Activating this feature will stop all interaction and design based motions and animations on the site for the duration of your visit Activating this feature will create a focus area on the screen that will follow your mouse to eliminate distractions With a background in project management and civil engineering Bruna has focused her career on climate-resilient One of Jacobs’ newest Global Solutions Directors Bruna brings more than a decade of experience advising clients in transportation Bruna’s role is focused on integrating sustainability across Jacobs’ end markets and helping clients adapt to and mitigate the impacts of climate change.  Bruna’s experience includes the first major flood protection project in lower Manhattan climate change vulnerability assessments and resilience planning for the U.S Bruna brings a holistic and interdisciplinary understanding of climate – including risk sustainability and decarbonization – that she brings to civil engineering Bruna holds a bachelor's degree in civil engineering and master's degree in civil engineering with a focus on sustainable design and construction from Stanford University.  She also studied cooking and architecture in France – a perfect opportunity to blend her love of food and design “Every engineer holds the ability to shape a greener tomorrow Sustainability and resilience are not just goals Jacobs Cross-Market Global Solutions Director for Sustainability and Climate Response climate-resilient and sustainable projects she’s worked on length of the first public wireless charging roadway in Detroit that she helped deliver Tell us a little bit about your career journey with Jacobs.  Jacobs is supporting the Port of Long Beach in evaluating the charging and refueling infrastructure needs to support the conversion of vehicles and boats to zero-emission technologies.  Your expertise is in climate change mitigation and adaptation What compelled you to pursue these areas as a career?  I owe the spark of my passion for civil engineering to he was leading a workforce preparation program which helped me obtain an internship at a local civil engineering company The experience was formative with the help of great mentors which led me to study civil engineering in college I knew I wanted to pursue climate-related work — I wanted to pursue a career that would have a positive impact on communities and the planet I have always been naturally curious about how we design and build the infrastructure that enables our everyday lives I pursued a graduate degree in sustainable design and construction because I knew focusing on climate would be exciting and dynamic At the time there were limited classes focused on climate so I worked with my professors to craft independent studies to expand my knowledge on topics such as flood protection and sustainable materials.  Tell us about a project in the space that’s changing how we think about sustainability and climate response  Most people do not think about these mission critical pieces of infrastructure until supply chain issues arise and suddenly we are not getting our Amazon deliveries as quickly as we were previously and people start talking about and wondering why.  The Port of Long Beach is one of the busiest ports in the United States handling 90 million metric tons of cargo valued at $200 billion each year setting an ambitious goal of achieving zero emission operations by 2030 this transition must be thoughtfully done to prevent any impact on operations the shift to zero emissions often demands a large increase in electricity needs and requires close coordination with utilities and other stakeholders.  Port of Long Beach and Jacobs worked on a master plan to determine the electrical demand and supply needed for the transition we designed a concept for charging infrastructure that does not require a new substation from the local utility We were able to optimize the port’s operations to convert their fleet to zero emission We are now exploring the viability of other technologies to help manage cargo handling equipment as the Port looks to achieve its zero-emission goal by 2030.  where you can make a real impact on the planet and our communities What are you most proud of looking back on your career so far It is hard to pick, but perhaps it is the wireless EV charging project in Detroit I learned how to take a brand-new technology and work with different stakeholders to design and build it into a public roadway we had to be open to changing our processes and thinking about ways we could innovate There has never been a wireless EV charging project on a public roadway in the U.S. we were figuring out solutions as the project progressed.  Here’s an example: What should pavement markings look like on wireless charging EV roadways so drivers know that strip of road is electrified We worked with the state of Michigan and the U.S Federal Highway Administration to come up with a design to be approved and tested but those are the types of things that are really exciting because they literally pave the way into the future What is the most important thing you’ve learned on your career journey?  The power of an organization is working with others You need a robust network of thought partners and mentors and you need to work well with others to be successful I have learned the value of stepping outside of your comfort zone and honing your people and technical skills and the best solutions happen when we collaborate and brainstorm together sustainability requires you to work with a variety of disciplines and project team members You were selected two years ago as an American Council of Engineering Companies’ Young Professional of the Year Tell us a little bit about what that meant to you.  What was the most memorable to me is that 2022 was the first year in which all five national ACEC awardees were women What an honor to be named amongst these talented engineers and to showcase strong women leaders in engineering The award connected me to some impressive professionals and also small businesses throughout the ACEC network giving me a better understanding of how we partner and collaborate with other diverse companies The recognition has also fostered a responsibility to mentor young professionals in engineering bringing their different perspectives to the table What do you enjoy most about being part of #OurJacobs I gravitated toward Jacobs because of the people and landmark projects and I’ve stayed because of the high caliber of technical expertise my colleagues have as well as the fun we have in meetings and workshops All parts of our organization are focused on sustainability and climate response and I’m excited to be helping to deliver it.  ACEC: 2022 Young Professional of the Year Award Winners Environmental Analyst: Jacobs recognized for 3 Sustainability Delivery Awards and Commendations Dairy cattle with poor temperament can cause several inconveniences during milking, leading to labor difficulties, increasing the risk of accidents with animals and workers, and compromising milk yield and quality. This study aimed to estimate variance components and genetic parameters for milking temperament and its genetic correlations with milk yield in crossbred Holstein-Gyr cattle. Volume 4 - 2023 | https://doi.org/10.3389/fanim.2023.1187273 Introduction: Dairy cattle with poor temperament can cause several inconveniences during milking increasing the risk of accidents with animals and workers This study aimed to estimate variance components and genetic parameters for milking temperament and its genetic correlations with milk yield in crossbred Holstein-Gyr cattle Methods: Data were collected at three commercial farms resulting in 5,904 records from 1,212 primiparous and multiparous lactating cows measured as the milking temperament of each cow was assessed during pre-milking udder preparation (RP) and when fitting the milking cluster (RF) by ascribing scores from 1 (cow stands quietly) to 8 (the cow is very agitated with vigorous movements and frequent kicking) The number of steps and kicks were also recorded during pre-milking udder preparation (SRP and KRP respectively) and when fitting the milking cluster (SRF and KRF Milk yield (MY) was obtained from each farm database MY was recorded during the monthly milk control (that could or could not coincide with the date when the milking temperament assessments were carried out) and in the remaining farm MY was recorded on the same day that the milking temperament assessments were made Genetic parameters were estimated using the THRGibbs1f90 program applying a threshold model which included 89 contemporary groups as fixed effects animal age at the assessment day and the number of days in milking as covariates and direct additive genetic and residual effects as random effects Results and discussions: The heritability estimates were MT= 0.14 ± 0.03 (for both The repeatability estimates were 0.38 ± 0.05 The estimates of most genetic correlation coefficients between MTRP-MTRF were all strong and positive (MTRR-MTRF= 0.63 ± 0.10 and MTRF-KRF= 0.56 ± 0.34) except for MY (MTRP-MY= 0.26 ± 0.26 and MTRF-MY= 0.21 ± 0.23) Despite the low magnitude of MT heritability it can be included as a selection trait in the breeding program of Holsteins-Gyr cattle although its genetic progress will be seen only in the long term Due to the low accuracy of the genetic correlation estimates between MT and MY and the high range of the 95% posterior density interval it cannot be affirmed by this study that the selection of a milking temperament trait will infer on milk yield More data is therefore needed per cow and more cows need to be observed and measured to increase the reliability of the estimation of these correlations to be able to accurately interpret the results directly affecting the sustainability of this system little has been done for Bos indicus breeds regardless of the importance of using local breeds to improve profitability while reducing health and welfare issues it is important to develop additional studies addressing Bos indicus breeds and their crosses there is no information available in the literature regarding the estimation of the genetic parameters for milking temperament for Girolando cattle the present study contributes a novel approach for Brazilian dairy producers and other dairy systems that use Bos taurus and Bos indicus crossbreed cattle this study aimed to estimate the genetic and phenotypic parameters of milking temperament as well as its genetic correlation with milk yield in crossbred Holstein-Gyr (HG) cattle raised in Brazil The Committee of the Ethical Use of Animals of the Faculty of Agricultural and Veterinary Sciences The study was conducted at three commercial dairy farms associated with the Girolando Breeders Association (GIROLANDO) from April 2018 to May 2019 resulting in 5,904 records from 1,212 lactating Holstein-Gyr cows The Girolando Breeders Association provided the pedigree data containing 19,531 sires and 349,222 dams the cows were housed in a free stall housing environment The cows were milked twice daily (in the mornings and afternoons) they were milked in herringbone parlors with automatic cluster removal systems they were milked in a rotary/carrousel parlor the cows are separated from the calves following calving before 24 hours and integrated within the milking herds The replacement of animals in the dairy herd is from the calves themselves born on the farms All dairy cows evaluated in the present study were born between 2009 and 2017 Most of them were ¾ Holstein-Gyr (632 and only 67 (~6%) represented other Holstein-Gyr crosses Around sixty-six per cent (803) cows were primiparous and 409 (~34%) were multiparous 314 (~39%) were 1/2 Holstein-Gyr and 37 (~5%) were other Holstein-Gyr crosses; of the multiparous cows and 37 (~9%) were other Holstein-Gyr crosses Cow birth seasons were classified as rainy (September to February) and dry (March to August) The average age at first calving was 32 ± 14 months (ranging from 21.8 to 61.3 months) Lactation days were calculated as the number of days in lactation that the cow was at the time of the milking temperament measurement with 408 cows at the beginning of lactation (9 to 100 days) 595 at the middle of lactation (100 to 200 days) and 209 cows at the end of lactation (200 to 305 days) with an average milk yield of 20.5 ± 6.3 L/day (ranging from 3.0 to 59.4 L/day) After pre-milking udder preparation and before the commencement of the milking process 298 (~25%) cows received an application of exogenous oxytocin and 914 (~75%) did not Of the cows that received an injection of exogenous oxytocin 159 (~53%) were primiparous and 139 (~47%) were multiparous; of those cows that did not receive an application of exogenous oxytocin 644 (~70%) were primiparous and 270 (~30%) were multiparous Table 1 The number of cows and respective percentages according to the number of records of milking temperament measurement events Milking temperament was scored during pre-milking udder preparation (MTRP) and when fitting the milking cluster (MTRF) by assigning one of the scores described in Table 2 Table 2 Description of the milking temperament scores used to assess Holstein-Gyr cows’ milking temperament during pre-milking udder preparation and when fitting the milking cluster Milk yield (MY) was obtained from the farms’ database MYs were recorded during the monthly milk recording records (that could or could not coincide with the days on which the milking temperament assessments were carried out) MYs were recorded on the same days that milking temperament assessments were made These differences concerning the recording of MY may have contributed to errors in the genetic correlation estimates of MY with MTRP and MTRF scores from 1 to 7 were considered (score 8 was eliminated due to only having a few recorded instances The number of steps and kicks during pre-milking udder preparation (SRP and KRP) and when fitting the milking cluster (SRF and KRF) and MY were considered continuous variables The animal model used included direct additive genetic and residual effects as random effects and CG as a fixed effect; the animal age at the time of milking temperament scoring (with linear and quadratic regressions) and the number of days in milk (linear regression) were included as covariates for all traits The matrix presentation of the general model used is as as follows: where: y is the vector of observations; β is the vector of fixed effects; a is the vector of the direct additive genetic effect of the animal; pe is the vector related to permanent environment random effects of the animal (each daily milking temperament measurement considered as repeated measurements on the cow); X and W are known incidence matrices relating β and pe to y; and e is the vector of residuals It was assumed that E[y] = Xβ; Var(a) = A⊗G; Var(pe)= I⊗PE; Var(e) = I⊗R where A is the relationship matrix among all animals in the pedigree file containing 19,531 sires and 349,222 dams G is the (co)variance matrix of direct additive genetic effects PE is the (co)variance matrix of permanent environmental effects and R is the (co)variance matrix of residual effects The vectors β, a, and pe are location parameters from the conditional distribution. A uniform distribution of β was assumed a priori, which reflects a vague prior knowledge about this vector. For the (co)variance matrices of random effects, inverted Wishart distributions were defined as prior distributions. Thus, the distribution of y given the parameters of location and scale was assumed (Van Tassell and Van Vleck, 1996): chains of 1,200,000 iterations were generated The first 300,000 iterations were discarded as fixed burn-in 45,000 samples were used for parameter estimations Data convergence was checked through the criteria proposed by Geweke (1992) and Heidelberger and Welch (1983) using the R software with the Bayesian Output Analysis (BOA) package in R 4.1.0 software (The R Development Core Team) After obtaining the correctly converged variances heritability (h2) and repeatability (R) for milking temperament and phenotypic (rP1P2) and genetic (rA1A2) correlations between milking temperament and milk yield during pre-milking udder preparation and when fitting the milking cluster were estimated as: where: σ2a is additive genetic variance; σ2pe is permanent environmental variance (due to repeated measurements of milking temperament records per cow); σ2 is residual variance; Co(P1 P2) is phenotype co(variance) between two traits; Cov(A1 A2) is genetic co(variance) between two traits; σP1 and σP2 are phenotypes standard deviation of traits 1 and 2; and σA1 and σA2 are genetic standard deviations of traits 1 and 2 and maximum (Max) values of the coefficients of variation (CV and number of steps and kicks during pre-milking udder preparation (MTRP and the number of steps and kicks when fitting the milking cluster (MTRF respectively) and milk yield (MY) in the dataset of Holstein-Gyr cross cattle According to the convergence criteria applied in this study for all trait analyses, the number of remaining Markov chains (45,000) was adequate for obtaining the convergence of all parameters estimated. Table 4 shows the posterior means of additive genetic and heritability and repeatability obtained for milking temperament-related traits and milk yield Table 4 Descriptive statistics of posterior density (95% highest posterior density intervals heritability (h2) and repeatability (R) estimates for milking temperament and milk yield of Holstein-Gyr cross cattle The estimated mean heritability for the number of steps (0.05 ± 0.03) and kicks (0.14 ± 0.05) was estimated during pre-milking udder preparation and the estimations for the number of steps and kicks when fitting the milking clusters were 0.10 ± 0.05 and 0.32 ± 0.16 It should be noted that the only literature currently available for discussion regarding the estimation of genetic parameters for milking temperament is based entirely on Bos taurus dairy cattle herds while in this study results from Bos taurus x Bos indicus dairy crosses are presented The MY heritability in our study was of lower magnitude (0.11 ± 0.08) than that obtained in other studies with Holstein, Gyr, and Brown Swiss breeds (0.20, 0.22, and 0.24; Rennó et al., 2003; Lagrotta et al., 2010; Campos et al., 2015, respectively), as well as than the estimate reported by the national breeding program for Girolando cattle (h2MY = 0.29) (da Silva et al., 2020) The repeatability estimates of this study were moderate for milking temperament and the number of steps and kicks during pre-milking udder preparation and when fitting the milking cluster, ranging from 0.30 to 0.52 (Table 5). Similar results were reported by Erf et al. (1992); Kramer et al. (2013), and Wethal and Heringstad (2019) These authors estimated values ranging from 0.32 to 0.56 in Holstein Table 5 Posterior estimates of genetic (above diagonal) and phenotypic (below diagonal) correlations (mean ± standard deviation) and the highest posterior density interval containing 95% of the observations (inside brackets) between milking temperament and milk yield traits of Holstein-Gyr cross cattle The phenotypic and genetic correlation estimates between milking temperament and milk yield cannot allow the orientation and degree of the phenotypic and genetic correlations to be inferred since the estimated value of the standard deviations and the highest posterior density interval containing 95% have a very high range the values of the correlations estimated can be negative and more cows need to be measured to increase the reliability of the estimation of these correlations to be able to accurately interpret the results Although the heritability estimated for milking temperament and the number of steps and kicks during pre-milking udder preparation and when fitting the milking cluster reached low magnitude there is a possibility that if the selection is made through this trait the estimations of heritability and repeatability for milking temperament justify the inclusion of this trait as a selection criteria trait for the Holstein-Gyr cross in Brazil This study confirms that milking temperament during pre-milking udder preparation has a positive and high genetic correlation with milking temperament when fitting the milking cluster a positive genetic correlation also exists between milking temperament and counting the steps and kicks during pre-milking udder preparation and when fitting the milking cluster Animals with high milking temperaments are known to express more steps and kicks during the milking process making handling difficult Counting steps during milking is an appropriate measurement for including milking temperament in selection indexes for the Holstein-Gyr cross because it is easy and inexpensive to measure and it can be used to assess milking temperament objectively More records are needed to estimate the genetic and phenotypic correlations between milking temperament and milk yield more accurately since they could not be affirmed in this study due to the high standard errors of the estimates as well as the high range of the 95% posterior density interval The original contributions presented in the study are included in the article/supplementary materials Further inquiries can be directed to the corresponding author/s The animal study was reviewed and approved by Committee of the Ethical Use of Animals of the Faculty of Agricultural and Veterinary Sciences Written informed consent was obtained from the owners for the participation of their animals in this study MdC and TV contributed to the conception and design of the study and wrote the first draft of the manuscript TV and MdS performed the statistical analysis MC organized the database All authors contributed to the article and approved the submitted version This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 The study was part of the doctoral thesis of the first author (Paula A prepared for the Graduate Program in Genetics and Animal Breeding at São Paulo State University (UNESP) Faculty of Agricultural and Veterinary Sciences Special appreciation is expressed to the owners or managers of the farms Santa Luzia (Mauricio Silveira Coelho) Calciolândia (Jordane Silva and Ronaldo Lazzarini Santiago) and Boa Fé (Jônadan Ma) and their staff for their support and making it possible to carry out this research The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher Behavioural response to humans and the productivity of commercial dairy cows Genetic parameters for linear type traits and milk and protein production in Holstein cows in Brazil Incorporating temperament traits in dairy cattle breeding programs: challenges and opportunities in the phenomics era Genetic parameters for traits other than production in purebred and crossbred new Zealand dairy cattle CrossRef Full Text | Google Scholar Google Scholar Social dominance and temperament of Holstein cows CrossRef Full Text | Google Scholar Inheritance and relationships of workability traits and yield for holsteins doi: 10.3168/jds.S0022-0302(92)77959-4 CrossRef Full Text | Google Scholar Ferreira A., Cordeiro M., Ribeiro C. 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A non-parametric local projection approach Volume 3 - 2021 | https://doi.org/10.3389/ftox.2021.802542 This article is part of the Research TopicWomen in Neurotoxicology: 2021View all 5 articles The embryonic stage is the most vulnerable period for congenital abnormalities the central nervous system (CNS) is susceptible to numerous genetic the CNS is more vulnerable to external influences such as environmental tobacco smoke (ETS) increasing the risk for delayed fetal growth This study aimed to evaluate the effects of in utero exposure to ETS on neuroinflammation in the offspring of pregnant mice challenged or not with lipopolysaccharide (LPS) After the confirmation of mating by the presence of the vaginal plug until offspring birth pregnant C57BL/6 mice were exposed to either 3R4F cigarettes smoke (Kentucky University) or compressed air Enhanced glial cell and mixed cell cultures were prepared from 3-day-old mouse pups both cells were stimulated with LPS or saline minocycline was added to the mixed cell culture media 24 h before LPS challenge To verify the influence of in utero exposure to ETS on the development of neuroinflammatory events in adulthood a different set of 8-week-old animals was submitted to the Autoimmune Experimental Encephalomyelitis (EAE) model The results indicate that cells from LPS-challenged pups exposed to ETS in utero presented high levels of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNFα) and decreased cell viability Such a proinflammatory environment could modulate fetal programming by an increase in microglia and astrocytes miRNA155 This scenario may lead to the more severe EAE observed in pups exposed to ETS in utero and tumor necrosis factor-alpha (TNFα) such a phenomenon might result from astrocytes and microglia response This study aimed to evaluate whether in utero exposure to ETS could modulate glial cells biology and its response to a systemic inflammatory challenge in the offspring It also investigated whether these alterations are maintained throughout the course of life as well as their impact on the development of neuroinflammatory/neurodegenerative diseases such as MS where microglia activation is crucial for disease pathogenesis Our results show that in utero exposure to ETS leads to fetal reprogramming with increased neuroinflammatory response to systemic stimuli which may have consequences until adulthood the animals from both sexes were decapitated and two types of cultures were prepared from the whole brain of neonates: an enhanced glial cells culture (enhanced glial cell culture) and a mixed glia/neuron cell culture (mixed cell culture) Each neonate was considered an individual experimental number cultures were stimulated with 100 ng/ml of lipopolysaccharide (LPS) from Escherichia coli (0111: B4 Experiments were performed after 24 h The mixed cell culture was incubated with minocycline (10 μM) 24 h before LPS stimulation to inhibit microglia activation conducted with the following experimental groups: ✓ Control Group (CO) – whole-brain cell cultures of the offspring of females exposed in utero to compressed air and challenged with culture medium ✓ Smoker Group (ETS) – whole-brain cell cultures of the offspring of females exposed in utero to environmental tobacco smoke and challenged with culture medium ✓ Group LPS (LPS) – whole-brain cell cultures of the offspring of females exposed in utero to compressed air and challenged with LPS ✓ Smoker Group + LPS (EPS) – whole-brain cell cultures of the offspring of females exposed in utero to environmental tobacco smoke and challenged with LPS Cell cultures were prepared according to Saura et al. (2003) After removing the brains of 3-day-old animals the meninges were extracted with the aid of a magnifying glass Brain tissue was incubated with trypsin (0.25%) for 10 min at 37°C supplemented with 10% fetal bovine serum (FBS) and processed with pipettes of different size for mechanical digestion and the supernatant was collected and filtered through a 70 μM membrane The suspension was then centrifuged at 450 × g for 5 min at room temperature and the pellet was resuspended in DMEN-F12 containing 10% FBS and 1% antibiotic (penicillin and streptomycin) Cells were grown on plates of different diameters according with the experiment to be performed plates were previously treated with Poly-L-lysine for 3 h in a 37°C oven and later conditioned with the medium (DMEN-F12 containing 10% FBS and 1% antibiotic (penicillin and streptomycin)) for 30 min cells were plated and incubated for 7 days at 37°C with 5% CO2 The enhanced glial cell culture was plated and incubated at 37°C with 5% CO2 for approximately 21 days until confluence Pregnant mice were weighed from the first day of exposure (day 0) until the 20th gestational day Water and food intake were evaluated every 5 days The average intake throughout the experimental period was determined per animal per box Neonates were individually weighed on the 3rd day of life before euthanasia 1 × 106 cells from enhanced glial cell culture (n = 6) were resuspended in 80 μl blocking solution (Fc blocking) and incubated for 20 min at 4°C cells were centrifuged at 450 × g and incubated with the CD80 – PE and CD11b – APC antibodies for 20 min at 4°C After being washed twice with 200 μl PBS and centrifuged at 450 × g at 4 °C for 5 min samples were resuspended in 100 μl of Fixperm® incubated for 20 min at 4°C washed with 100 μl Permwash® and centrifuged at 450 × g at 4°C for 5 min Cells were then incubated with the desired antibody (GFAP - FITC) centrifuged at 450 × g at 4°C for 5 min and resuspended in 200 μl 1% paraformaldehyde apoptosis was verified by Annexin and propidium iodide (PI) assay Cells incubated with trypsin (0.25%) were collected and resuspended in binding buffer (10 mM Hepes - pH 7.4 and 1.8 mM CaCl2) containing annexin V-FITC and PI at 1:20 dilution (in a final volume of 20 μl) After being incubated at room temperature for 20 min in the dark 80 μl of the binding buffer was added to the samples and TNF-α cytokines in cell supernatants were quantified in both enhanced glial cell culture (n = 6) and mixed cell culture (n = 5) using the cytometry beads array (CBA) according to the manufacturer’s instructions (BD®) Samples were collected and analyzed on a FACS Accuri C6® flow cytometer (Becton & Dickinson culture medium was removed and cells were washed three times with PBS fixed with methanol 100% for 20 min at room temperature cells were incubated in 40 mM glycine solution in PBS for 5 min and washed Non-specific sites were blocked with 0.05% Triton 1% bovine serum albumin (BSA) in PBS for 2 h at room temperature cells were incubated with specific primary antibodies (GFAP anti-mouse-Alexa fluorine 594 (1:500); MAP2 anti-chicken-Alexa fluorine 555 (1:250); and microglia tomato-lectin-FITC (1:500) diluted in blocking solution) overnight at 4°C on a dark chamber cells were washed five times with PBS for 5 min and incubated for 2 h at room temperature on a dark chamber with a specific secondary antibody After washing the cells five times with PBS 150 μl DAPI (1:100.000) was added to samples for nuclear visualization Slides were analyzed using the ZOE™ Fluorescent Cell Imager microscope The mixed cell culture supernatant (n = 5) and the Griess reagent (0.1% N-1 Naphthyl-ethylenediamine 2.5% H3PO4) were added to a 96-well plate in equal proportions (1:1) Samples were incubated for 10 min at room temperature and absorbance was measured at 550 nm The analyses were compared with a NaNO2 (0–100 μM) standard curve For quantitative real-time polymerase chain reaction (qRT PCR) total mRNA was isolated from enhanced glial cell culture (n = 3) with Triazole mRNA (1 µg/ml) was converted into cDNA and diluted in DEPC-treated water in the proportion of 1:5 using Applied Biosystems Taqman probes with Taqman Universal Master Mix II according to the manufacturer’s instructions The qPCR was performed on the Applied Biosystems QuantStudio™ 3 for IL-6 (Mm00446190_m1) TLR4 (Mm00445273_m1) and Chuk (IKKα - Mm00432543_g1) considering β-actin (Mm02619580_g1) as a housekeeping gene The relative fold expression of mRNA was determined using the ∆∆Ct method RNA from the enhanced glial cell culture (n = 3) was extracted as described above The cDNA synthesis was performed on 130 ng of total RNA by means of a polyadenylation reaction followed by a reverse transcription reaction Gene expression was quantified by real-time PCR using the Stratagene Mx3005P equipment from Agilent Technologies (Santa Clara SYBR Green as fluorescent marker (Luna® Universal qPCR Master Mix #M3003L) and universal antisense primer (each miRNA specific primer was used as sense) miR-155 and miR-223 were quantified using the comparative cycle threshold (Ct) method and results were normalized with the constitutive gene RNU43 8-week-old male mice exposed (n = 9) or not (n = 11) to ETS in utero were subcutaneously injected into the hind flank with 150 μg MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK synthesized by Proteimax Biotechnology - São Paulo - SP - Brazil) emulsified in complete Freund’s adjuvant (v/v) containing 5 mg/ml of BCG (Bacillus Calmette-Guérin) On the day of inoculation and 2 days after mice received intraperitoneal injections of 200 ng pertussis toxin Mice were monitored daily for symptoms of EAE classified according to an arbitrary scale ranging from 0 to 5 whereby 0 indicates no symptoms; 1 indicates flaccid tail; 2 hindlimb weakness or abnormal gait; 3 total hindlimb paralysis; 4 total hindlimb paralysis with forelimb weakness or paralysis; and 5 moribund or deceased mice After euthanasia with ketamine (500 mg/kg i.p.) and xylazine (50 mg/kg i.p), spinal cord samples were removed from animals and fixed in buffered formalin. Inflammation was evaluated by analyzing inflammatory infiltrates in Hematoxylin & Eosin (H&E) staining sections and demyelination by Luxol Fast Blue (LFB) staining. The semiquantitative evaluation was adapted from Soellner et al. (2013) In vivo animals and the enhanced glial cell culture were analyzed by means of a two-way analysis of variance (ANOVA) with the subject factor’s “exposure” (ETS or compressed air in utero) and “challenge.” Microglia contribution in mixed cell culture results was evaluated using a tree-way ANOVA considering the factors “exposure,” “challenge,” and “minocycline” (present or absent) Multiple comparisons were performed by the Tukey’s post-hoc test and p < 0.05 was considered as statistically significant Values are expressed as mean ± standard error of the mean (SEM) During exposure, CO levels in the chamber were equal to 470.2 ± 90.93 ppm and exposure biomarkers measurements were as follows: COHb: 21.62 ± 1.80%; plasma cotinine: 139.94 ± 13.02 ng/ml; and plasma 3-hydroxycotine: 113.65 ± 16.78 ng/ml. Supplementary Figure S1 shows the food intake of pregnant mice as well as their weight during pregnancy and the weight of the offspring When compared with mice exposed to compressed air pregnant females exposed to tobacco smoke ingested 24% less food and gained less weight during gestation the offspring exposed in utero to ETS showed less weight gain than the control group As shown in Supplementary Figure S2 the enhanced glial cell culture was composed of 70% astrocytes and 30% microglia cells Enhanced glia cell culture from C57Bl/6 mice offspring exposed or not to environmental tobacco smoke in utero (A) Flow cytometry analysis of microglia (CD11b) and astrocytes (GFAP) in enhanced glia cell culture graphs for the total number of cells; (B) mean fluorescence intensity (MFI) for CD80 and CD86; (C) Gene expression analysis by PCR; (D) Cytokine’s quantification in culture supernatant; (E) Gate representative of cell frequency in enhanced glia cell culture Statistical analysis: Two-way ANOVA and Tukey’s post-hoc ***p < 0.001 ****p < 0.0001 The black bar represents exposure to compressed air and gray bar represents exposure to ETS Complementing the results obtained by the qPCR, Figure 2D shows the results of the two-way ANOVA (Supplementary Table S1) for cytokines release Tukey’s post-hoc analysis revealed an increase in IL-6 and TNFα release in ETS and EPS groups in relation to their respective controls (p < 0.0001; p < 0.001 thus suggesting that cells exposed to ETS before LPS challenge have a higher inflammatory capacity ETS-exposed groups challenged or not with LPS presented a decreased of cytokine IL-12 – which plays an essential role in the polarization of TH1 CD4+ T lymphocytes – in relation to control (p < 0.0001; p < 0.0001 respectively) and LPS group (p < 0.0001; p < 0.0001 we observed lower levels of IL-12 release in groups challenged with LPS (p < 0.05) we verified an increased production of IL-10 in the LPS group when compared with the control group (p < 0.0001) the cytokine production decreased in ETS-exposed groups (ETS and EPS) when compared with control (p < 0.001 p < 0.01) and LPS (p < 0.0001 IL-10 release was reduced in the EPS group in relation to LPS group (p < 0.0001) These findings suggest that ETS exposure can increase the number of glial cells and their activation as well as the production and release of proinflammatory cytokines after LPS challenge After verifying an increased inflammatory response by glial cells in ETS-exposed groups challenged with LPS, we investigated whether this inflammation process could change cellular viability in a mixed cell culture composed of 3% microglia cells, 39% astrocytes, and 58% neurons (Supplementary Figure S3) To investigate the role of immune cells in this phenomenon the culture was treated with minocycline 24 h before LPS stimulation experiments were conducted in the presence or absence of microglia These results are in line with the increased percentage of apoptotic cells (p < 0.0001) in LPS (p < 0.01) and EPS (p < 0.0001) in relation to control the EPS group showed a decrease in cell viability when compared with ETS groups (p < 0.0001) the number of apoptotic cells was higher in the EPS group than in the ETS group (p < 0.0001) These results suggest that previous exposure to ETS reduce cell viability and increase apoptosis in relation to LPS challenge alone Mixed cell culture (mixed neuron-glia culture) from animals exposed or not to environmental tobacco smoke cells were treated with minocycline 24 h before LPS challenge (A) Gate representative of Annexin and PI marking without minocycline (B) Gate representative of Annexin and PI marking with minocycline graph for percentage of live cells and apoptosis (D) Cytokine measurement in the supernatant Statistical analysis: Three-way ANOVA and Tukey’s post-hoc # shows difference between negative and positive minocycline To understand the mechanism controlling microglia apoptosis, we measured the production of TNFα, CCL2 (Figure 3D), and NO (Figure 3E) with and without the presence of minocycline Tukey’s post-hoc test demonstrated an increase in CCL2 (p < 0.0001) and TNF-α release (p < 0.001) in the LPS-challenged group with minocycline when compared with its respective experimental group without minocycline showing the effect of astrocyte in releasing these cytokines in the absence of microglia The same result was observed regarding the EPS group for CCL2 (p < 0.0001) and TNF-α (p < 0.0001) CCL2 release increases in the LPS group when compared with CO (p < 0.01) and ETS groups (p < 0.001) When assessing NO production, the Tukey’s post-hoc test showed an increased release in the EPS group in relation to control (p < 0.01) and LPS groups (p < 0.05), suggesting that the combination of ETS and LPS increases the activity of microglia cells (Figure 3E) NO release decreased when microglia cells were inhibited by minocycline (p < 0.01) we may infer that apoptosis is caused by microglia cells ability to release NO against stimuli The miRNAs quantification was performed on enhanced glial cell culture cells. Supplementary Table S1 shows the results of the two-way ANOVA. We verified an increase of miR-146 transcription in the ETS-exposed group when compared with the control group (p < 0.05), and of miR-155 compared to both control (p < 0.05) and LPS groups (p < 0.05) (Figure 4) the ETS-exposed group challenged with LPS showed an increase in miRNA155 transcription in relation to control (p < 0.05) and LPS groups (p < 0.05) thus indicating the effect only of ETS in these miRNAs we observed an increased transcription only in the LPS group in relation to control (p < 0.05) Evaluation of miRNA in enhanced glial cell culture from C57Bl/6 mice offspring exposed or not to environmental tobacco smoke in utero To verify the influence of in utero exposure to ETS in the development of neuroinflammatory events during adulthood animals were submitted to the EAE protocol – a murine model of MS These data suggest that the susceptibility of glial cells to inflammatory stimuli caused by in utero exposure remains in adulthood and may favor the development of neuroinflammatory diseases such as MS EAE clinical score in animals exposed or not to environmental tobacco smoke (A) Curve up to the peak (15th day); (B) Area under the curve Statistical analysis: Student’s t-test *p < 0.05 ***p < 0.0001 n = 12; (C–F) Hematoxylin & Eosin or Luxol Fast Blue staining n = 3 (C,E) represents control groups and (D,F) exposed groups Despite all efforts exerted by public health systems worldwide in utero exposure to ETS remains a major concern this study investigated the effects of in utero exposure to ETS using enhanced glial cell culture and mixed cell culture to understand how these cell types react in the presence or absence of an inflammatory stimulus We verified increased levels of proinflammatory cytokines such as IL-6 and TNFα as well as decreased viability in the cells of ETS-exposed offspring challenged with LPS indicating an increase in neuroinflammation pups exposed to ETS presented with more severe EAE suggesting a fetal reprograming possibly related to the increase in miR-155 observed on the enhanced glial cell culture exposed to ETS the authors found that ETS exposure during the early postnatal period decreased 18fluorine-fluorodeoxyglucose (18F-FDG) uptake in several brain areas of males and females from late infancy to early adulthood serum cotinine levels can be used to discriminate passive and active smoking whereby passive smokers can have serum cotinine levels between 0.05 and 10 ng/ml and active smokers >10 ng/ml this classification was based on children and not rodents – which explains why the cotinine levels observed in our study could be considered as the produced by active smokers the study did not mention how long after cigarette exposure the blood was collected which is an important issue to be considered blood samples for biological marker quantification were collected immediately after ETS exposure so that cotinine levels reflected the peak as plasma nicotine half-life in rodents is 0.9–1.1 h Low birthweight may have significant negative effects on infants being associated with higher morbidity and mortality rates Although this study focused on evaluating the effects of in utero exposure to ETS in CNS cells in vitro we detected many features present in a neuroinflammatory response such as the increased expression of the microglia activation marker B7.2 (CD86) as well as the increase in apoptosis of IL-6 and the decrease of live cells in the ETS-exposed group challenged with LPS These findings suggest that in utero exposure to ETS increases microglia proinflammatory response in the offspring Transient cell activation in the CNS is beneficial to the organism. For example, IL1 receptor activation is essential for the repopulation of depleted microglia in the CNS (Bruttger et al., 2015). However, the responsive cells persistent activation in the CNS can be harmful, often leading to neuronal death, as observed in neurodegenerative diseases such as Alzheimer’s and MS (Pekny et al., 2016) with higher incidences in the ages between 25 and 64 years These factors together corroborate our choice to work with male animals the authors reported that miR-155 inhibition or deletion in the murine model of EAE is neuroprotective for the activation of helper and cytotoxic T cells decreases as well as the production and release of cytokines A previous study has already described the association between stress during intrauterine life and increased risk of diseases in adulthood (Stevenson et al., 2020) Our study shows that in utero exposure to ETS stimulates aspects of neuroinflammatory response in vitro This scenario may increase the expression of pro-inflammatory miR-155 in microglia and astrocytes the increase in a fetal programming modulator may also be involved in the more severe outcome observed for ETS-exposed offspring during the EAE protocol The raw data supporting the conclusions of this article will be made available by the authors The animal study was reviewed and approved by the Ethics Committee of the School of Pharmaceutical Sciences (CEUA/FCF/486) (University of São Paulo) AD and WB: Designed and performed the experiments and wrote the manuscript; VB and LS: Performed statistical analyses; SD: Performed the exposure to cigarette smoke; NS: Prepared the mixed glial culture; BP and MY: Performed and supervised the dosage of cotinine and nicotine; NZ: Performed NO quantification; JP: Designed the experiments and reviewed the manuscript; CM: Discussed the results and reviewed the manuscript; TM: Supervised the project This study was supported by the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo–(FAPESP) the Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–(CAPES) and by the National Council of Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico–(CNPq)) The authors would like to thank the professor Alexandrina Sartori and the research fellow Luiza Ayumi Nishiyama Mimura from the Vaccines and Immunomodulation Laboratory - IBB for their help with histological techniques; as well as the professor Silvana Auxiliadora Bordin da Silva and PhD student Tanyara Baliani Payolla from the Molecular Biology Laboratory of the Institute of Biomedical Sciences (ICB-USP) for their help with miRNA techniques The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ftox.2021.802542/full#supplementary-material CrossRef Full Text | Google Scholar The Gestational Environment and Parkinson's Disease: Evidence for Neurodevelopmental Origins of a Neurodegenerative Disorder PubMed Abstract | CrossRef Full Text | Google Scholar Exposure to Smoke during Development: Fetal Programming of Adult Disease PubMed Abstract | CrossRef Full Text | Google Scholar Simplified Determination of Carboxyhemoglobin PubMed Abstract | CrossRef Full Text | Google Scholar Female Sexuality in Multiple Sclerosis: The Multidimensional Nature of the Problem and the Intervention PubMed Abstract | CrossRef Full Text | Google Scholar Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System PubMed Abstract | CrossRef Full Text | Google Scholar Centers for Disease Control and Prevention (2021) Tips from Former Smokers®. 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Spelta, Duro, Barreto dos Santos, Paranhos, Zanluqui, Yonamine, Pierre Schatzmann Peron, Munhoz and Marcourakis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use *Correspondence: Carolina Demarchi Munhoz, Y2RtdW5ob3pAdXNwLmJy; Tania Marcourakis, dG1hcmNvdXJAdXNwLmJy ‡The authors dedicate the work to Nilton Barreto dos Santos an esteemed colleague who passed away at the age of 34 from complications of COVID-19 †These authors have contributed equally to this work and share first authorship The new halls are being promoted by Temprano Capital Partners a private investor that was created in 2013 is based in Spain and which has joined forces with the British company Collegiate in Portugal Collegiate is now promoting this new student accommodation in Porto and information can be consulted on the website and is even included in the studies of certain real estate consultants like Perspetiva Imobiliária/CBRE said that "the operator of the residence Porto Campus will be confirmed in the coming months" and that they "use a variety of international operators for our student residences" a few metres from the Economics and Engineering faculties of the University of Porto the new student halls will have significantly lower rental prices than those in the Collegiate Marquês de Pombal residence in Lisbon its "current expectation is that the prices of the Campus da U amenities and high-speed internet connection" At the Collegiate Marquês de Pombal residence the prices charged on its opening date in January 2018 ranged from 587 euros to 1,400 euros per month At the moment, according to the rates available on the Collegiate Marquês de Pombal website the prices of studios or single rooms for students range from 812 euros up to 1,600 euros for a penthouse studio with a terrace Monthly rents for single rooms in these facilities include services and other amenities Collegiate highlights that the exact cost of renting one of these studios in the new halls of residence will soon be available as well as other information and invites those interested to register on the website idealista/news spoke to some students who were looking for more information from Collegiate by phone They were told that "the values would be lower than those in Lisbon" the reason being that "the residence is bigger" and that the "reality of the Porto market is different" On Temprano's website, the new development is presented as "an 18,000 m2 project located on the university campus of Asprela". It adds that the project "will provide state-of-the-art accommodation for students" and includes, in addition to the flats, " an outdoor swimming pool within the landscaped inner courtyard, a movie theatre, library, gymnasium/spa, chill-out areas, common rooms, and private dinner party rooms.” It also says that "the residences have a 24-hour concierge and the studios are fully equipped", having for example "household appliances in kitchens and premium bathrooms". The Collegiate Porto Campus student halls will be next door to another private student accommodation residence built by the Austrian company Milestone. Interestingly enough, the two private residences are located just a few metres away from each other, on Rua Dr. Manuel Pereira da Silva, and are scheduled to open at the beginning of the 2019/2020 school year. The developers of the two residences have invested in two plots of land that were still open, but which have a location very close to the various university faculties – both public and private – and good public transport links, especially via Metro. In Portugal, Temprano has two professionals with extensive experience in the market, both connected for decades to the Sonae Group. They are José Manuel Quintela da Fonseca, the architect who designed most of Sonae Sierra's shopping centre projects both in Portugal and abroad and who is now responsible for the concept and design of the project at Temprano, and João Pessoa Jorge, who has over 35 years of experience in the real estate sector in Portugal, Brazil, Colombia and China. 6 Portuguese universities amongst the best in the world Where are the best universities in Portugal 6 Portuguese universities have once again been included among the top 1,000 universities in the world in 2022 according to the Shanghai Ranking with Harvard appearing at the top of the list