incentivization of IP ownership by all researchers, further commercialization and entrepreneurship are formed. Third, this IP policy works hand in hand with world-renowned business incubators such as the Accelerator Centre in the dedicated research and technology park and velocity, a 14-year-old facility that equips and guides founders to build and scale companies. Communitech, a 25-year-old provincially backed facility in the region, also works closely with the University of Waterloo to build strong teams, access capital, and commercialize products. Fourth, Waterloo’s co-operative education program contributes 31% of all co-op participants to the Canadian economy. Home to the world’s largest co-operative education program, data shows that over 7,000 from around the world recruit Waterloo students for short- and long-term placements- directly contributing to the student’s ability to learn and optimize essential employment skills when they graduate. Finally, the students themselves at Waterloo are exceptional. The entrance average ranges from the low 80s to the mid-90s depending on the program. In computer, electrical, mechanical, mechatronics, and systems design engineering, to have a 66% chance of acceptance, the applicant’s average must be 95% or above. Singularly, none of these five attributes could lead to the university’s outstanding track record of innovative creativity, but when bundled up into a 1000 acre- 100 building main campus with 6 academic faculties, 40,000+ students, and over 1300 world-class faculty, the recipe for success becomes quite evident.

Keywords: IP policy, higher education, economy, innovation

Procedia PDF Downloads 45

Authors: Teresita Lopez Joy, Jose A. Acevedo Suarez, Martha I. Gomez Acosta, Ana Julia Acevedo Urquiaga

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Current trends in business competitiveness indicate the need to manage businesses as supply chains and not in isolation. The use of strategies aimed at maximum satisfaction of customers in a network and based on inter-company cooperation; contribute to obtaining successful joint results. In the Cuban economic context, the development of productive linkages to achieve integrated management of supply chains is considering a key aspect. In order to achieve this jump, it is necessary to develop acting capabilities in the entities that make up the chains through a systematic procedure that allows arriving at a management model in consonance with the environment. The objective of the research focuses on: designing a model and procedure for the development of integrated management of supply chains in economic entities. The results obtained are: the Model and the Procedure for the Development of the Supply Chains Integrated Management (MP-SCIM). The Model is based on the development of logistics in the network actors, the joint work between companies, collaborative planning and the monitoring of a main indicator according to the end customers. The application Procedure starts from the well-founded need for development in a supply chain and focuses on training entrepreneurs as doers. The characterization and diagnosis is done to later define the design of the network and the relationships between the companies. It takes into account the feedback as a method of updating the conditions and way to focus the objectives according to the final customers. The MP-SCIM is the result of systematic work with a supply chain approach in companies that have consolidated as coordinators of their network. The cases of the edible oil chain and explosives for construction sector reflect results of more remarkable advances since they have applied this approach for more than 5 years and maintain it as a general strategy of successful development. The edible oil trading company experienced a jump in sales. In 2006, the company started the analysis in order to define the supply chain, apply diagnosis techniques, define problems and implement solutions. The involvement of the management and the progressive formation of performance capacities in the personnel allowed the application of tools according to the context. The company that coordinates the explosives chain for construction sector shows adequate training with independence and opportunity in the face of different situations and variations of their business environment. The appropriation of tools and techniques for the analysis and implementation of proposals is a characteristic feature of this case. The coordinating entity applies integrated supply chain management to its decisions based on the timely training of the necessary action capabilities for each situation. Other cases of study and application that validate these tools are also detailed in this paper, and they highlight the results of generalization in the quantitative and qualitative improvement according to the final clients. These cases are: teaching literature in universities, agricultural products of local scope and medicine supply chains.

Keywords: integrated management, logistic system, supply chain management, tactical-operative planning

Procedia PDF Downloads 120

Authors: A. Gonzalez Aguna, J. M. Santamaria Garcia, J. L. Gomez Gonzalez, R. Barchino Plata, M. Fernandez Batalla, S. Herrero Jaen

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Background: Computer Science is a field that transcends other disciplines of knowledge because it allows to support all kinds of physical and mental tasks. Health centres have a greater number and complexity of technological devices and the population consume and demand services derived from technology. Also, nursing education plans have included competencies related to and, even, courses about new technologies are offered to health professionals. However, nurses still limit their performance to the use and evaluation of products previously built. Objective: Develop a teaching-learning methodology for acquiring skills on designing applications for care. Methodology: Blended learning teaching with a group of graduate nurses through official training within a Master's Degree. The study sample was selected by intentional sampling without exclusion criteria. The study covers from 2015 to 2017. The teaching sessions included a four-hour face-to-face class and between one and three tutorials. The assessment was carried out by written test consisting of the preparation of an IEEE 830 Standard Specification document where the subject chosen by the student had to be a problem in the area of care. Results: The sample is made up of 30 students: 10 men and 20 women. Nine students had a degree in nursing, 20 diploma in nursing and one had a degree in Computer Engineering. Two students had a degree in nursing specialty through residence and two in equivalent recognition by exceptional way. Except for the engineer, no subject had previously received training in this regard. All the sample enrolled in the course received the classroom teaching session, had access to the teaching material through a virtual area and maintained at least one tutoring. The maximum of tutorials were three with an hour in total. Among the material available for consultation was an example of a document drawn up based on the IEEE Standard with an issue not related to care. The test to measure competence was completed by the whole group and evaluated by a multidisciplinary teaching team of two computer engineers and two nurses. Engineers evaluated the correctness of the characteristics of the document and the degree of comprehension in the elaboration of the problem and solution elaborated nurses assessed the relevance of the chosen problem statement, the foundation, originality and correctness of the proposed solution and the validity of the application for clinical practice in care. The results were of an average grade of 8.1 over 10 points, a range between 6 and 10. The selected topic barely coincided among the students. Examples of care areas selected are care plans, family and community health, delivery care, administration and even robotics for care. Conclusion: The applied methodology of learning-teaching for the design of technologies demonstrates the success in the training of nursing professionals. The role of expert is essential to create applications that satisfy the needs of end users. Nursing has the possibility, the competence and the duty to participate in the process of construction of technological tools that are going to impact in care of people, family and community.

Keywords: care, learning, nursing, technology

Procedia PDF Downloads 104

Authors: S. Delgado, C. Cerrada, R. S. Gómez

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This research introduces an approach to voxelizing the surfaces of triangular meshes with efficiency and accuracy. Our method leverages parallel equidistant scan-lines and introduces a Gap Detection technique to address the limitations of existing approaches. We present a comprehensive study showcasing the method's effectiveness, scalability, and versatility in different scenarios. Voxelization is a fundamental process in computer graphics and simulations, playing a pivotal role in applications ranging from scientific visualization to virtual reality. Our algorithm focuses on enhancing the voxelization process, especially for complex models and high resolutions. One of the major challenges in voxelization in the Graphics Processing Unit (GPU) is the high cost of discovering the same voxels multiple times. These repeated voxels incur in costly memory operations with no useful information. Our scan-line-based method ensures that each voxel is detected exactly once when processing the triangle, enhancing performance without compromising the quality of the voxelization. The heart of our approach lies in the use of parallel, equidistant scan-lines to traverse the interiors of triangles. This minimizes redundant memory operations and avoids revisiting the same voxels, resulting in a significant performance boost. Moreover, our method's computational efficiency is complemented by its simplicity and portability. Written as a single compute shader in Graphics Library Shader Language (GLSL), it is highly adaptable to various rendering pipelines and hardware configurations. To validate our method, we conducted extensive experiments on a diverse set of models from the Stanford repository. Our results demonstrate not only the algorithm's efficiency, but also its ability to produce 26 tunnel free accurate voxelizations. The Gap Detection technique successfully identifies and addresses gaps, ensuring consistent and visually pleasing voxelized surfaces. Furthermore, we introduce the Slope Consistency Value metric, quantifying the alignment of each triangle with its primary axis. This metric provides insights into the impact of triangle orientation on scan-line based voxelization methods. It also aids in understanding how the Gap Detection technique effectively improves results by targeting specific areas where simple scan-line-based methods might fail. Our research contributes to the field of voxelization by offering a robust and efficient approach that overcomes the limitations of existing methods. The Gap Detection technique fills a critical gap in the voxelization process. By addressing these gaps, our algorithm enhances the visual quality and accuracy of voxelized models, making it valuable for a wide range of applications. In conclusion, "Closing the Gap: Efficient Voxelization with Equidistant Scan-lines and Gap Detection" presents an effective solution to the challenges of voxelization. Our research combines computational efficiency, accuracy, and innovative techniques to elevate the quality of voxelized surfaces. With its adaptable nature and valuable innovations, this technique could have a positive influence on computer graphics and visualization.

Keywords: voxelization, GPU acceleration, computer graphics, compute shaders

Procedia PDF Downloads 39

Authors: Emilio Galdeano-Gómez, Juan C. Pérez-Mesa, Laura Piedra-Muñoz, María C. García-Barranco, Jesús Hernández-Rubio

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The situation in which firms engage in simultaneous cooperation and competition with each other is a phenomenon known as co-opetition. This scenario has received increasing attention in business economics and management analyses. In the domain of supply chain networks and for small and medium-sized enterprises, SMEs, these strategies are of greater relevance given the complex environment of globalization and competition in open markets. These firms face greater challenges regarding technology and access to specific resources due to their limited capabilities and limited market presence. Consequently, alliances and collaborations with both buyers and suppliers prove to be key elements in overcoming these constraints. However, rivalry and competition are also regarded as major factors in successful internationalization processes, as they are drivers for firms to attain a greater degree of specialization and to improve efficiency, for example enabling them to allocate scarce resources optimally and providing incentives for innovation and entrepreneurship. The present work aims to contribute to the literature on SMEs’ internationalization strategies. The sample is constituted by a panel data of marketing firms from the Andalusian food sector and a multivariate regression analysis is developed, measuring variables of co-opetition and international activity. The hierarchical regression equations method has been followed, thus resulting in three estimated models: the first one excluding the variables indicative of channel type, while the latter two include the international retailer chain and wholesaler variable. The findings show that the combination of several factors leads to a complex scenario of inter-organizational relationships of cooperation and competition. In supply chain management analyses, these relationships tend to be classified as either buyer-supplier (vertical level) or supplier-supplier relationships (horizontal level). Several buyers and suppliers tend to participate in supply chain networks, and in which the form of governance (hierarchical and non-hierarchical) influences cooperation and competition strategies. For instance, due to their market power and/or their closeness to the end consumer, some buyers (e.g. large retailers in food markets) can exert an influence on the selection and interaction of several of their intermediate suppliers, thus endowing certain networks in the supply chain with greater stability. This hierarchical influence may in turn allow these suppliers to develop their capabilities (e.g. specialization) to a greater extent. On the other hand, for those suppliers that are outside these networks, this environment of hierarchy, characterized by a “hub firm” or “channel master”, may provide an incentive for developing their co-opetition relationships. These results prove that the analyzed firms have experienced considerable growth in sales to new foreign markets, mainly in Europe, dealing with large retail chains and wholesalers as main buyers. This supply industry is predominantly made up of numerous SMEs, which has implied a certain disadvantage when dealing with the buyers, as negotiations have traditionally been held on an individual basis and in the face of high competition among suppliers. Over recent years, however, cooperation among these marketing firms has become more common, for example regarding R&D, promotion, scheduling of production and sales.

Keywords: co-petition networks, international supply chain, maketing agrifood firms, SMEs strategies

Procedia PDF Downloads 49

Authors: Alberto Fina, Samuele Colonna, Maria del Mar Bernal, Orietta Monticelli, Mauro Tortello, Renato Gonnelli, Julio Gomez, Chiara Novara, Guido Saracco

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Thermally conductive polymer nanocomposites are of high interest for several applications including low-temperature heat recovery, heat exchangers in a corrosive environment and heat management in electronics and flexible electronics. In this paper, the preparation of thermally conductive nanocomposites exploiting graphene-related materials is addressed, along with their thermal characterization. In particular, correlations between 1- chemical and physical features of the nanoflakes and 2- processing conditions with the heat conduction properties of nanocomposites is studied. Polymers are heat insulators; therefore, the inclusion of conductive particles is the typical solution to obtain a sufficient thermal conductivity. In addition to traditional microparticles such as graphite and ceramics, several nanoparticles have been proposed, including carbon nanotubes and graphene, for the use in polymer nanocomposites. Indeed, thermal conductivities for both carbon nanotubes and graphenes were reported in the wide range of about 1500 to 6000 W/mK, despite such property may decrease dramatically as a function of the size, number of layers, the density of topological defects, re-hybridization defects as well as on the presence of impurities. Different synthetic techniques have been developed, including mechanical cleavage of graphite, epitaxial growth on SiC, chemical vapor deposition, and liquid phase exfoliation. However, the industrial scale-up of graphene, defined as an individual, single-atom-thick sheet of hexagonally arranged sp2-bonded carbons still remains very challenging. For large scale bulk applications in polymer nanocomposites, some graphene-related materials such as multilayer graphenes (MLG), reduced graphene oxide (rGO) or graphite nanoplatelets (GNP) are currently the most interesting graphene-based materials. In this paper, different types of graphene-related materials were characterized for their chemical/physical as well as for thermal properties of individual flakes. Two selected rGOs were annealed at 1700°C in vacuum for 1 h to reduce defectiveness of the carbon structure. Thermal conductivity increase of individual GNP with annealing was assessed via scanning thermal microscopy. Graphene nano papers were prepared from both conventional RGO and annealed RGO flakes. Characterization of the nanopapers evidenced a five-fold increase in the thermal diffusivity on the nano paper plane for annealed nanoflakes, compared to pristine ones, demonstrating the importance of structural defectiveness reduction to maximize the heat dissipation performance. Both pristine and annealed RGO were used to prepare polymer nanocomposites, by melt reactive extrusion. Thermal conductivity showed two- to three-fold increase in the thermal conductivity of the nanocomposite was observed for high temperature treated RGO compared to untreated RGO, evidencing the importance of using low defectivity nanoflakes. Furthermore, the study of different processing paremeters (time, temperature, shear rate) during the preparation of poly (butylene terephthalate) nanocomposites evidenced a clear correlation with the dispersion and fragmentation of the GNP nanoflakes; which in turn affected the thermal conductivity performance. Thermal conductivity of about 1.7 W/mK, i.e. one order of magnitude higher than for pristine polymer, was obtained with 10%wt of annealed GNPs, which is in line with state of the art nanocomposites prepared by more complex and less upscalable in situ polymerization processes.

Keywords: graphene, graphene-related materials, scanning thermal microscopy, thermally conductive polymer nanocomposites

Procedia PDF Downloads 244

Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

Procedia PDF Downloads 318