Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-02-13T14:53:36Z No. of bitstreams: 1 Clarissa Sampaio de Oliveira Lima.pdf: 835685 bytes, checksum: 2d915cd2ec8e3deb3719d91c16b3073f (MD5) / Made available in DSpace on 2017-02-13T14:53:36Z (GMT). No. of bitstreams: 1 Clarissa Sampaio de Oliveira Lima.pdf: 835685 bytes, checksum: 2d915cd2ec8e3deb3719d91c16b3073f (MD5) Previous issue date: 2016-01-26 / Blood cholesterol excess can be responsible for the onset of cardiovascular disease, especially atherosclerosis. The inclusion of carotenoids in the diet of animals can benefit human health by changing the concentration of cholesterol and fatty acid profile, due antioxidant action. The urucunzeiro (Bixa orellana L.) features a powerful natural carotenoid. Aimed to evaluate plasma lipids and fatty acids in dairy cows fed diets containing annatto. 32 Holstein cows were used (550 kg), distributed in a completely randomized design allocated in individual pens submitted to the following treatments: (1) diet without spice, (2) 0.08 g of spice / kg DM, (3) 0 , 12g of spice / kg DM and (4) 0.16 g of spice / kg DM. The blood sample was collected via the epigastric vein puncture, centrifuged and frozen for later analysis. The results showed that the inclusion of annatto not promote improvements in the quality of serum cholesterol, increase HDL and LDL decreased (P> 0.05). The results indicate that the use of annatto in cows diet does not allow improvements in total cholesterol concentration, however leads to changes in plasma fatty acid profile (p <0.05). / Excesso de colesterol sanguíneo pode ser responsável pelo aparecimento das doenças cardiovasculares, especialmente a aterosclerose. A inclusão de carotenoides na dieta dos animais pode trazer benefícios à saúde humana, alterando a concentração de colesterol e perfil de ácido graxo, devido ação antioxidante. O urucunzeiro (Bixa orellana L.) apresenta um potente carotenoide natural. Objetivou-se avaliar o perfil lipídico plasmático e de ácidos graxos de vacas leiteiras que receberam dietas contendo urucum. Foram utilizadas 32 vacas Holandesas (550 kg), distribuídas em delineamento inteiramente casualizado alocadas em baias individuais submetidas aos seguintes tratamentos: (1) dieta sem inclusão de colorífico, (2) 0,08g de colorífico/kg de MS, (3) 0,12g de colorífico/kg de MS e (4) 0,16g de colorífico/kg de MS. A amostra de sangue foi coletada via punção da veia epigástrica, centrifugada e congelada para análise posterior. Os resultados mostraram que a inclusão do urucum não promove melhorias na qualidade do colesterol plasmático, aumento do HDL e diminuição do LDL (P>0,05). Os resultados indicam que a utilização de urucum na dieta de vacas não possibilita a melhoria na concentração de colesterol total, entretanto promove alteração no perfil de acido graxo plasmático (p<0,05).

Objetivou-se avaliar o efeito da maturação (14 dias) e do escore de marmorização (escore visual 4 ou maior e escore visual 3 ou menor) sobre a qualidade física e química, sensorial, microbiológica e perfil de ácidos graxos da carne suína. Foram utilizados 40 lombos (m. longissimus) oriundos de suínos mestiços comerciais, fêmeas, com peso corporal médio final de 130 kg e aproximadamente 180 dias de idade, provenientes de cruzamento de linhagem paterna Duroc, submetidos à mesma condição alimentar. Os lombos foram coletados em frigorifico comercial e separados de acordo com o escore visual de marmorização, por meio de cartões fotográficos (variando o valor da escala visual de 1 a 6 e 10). Foi considerada alta marmorização (A) os lombos com pontuações acima ou igual a 4 e baixa (B) com pontuações abaixo ou igual a 3. Em seguida, os lombos foram divididos em: 1- lombos sem maturação e com marmorização abaixo ou igual a 3; 2- lombos sem maturação e com marmorização acima ou igual a 4; 3- lombos maturados por 14 dias e com marmorização abaixo ou igual a 3 e 4- lombos maturados por 14 dias e com marmorização acima ou igual a 4. Ainda, foi avaliada a área de olho de lombo (AOL) e espessura de gordura subcutânea (EGS). Foram coletadas amostras de 2,54 cm e de 1,0 cm, embaladas a vácuo e maturadas por zero (sem maturação) e 14 dias para análises de pH, cor, maciez objetiva (FC), perdas por cocção (PPC), lipídeos totais (LT), perfil de ácidos graxos, colesterol total (CT), oxidação lipídica (TBARS), análise sensorial e microbiológica. Os LT diferiram-se (P &lt; 0,05) entre alta e baixa marmorização (3,42% e 2,39%, respectivamente). Os lombos com alta marmorização apresentaram menores valores de AOL e maiores valores de EGS (P &lt; 0,05). A maturação por 14 dias reduziu os valores de pH dos lombos em comparação aos não maturados (P &lt; 0,01). As carnes maturadas por 14 dias apresentaram-se mais claras em comparação às não maturadas (P &lt; 0,01). O escore de marmorização e a maturação não influenciaram a FC, PPC e TBARS. As carnes maturadas apresentaram maior contagem de bactérias ácido-láticas e psicrotróficas (P &lt; 0,01), no entanto, sem risco para a saúde humana. Com o aumento do escore de marmorização houve redução nas concentrações dos ácidos graxos poliinsaturados e na relação AGPI:AGS e aumento na concentração dos ácidos graxos saturados (P &lt; 0,05). O escore de marmorização não alterou o CT nos lombos. O atributo sabor, avaliado em análise sensorial foi influenciado pelo escore de marmorização (P &lt; 0,05). Os consumidores avaliaram a carne de alta marmorização como sendo a mais saborosa. A marmorização na carne suína pode ser um atrativo ao consumidor mais exigente em sabor, contudo, pode impactar no valor nutricional da carne, o que pode prejudicar a intenção de compra. / The aim of the study was to evaluate the effect of aging (14 days) and marbling score (visual marbling scores less than 3 and greater than 4) on sensory attributes, microbiological, fatty acid profile, physical and chemical analysis of pork loin. A total of 40 pork loins (longissimus muscle, LM) were obtained by commercially raised crossbred gilts (average carcass weight = 130 kg, 180 days of age) from a crossbred Duroc paternal lineage. Pigs were fed similar based diet. The LM were collected in a commercial packing plant and separated according to the visual marbling score, (visual scores from 1 to 6 and 10). the loins were divided into higher (HMAR) and lower marbling (LMAR): 1- LM without aging and marbling score less than or equal 3; 2- LM without aging and marbling score greater than 4; 3- LM aged for 14 days and marbling score less than 3 or equal; 4- LM aged for 14 days and marbling score greater than 4 or equal. Then, the loin eye area (LMA) and subcutaneous fat thickness (SFT) were evaluated. Samples of 2.54 and 1.0 cm were collected and aging for zero and 14 days to pH, color, shear force (SF), cooking loss (CL), total lipids (TL), fatty acid profile, cholesterol content (CHL), thiobarbituric acid-reactive substance (TBARS), sensory and microbiological analysis. The TL differed (P &lt; 0.05) between high and low marbling score (3.42% and 2.39%, respectively). Moreover, the loins with high visual marbling obtained lower values of LMA and high values of SFT (P &lt; 0.05). The aging for 14 days reduced the pH and the samples were lighter compared to without aging (P &lt; 0.01). In addition, the marbling score and aging did not influence SF, CL and TBARS. The aging loins had higher of acid-lactic and psychrotrophic counts (P &lt; 0.01), however, it does not present health risks. When increasing marbling score, there was a decrease in the concentrations of polyunsaturated fatty acids (PUFA), relationship PUFA: SFA ratio and an increase in SFA (P &lt; 0.05). The marbling score did not change the CHL content. The flavor attribute was influenced by the marbling score (P &lt; 0.05). The consumers evaluated the HMAR as the tastiest. The marbling score may be a more consumer-demanding taste, but it can impact the nutritional values of meat and reduce the purchase intention.

Os ácidos graxos poli-insaturados da família &#937;-3 são classificados como possíveis agentes anti-inflamatórios devido à formação de eicosanóides inflamatórios menos potentes. Cães no período pós-cirúrgico ou com câncer podem desenvolver resposta inflamatória exacerbada, o que pode ser danoso ao organismo. Assim, a intervenção nutricional é parte da terapia para suporte do sistema imune e da modulação da inflamação. O objetivo deste estudo foi verificar a modulação da resposta inflamatória, o status nutricional e a composição corporal de cadelas no período pós- cirúrgico consumindo dieta com alto teor de proteína, gordura, EPA e DHA. Doze animais foram incluídos e divididos em dois grupos experimentais que receberam dietas distintas: grupo A - ração para cão adulto em manutenção sem EPA e DHA; grupo B - ração teste com alto teor de proteínas, gordura e EPA/DHA por meio de enriquecimento da dieta com óleo de peixe. Mensurações séricas de TNF-&#945;, IL-6, IL-10, IGF-1, proteína C reativa (PCR); determinação da composição corporal por deutério; e parâmetros nutricionais laboratoriais foram realizados em intervalos periódicos ao longo de 51 dias. Testes estatísticos paramétricos e não paramétricos foram utilizados para comparar a quantidade de nutrientes ingeridos e efeito da ingestão de óleo de peixe nos parâmetros inflamatórios, nutricionais e na porcentagem de massa muscular (MM) e gordura corporal (GC). Não foram observadas diferenças nas concentrações de citocinas (pgt;0,05) nem de PCR (p= 0,51) entre as dietas. Quando considerado o tipo de dieta independente do tempo de análise, pôde-se constatar que o grupo B teve maior concentração de IGF-1 (p=0,04), MM (p&lt;0,01) e menor GC (p&lt;0,01), no qual os dois últimos parâmetros tiveram correlação positiva com escore de condição corporal e muscular avaliados no exame físico. Conclui-se que o teor de EPA e DHA na concentração avaliada neste estudo não foi suficiente para modular a inflamação, mas apresenta potencial efeito benéfico na manutenção da massa muscular. / Omega 3 polyunsaturated fatty acids family are said to be anti-inflammatory agents due to the formation of less potent inflammatory eicosanoids. Dogs in the post-surgical or with cancer can develop an unwanted inflammatory response, which can be harmful to these. Thus, nutritional intervention is part of the therapy for the support of the immune system and the modulation of inflammation. The objective of this study was to verify the inflammatory response modulation, nutritional status and body composition of bitches in the post-surgical period consuming diet with high protein, fat, EPA and DHA content. Twelve bitches were included and divided into two groups receiving different diets: group A extrused dry feed for adult dogs in maintenance without EPA and DHA; Group B - extrused dry feed with high protein, fat and EPA/DHA levels through dietary enrichment with fish oil. Serum measurements of TNF-&#945;, IL-6, IL-10, IGF-1, C-reactive protein (CRP); determination of body composition by deuterium; and nutritional laboratories parameters were performed at periodic intervals over 51 days. Parametric and non-parametric statistical tests were used to compare the amount of nutrients ingested and the effect of fish oil intake on inflammatory, nutritional parameters and percentage of muscle mass (MM) and body fat (BF). There were no differences in cytokines (p&gt; 0.05) or CRP (p = 0.51) concentrations between diets. When considering the type of diet independent of the time of analysis, it can be seen that group B had a higher concentration of IGF-1 (p = 0.04), MM (p &lt; 0.01) and lower BF (p &lt;0.01), which the last two parameters had a positive correlation with body and muscle condition score assessed on physical examination. It is concluded that the EPA and DHA content of this study can not modulate the inflammation, but has a potential beneficial effect on the maintenance of the body composition.

Made available in DSpace on 2016-04-28T14:59:46Z (GMT). No. of bitstreams: 1 2009 - Renata de Oliveira Santos Ramalho.pdf: 151905 bytes, checksum: 3e6bc90df190c2dc80a5b2ac16eb37fd (MD5) Previous issue date: 2009-02-13 / The objective of the present study was to compare the fatty acids (FA) profile in Longissimus dorsi (LDM) muscle of bovine Nelore and F1 Sindi Nelore. A completely randomized experimental design in 2 x 2 factorial arrangements (two genetic groups and two ages - 36 and 48 months) was used. Sixteen young bulls (eight Nelore and eight F1 Sindhi Nellore), castrated were used. Animals were fed the same diet and were slaughtered at average live weight of 460.0 (10.1) kg. The FA profile was determined by gas chromatography with high resolution, within a universe of 14 FA, and monounsaturated fatty acids (MFA), polyunsaturated fatty acids (PUFAs), total saturated fatty acids (SFA) and total unsaturated fatty acids (UFA). The animals F1 Sindi Nelore presented smaller concentration of the fatty acid saturated mirystic (0.88%), in comparison with the Nelore (1.31%). There was not interaction (P>0.05) among genetic group and age for studied SFA. The palmitic acid presented larger concentration among SFA. Among the monounsaturated there was significant difference (P <0.05) among the genetic groups for the palmitoleic acid, that had 0.63 percentage points the more for the animals Nelore. The oleic acid presented larger concentration in the meat of the two genetic groups, with 41.44% for the Nelore and 41.38% for F1 Sindi Nelore, followed by the linoleic acid , with 6.15% for the Nelore and 4.86% for F1 Sindi Nelore. There was not statistical difference (P>0.05) for monounsaturated total or for the unsaturated total among the genetic groups. There was no interaction (P>0.05) among genetic group and age for the monounsaturated and polyunsaturated. There was difference (P <0.05) for the ration &#969;6: &#969;3. F1 Sindi Nelore obtained smaller ration &#969;6: &#969;3. the smallest concentration of acid mirystic and the smallest ration &#969;6: &#969;3 in the meat of bovine F1 Sindi Nelore checks makes the meat of this breed healthier considering the fatty acids profile. / O objetivo do presente trabalho foi comparar o perfil de ?cidos graxos (AG) do m?sculo Longissimus dorsi (MLD) de bovinos Nelore e F1 Sindi Nelore. O delineamento experimental foi o inteiramente casualizado em um esquema fatorial 2 x 2 (dois grupos gen?ticos e duas idades - 36 e 48 meses). Foram utilizados 16 novilhos, 8 Nelore e 8 F1 Sindi Nelore, castrados, que receberam a mesma dieta e foram abatidos com peso vivo m?dio de 460,0 (10,1) kg. O perfil de AG foi determinado por cromatografia gasosa de alta resolu??o, dentro de um universo de 14 AG, entre ?cidos graxos monoinsaturados (AGM), ?cidos graxos poliinsaturados (AGPI), total de ?cidos graxos saturados (AGS) e total de ?cidos graxos insaturados (AGI). Os animais F1 Sindi Nelore apresentaram menor concentra??o do ?cido graxo saturado mir?stico (0,88%), em compara??o ao Nelore (1,31%). N?o houve intera??o (P>0,05) entre grupo gen?tico e idade para os AGS estudados. O ?cido palm?tico foi o que apresentou maior concentra??o entre os AGS. Dentre os monoinsaturados houve diferen?a significativa (P<0,05) entre os grupos gen?ticos apenas para o ?cido palmitoleico, que teve 0,63 pontos percentuais a mais para os animais Nelore. O ?cido ol?ico foi o que apresentou maior concentra??o na carne dos dois grupos gen?ticos, com 41,44 % para os Nelore e 41,38 % para os F1 Sindi Nelore, seguido do ?cido linoleico, com 6,15 % para os Nelore e 4,86 % para os F1 Sindi Nelore. N?o houve diferen?a estat?stica (P>0,05) para total de monoinsaturados ou para o total de insaturados entre os grupos gen?ticos. N?o houve intera??o (P>0,05) entre grupo gen?tico e idade para os monoinsaturados e poliinsaturados. Houve diferen?a (P<0,05) para a rela??o &#969;6:&#969;3, sendo que F1 Sindi Nelore obtiveram menor rela??o &#969;6:&#969;3. A menor concentra??o de ?cido mir?stico e a menor rela??o &#969;6:&#969;3 na carne de bovinos F1 Sindi Nelore confere a esta ra?a uma carne mais saud?vel no que se refere ao perfil de ?cidos graxos.

In both the legume symbiont Sinorhizobium meliloti and the mammalian pathogen Brucella abortus, the inner membrane BacA protein is essential for host persistence. In free-living S. meliloti and B. abortus loss of the BacA protein also results in an increased resistance to the glycopeptide bleomycin and a ~ 50% decrease in the lipopolysaccharide (LPS) very-long-chain-fatty-acid (VLCFA) content. Consequently, it was proposed that BacA may be involved in transport of peptides into the cell and/or that BacA may be involved in the VLCFA modification of the LPS. During this work it was determined that the increased resistance observed in an S. meliloti DbacA mutant to bleomycin and to the truncated eukaryotic peptide Bac7(1-16), is independent of the VLCFA modification. These data support a model for BacA having multiple non-overlapping functions. Using flow cytometry studies with fluorescently labelled forms of bleomycin and Bac7(1-16) it was found that the BacA protein plays a role in the uptake of bleomycin. However, BacA was shown to be essential for the uptake of Bac7(1-16). Additionally, it was determined that two symbiotically defective bacA site directed mutants with known reductions in their VLCFA could still take up Bac7, suggesting that the BacA function that leads to the VLCFA modification could also play a key role in host persistence. To investigate further the role of BacA in the VLCFA modification and where in the cell envelope the lipid A is modified with the VLCFA, the role of the putative lipid trafficking protein MsbA2 was investigated. Interestingly, it was discovered that S. meliloti lacking the MsbA2 protein, is unable to enter host cells and induces a plant defence response more characteristic of a pathogen. To investigate the importance of the VLCFA modification during the symbiosis S. meliloti mutants lacking either the AcpXL (VLCFA acyl carrier protein) or LpxXL (VLCFA acyl transferase protein) were characterized in the host. Although not essential for host persistence, loss of each of the proteins did result in distinct defects, suggesting the VLCFA modification is important during the symbiosis. Since there are hundreds of nodule specific cysteine-rich peptides produced by the host plant Medicago truncatula, the BacA mediated uptake of one of these peptides combined with the VLCFA modification may account for the essential role of the BacA protein in the legume symbiosis.

Dietary fish oil supplementation has long been shown to have significant health benefits, largely stemming from the anti-inflammatory activity of the ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) present in fish oils. The anti-inflammatory properties of these fatty acids has been linked to beneficial health effects, such as protecting the heart, in individuals consuming diets rich in fish, or supplemented with fish oils. These effects are highly notable in the Māori people native to coastal regions of New Zealand; the significantly lower rates of heart problems compared to the inland populous has been attributed to the consumption of the green lipped mussel Perna Canaliculus. Commercially available health supplements based on the New Zealand green lipped mussel include a freeze-dried powder and a lipid extract (Lyprinol®), the latter of which has shown anti-inflammatory properties comparable to classical non-steroidal anti-inflammatory drugs (NSAIDs) such as Naproxen. GCMS analysis of Lyprinol by Murphy et al. showed the presence of a class of ω-4 and ω-6 PUFAs bearing a highly electron rich tri- or tetra-alkyl furan ring, which were designated furan fatty acids (F-acids). Due to their instability, isolation of F-acids from natural sources cannot be carried out and a general synthetic route toward this class of natural products was required. To accomplish this, the biosynthesis of F-acids was mimicked by utilising an oxidation of 1,3-dienes, followed by a dehydration/aromatisation to generate the heterocyclic furan ring. Singlet oxygen was chosen as the means of oxidising the conjugated dienes giving endoperoxides. To mimic the biological aromatisation of the peroxide intermediates the Appel reagent was chosen and, in a novel application of the reagent, was exploited as a mild, metal free method of dehydrating the cyclic peroxides to their corresponding furans. The biomimetic furan synthesis was applied toward a selection of 1,3-diene substrates bearing a range of pre-installed functionalities and substitution patterns including alkyl, aryl, alkenes, cyclopropyl rings, silyl ethers, and esters, alongside being applied to the total synthesis of the furan fatty acid F5. A brief exploration of the possibility of performing the aromatisation reaction under catalytic conditions was carried out, to determine whether endoperoxides could be converted to furans without needing a stoichiometric quantity of Appel reagent, by harnessing a catalytic quantity of triphenylphosphine oxide and regenerating the active P(V) species via reaction with oxalyl chloride. Furthermore, an optimisation study was carried out using a simple design of experiments procedure to ascertain the ideal conditions for carrying out the Appel-type dehydration of endoperoxides. Finally, the scope of the reaction sequence was expanded to be performed in a continuous flow reactor, with telescoping of the singlet oxygen diene oxidation and Appel-type aromatisation to increase oxidation yields and to omit the requirement for isolation of peroxide intermediates, and was applied to the synthesis of a selection of 2,5-diaryl furan motifs.

The purpose of this study was to determine if supplementation of two grams of fish oil for 90 days would significantly lower cardiovascular disease (CVD) risk in Hispanics with type 2 diabetes. The Hispanic American population is at an increased risk for CVD. Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) in fish oil have been found to reduce risk of CVD. Subjects were randomly divided into two groups and received either two grams of fish oil or corn oil (control) per day for 90 days. Before and after the trial, participant blood lipids and plasma fatty acids were evaluated. Respired air samples were obtained to evaluate plasma fatty acids. Although analysis of blood lipids and plasma fatty acids did not show sufficient evidence to disprove the null hypothesis, this study is an important model for future studies concerning fish oil to lower CVD risk in Hispanics with type 2 diabetes.

Many factors influence the nutrient composition of breast milk (BRM) particularly within the fatty acids composition. In this study BRM between 2-14 weeks of lactation was collected from 44 Caucasian women (24 normal weight (NW, BMI 18.5-24.9kg/m2) and 20 overweight (OW, BMI >25.0kg/m2). BRM was subjected to proximate analysis and participants completed food frequency questionnaires (FFQ) to estimate fruit, vegetable, and fat intake. BMI differed between NW and OW groups, 22.03 vs 33.86kg/m2 (p0.10) were identified for total calories (5,581.8 vs 5562.9cal/g), %fat (39.6 vs 43.9), or %protein (9.8 vs 8.9) for BRM in NW versus OW women as determined by proximal analysis. Gas chromatography of fatty acid methyl esters demonstrated NW BRM contained higher levels of omega-3, mono-unsaturated, and less palmitic acid fatty acids (p>0.10). The increased palmitic acid level seen in OW BRM may support increased de novo synthesis of fatty acids.

N-acylethanolamines (NAEs) are derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine and are hydrolyzed by fatty acid amide hydrolases (FAAH) into free fatty acid (FFA) and ethanolamine in both plants and animals. In Arabidopsis, NAE plays an important physiological role in growth/development and response to stress. Although NAEs are reported in tomato, their metabolic pathway remains undiscovered. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To this extent, a putative gene that likely encodes for putative SlFAAH1 protein was identified, cloned, and heterologously expressed. Amidase activity was tested using radiolabeled NAE substrates. Furthermore, expression of putative SlFAAH1 transcripts and protein activity was quantified at different developmental stages to demonstrate endogenous amidase activity in tomato seedlings. In future, molecular and biochemical characterization of tomato FAAH will further test the conserved nature of NAE metabolic pathway in plants.

The increasing demand for healthy edible oil has generated the need to identify promising oil crops. Tree peony (Paeonia section Moutan DC.) is a woody oil crop with α-linolenic acid contributing for 45% of the total fatty acid (FA) content in seeds. Molecular and genetic differences that contribute to varied FA content and composition among the wild peony species are however, poorly understood. Analyses of FA content and composition during seed development in three tree peony species (P. rockii, P. potaninii, and P. lutea) showed varied FA content in the three species with highest in P. rockii, followed by P. potaninii, and P. lutea. Total FA content increased with seed development and reached its maximum in its final stage. Seed FA composition analysis of the three species also revealed that α-linolenic acid (C18:3) was the most abundant, followed by oleic (C18:1) and linoleic (C18:2) acids. Additionally, quantitative real-time RT-PCR analyses of 10 key seed oil synthesis genes in the three tree peony species revealed that FAD3, FAD2, β-PDHC, LPAAT and Oleosin gene expression levels positively correlate with total FA content and rate of accumulation. Specifically, the abundance of FAD3 transcripts in P. rockii compared with P. potaninii, and P. lutea suggests that FAD3 might play in an important role in synthesis of α-linolenic acid via phosphatidylcholine-derived pathway. Overall, comparative analyses of FA content and composition in three different peony species revealed correlation between efficient lipid accumulation and lipid gene expression during seed development. Further characterization and manipulation of these key genes from peonies will allow for subsequent improvement of tree peony oil quality and production.