44 research outputs found
Designing crossing and selection strategies to combine diagnostic markers and quantitative traits
Tese de doutoramento em BiociĂȘncias, ramo de especialização em Toxicologia, apresentada Ă Faculdade de CiĂȘncias e Tecnologia da Universidade de CoimbraDoxorubicin (DOX) is one of the most potent antineoplastic drugs. Although
possessing a superior anti-Âââcancer activity, a broader clinical use of DOX is limited by
a dose-Âââdependent, constant and cumulative cardiomyopathy involving deterioration
of mitochondrial function.
Although acute effects of DOX treatment often disappear when treatment finishes,
chronic effects often result in a persistent cardiotoxicity, including a progressive
deterioration of mitochondrial metabolism, the development of cardiomyopathy and
ultimately congestive heart failure. Important in the context of DOX-Âââinduced
cardiotoxicity, mitochondrial disruption has been observed in different models. This
alteration of mitochondrial function is often sub-Âââclinical and is only manifested as
cardiomyopathy when other factors are combined, including age or different types of
cardiovascular stress. Also, metabolic alterations in the cardiac cell occur, which
contribute to the ability of the heart to withstand increased workloads. Although
mitochondrial disruption is an early and sensitive marker of DOX cardiotoxicity,
how metabolic stress contributes to the development of cardiomyopathy remains to
be determined. Because of this gap in knowledge, the objective of this work was to
use of model of metabolic inhibition in perfused hearts from saline (SAL) and DOX-Âââ
treated rats in order to identify metabolic alterations caused by an acute and sub-Âââ
chronic treatment.
Our assumption for this strategy is that a lower susceptibility to a determined
inhibitor during perfusion, would be a sign of a more robust (i.e. more capacity) of
the targeted pathway(s).
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For the acute treatment protocol, sixteen week-Âââold male Wistar rats were i.p. injected
with 20mg/Kg DOX or 1mg/Kg 0.9%NaCl and sacrificed 24 hours later. For sub-Âââ
chronic protocol, eight weeks-Âââold male Wistar rats received seven weekly s.c.
injections with DOX(2mg/Kg) or equivalent SAL solution and sacrificed one week
after the last injection. Following the protocol treatments, animals were sacrificed
and hearts were removed and perfused using a Langendorff apparatus with distinct
cardiac substrates (glucose, galactose plus glutamine -Âââ GG or octanoate plus malate â
OM). Glycolytic (iodoacetate) and oxidative phosphorylation (rotenone-Âââ Rot or
cyanide-Âââ KCN) inhibitors were separately added to the different metabolic
perfusates, aiming to detect undercover mitochondrial defects in the DOX-Âââtreated
group. In non-Âââperfused hearts, or hearts perfused in the absence (time controls, TC)
or presence of inhibitors, selected metabolic and mitochondrial proteins were semi-Âââ
quantified by Western blotting, and mRNA levels were quantified by RT-ÂââPCR.
In the acute DOX treatment model, hearts perfused with glucose as substrate
suffered a decline in the number of heart beat and rate pressure product (RPP) when
iodoacetate was added, contrarily to Rot or KCN which had no effect. With GG,
inhibitor titration decreased the heart rate, despite that the decrease in the RPP was
more evident in SAL vs. DOX group with iodoacetate and KCN. Perfusion with OM
resulted in decreased heart rate an RPP in the presence of the inhibitors, showing
equal response between treatments. When glycolytic and mitochondrial proteins
were semi-Âââquantified by Western blotting, alterations in proteins involved in
mitochondrial biogenesis and autophagy were observed in DOX hearts perfused
with inhibitors. The data from the acute protocol study, appears to suggest that
hearts from DOX-Âââtreated animals have improved function in the presence of
XVI
metabolic inhibitors, thus indicating that DOX triggers adaptations that allow the
hearts to be less susceptible to mitochondrial and glycolytic inhibition.
In the sub-Âââchronic model and using glucose as substrate, the DOX-Âââtreated group
showed again a better tolerability to inhibitors than SAL. With GG, titration with
iodoacetate caused a decrease in heart beat and on RPP in DOX group, when rot or
KCN was added the number of heart beat and RPP remains identical between the
two groups. Glycolytic and mitochondrial proteins semi-Âââquantification suggested an
impairment of autophagy in DOX perfused hearts perfused, more evident during GG
perfusion. The presence of inhibitors in the perfusion also generally decreased the
total amount of proteins detected by Western Blotting, although glycolytic proteins
were increased when hearts were perfused with glucose, contrarily to GG perfusion.
The results suggest that sub-Âââchronic DOX-Âââtreated rats suffered a metabolic
remodeling which is based on stronger glycolytic fluxes to maintain contractility,
although no overt mitochondrial defect was uncovered.
A surprising result is that regardless of the perfusion buffer used, no striking
differences between SAL and DOX hearts in terms of hemodynamic parameters were
found.
The present work suggests that metabolic remodeling during DOX acute and sub-Âââ
chronic treatment maintains cardiac function in the treated animals. This remodeling
is apparently based in a stronger contribution of glycolysis to overall metabolism.
Data from protein amount analyzed suggest that DOX treatment in both models
affect important regulators of autophagy, mitochondrial biogenesis as well as the
adenine nucleotide translocator. The results also suggest that a longer treatment
XVII
protocol or resting period should also be tested in order to uncover more profound
differences.A doxorrubicina (DOX) Ă© um dos fĂĄrmacos antineoplĂĄsicos mais potentes. Apesar de
possuir uma actividade anti-Âââcancro superior, uma mais ampla utilização clĂnica da
DOX Ă© limitada por uma dose-Âââdependente, constante e cumulativa cardiomiopatia
que envolve a deterioração da função mitocondrial.
Embora os efeitos agudos do tratamento DOX normalmente desaparece quando se
conclui o tratamento, os efeitos crĂłnicos resultam muitas vezes numa
cardiotoxicidade persistente, incluindo a deterioração progressiva do metabolismo
mitocondrial, o desenvolvimento de cardiomiopatia e por fim insuficiĂȘncia cardĂaca
congestiva. Importante no contexto da cardiotoxicidade induzida pela DOX,
perturbação mitocondrial foi observada em diferentes modelos. Esta alteração da
função mitocondrial Ă© muitas vezes sub-ÂââclĂnica e sĂł se manifesta como
cardiomiopatia quando outros factores sĂŁo combinados, incluindo a idade ou
diferentes tipos de estresse cardiovascular. Além disso, ocorrem alteraçÔes
metabĂłlicas na cĂ©lula cardĂaca, o que contribui para a capacidade do coração resistir
a maior demanda. Embora a perturbação mitocondrial seja um marcador precoce e
sensĂvel de DOX cardiotoxicidade, como o stress metabĂłlico contribui para o
desenvolvimento de cardiomiopatia permanece por determinar. Devido a essa
lacuna no conhecimento, o objetivo deste trabalho foi a utilização de um modelo de
inibição metabólica em coraçÔes perfundidos de ratos tratados com uma solução
salina (SAL) e ratos tratados com DOX, a fim de identificar alteraçÔes metabólicas
causadas por um tratamento agudo e sub-ÂââcrĂŽnico.
XIX
O nosso pressuposto para esta estratégia é que uma menor susceptibilidade a um
determinado inibidor durante a perfusĂŁo, seria um sinal de uma forma mais robusta
(ou seja, mais de capacidade) da via-Âââalvo (s).
Para o protocolo de tratamento agudo, ratos machos Wistar de 16 semanas de idade
foram injectados i.p. com 20 mg / kg de DOX ou de 1 mg / kg a 0,9% de NaCl e
sacrificados 24 horas mais tarde. Para o protocolo sub-ÂââcrĂŽnico, ratos machos Wistar
de oito semanas de idade, receberam sete injecçÔes s.c. semanais com DOX (2 mg /
kg) ou uma equivalente da solução SAL sendo sacrificados uma semana após a
Ășltima injecção. ApĂłs o protocolo de tratamentos, os animais foram sacrificados e os
coraçÔes foram perfundidos com um aparelho de Langendorff com substratos
cardĂacos distintos (glucose, galactose mais glutamina -Âââ GG ou octanoato mais
malato -Âââ OM). Inibidores glicolĂticos (iodoacetato) e inibidores da fosforilação
oxidativa (Rot-Âââ rotenona ou KCN-Âââ cianeto) foram adicionados separadamente nos
diferentes substratos metabĂłlicos, com o objetivo de detectar defeitos mitocondriais
no grupo tratado com DOX. Em coraçÔes não perfundidos, ou coraçÔes perfundidos
na ausĂȘncia (controlos de tempo, TC) ou na presença de inibidores, algumas
proteĂnas metabĂłlicas e proteĂnas mitocondriais foram semi-Âââquantificadas por
Western blotting, e os nĂveis de mRNA foram quantificados por RT-ÂââPCR.
No modelo de tratamento agudo DOX, coraçÔes perfundidos com glucose como
substrato sofreram um declĂnio no nĂșmero de batimentos cardĂacos e produto da
taxa de pressĂŁo (RPP), quando iodoacetato foi adicionado, ao contrĂĄrio da Rot ou
KCN, que não teve nenhum efeito. Com GG, a titulação com o inibidor diminuiu a
frequĂȘncia cardĂaca, apesar de que a diminuição da RPP foi mais evidente no grupo
SAL vs. DOX com iodoacetato e KCN. Perfusão com OM resultou em diminuição da
XX
frequĂȘncia cardĂaca e do RPP na presença dos inibidores, mostrando uma resposta
igual entre os tratamentos. Quando proteĂnas glicolĂticas e mitocondriais foram semi-Âââ
quantificadas por Western blotting, alteraçÔes de proteĂnas envolvidas na biogĂȘnese
mitocondrial e autofagia foram observados em coraçÔes DOX perfundidos com
inibidores. Os dados do protocolo de estudo agudo, parecem sugerir que os coraçÔes
provenientes de animais tratados com DOX na presença de inibidores, tĂȘm a função
metabólica melhorada, indicando assim que a DOX desencadeia adaptaçÔes que
permitem que os coraçÔes sejam menos susceptĂveis Ă inibição mitocondrial e
glicolĂtica.
No modelo sub-ÂââcrĂłnica e utilizando glucose como substrato, o grupo tratado com
DOX mostrou novamente um melhor tolerabilidade para inibidores que SAL. Com
GG, a titulação com iodoacetato causou uma diminuição no batimento cardĂaco e no
RPP em grupo DOX, quando rot ou KCN foi adicionado o nĂșmero de batimentos
cardĂacos e RPP permaneceram idĂȘnticos entre os dois grupos. A semi-Âââquantificação
de proteĂnas glicolĂticas e mitocondriais sugerem uma deficiĂȘncia da autofagia em
coraçÔes DOX perfundidos, mais evidente durante a perfusão com GG. A presença
de inibidores da perfusão geralmente também reduziu a quantidade total de
proteĂnas detectadas atravĂ©s de Western Blot, embora proteĂnas glicolĂticas
aumentaram quando os coraçÔes foram perfundidos com glucose, ao contrårio da
perfusĂŁo com GG.
Os resultados sugerem que ratos tratados sub-Âââcronicamente com DOX sofreram uma
remodelação metabĂłlica, que Ă© baseado em fluxos glicolĂticos mais fortes para
manter a contratilidade, embora nenhum defeito mitocondrial ostensivo foi
descoberto.
XXI
Um resultado surpreendente Ă© que, independentemente do tampĂŁo de perfusĂŁo
utilizado, não foram encontradas diferenças marcantes entre SAL e DOX coraçÔes em
termos de parĂąmetros hemodinĂąmicos.
O presente trabalho sugere que o remodelação metabólica durante o tratamento
agudo e sub-ÂââcrĂŽnico com DOX, mantĂ©m a função cardĂaca nos animais tratados. Esta
remodelação é, aparentemente, baseado numa contribuição mais forte da glicólise ao
metabolismo geral. Os resultados de quantidade de proteĂna analisados sugerem que
o tratamento com DOX em ambos os modelos afectam importantes reguladores da
autofagia e biogénese mitocondrial, bem como o translocador de nucleótidos de
adenina. Os resultados também sugerem que um protocolo de tratamento mais longo
ou com um perĂodo de repouso tambĂ©m devem ser testados a fim de descobrir
diferenças mais profundas
Genetic improvement of wheat for dry environments - a trait based approach
Item does not contain fulltext23 januari 201
Labrador retrievers under primary veterinary care in the UK: demography, mortality and disorders
Abstract Background Labrador retrievers are reportedly predisposed to many disorders but accurate prevalence information relating to the general population are lacking. This study aimed to describe demography, mortality and commonly recorded diseases in Labrador retrievers under UK veterinary care. Methods The VetCompassâą programme collects electronic patient record data on dogs attending UK primary-care veterinary practices. Demographic analysis covered all33,320 Labrador retrievers in the VetCompassâą database under veterinary care during 2013 while disorder and mortality data were extracted from a random sample of 2074 (6.2%) of these dogs. Results Of the Labrador retrievers with information available, 15,427 (46.4%) were female and 15,252 (53.6%) were male. Females were more likely to be neutered than males (59.7% versus 54.8%, Pâ<â 0.001). The overall mean adult bodyweight was 33.0 kg (SD 6.1). Adult males were heavier (35.2 kg, SD 5.9 kg) than adult females (30.4 kg, SD 5.2 kg) (Pâ<â 0.001). The median longevity of Labrador retrievers overall was 12.0 years (IQR 9.9â13.8, range 0.0â16.0). The most common recorded colours were black (44.6%), yellow (27.8%) and liver/chocolate (reported from hereon as chocolate) (23.8%). The median longevity of non-chocolate coloured dogs (nâ=â139, 12.1 years, IQR 10.2â13.9, range 0.0â16.0) was longer than for chocolate coloured animals (nâ=â34, 10.7 years, IQR 9.0â12.4, range 3.8â15.5) (Pâ=â0.028). Of a random sample of 2074 (6.2%) Labrador retrievers under care in 2013 that had full disorder data extracted, 1277 (61.6%) had at least one disorder recorded. The total number of dogs who died at any date during the study was 176. The most prevalent disorders recorded were otitis externa (nâ=â215, prevalence 10.4%, 95% CI: 9.1â11.8), overweight/obesity (183, 8.8%, 95% CI: 7.6â10.1) and degenerative joint disease (115, 5.5%, 95% CI: 4.6â6.6). Overweight/obesity was not statistically significantly associated with neutering in females (8.3% of entire versus 12.5% of neutered, Pâ=â0.065) but was associated with neutering in males (4.1% of entire versus 11.4% of neutered, Pâ<â0.001). The prevalence of otitis externa in black dogs was 12.8%, in yellow dogs it was 17.0% but, in chocolate dogs, it rose to 23.4% (Pâ<â0.001). Similarly, the prevalence of pyo-traumatic dermatitis in black dogs was 1.1%, in yellow dogs it was 1.6% but in chocolate dogs it rose to 4.0% (Pâ=â0.011). Conclusions The current study assists prioritisation of health issues within Labrador retrievers. The most common disorders were overweight/obesity, otitis externa and degenerative joint disease. Males were significantly heavier females. These results can alert prospective owners to potential health issues and inform breed-specific wellness checks
Effects of a single administration of prostaglandin F2alpha, or a combination of prostaglandin F2alpha and prostaglandin E2, or placebo on fertility variables in dairy cows 3â5 weeks post partum, a randomized, double-blind clinical trial
BACKGROUND: Delayed uterine involution has negative effects on the fertility of cows; use of prostaglandin F2alpha alone as a single treatment has not been shown to consistently improve fertility. Combined administration of PGF2alpha and PGE2 increased uterine pressure in healthy cows. We hypothesized, that the combination of both prostaglandins would accelerate uterine involution and have, therefore, a positive effect on fertility variables. In commercial dairy farming, the benefit of a single post partum combined prostaglandin treatment should be demonstrated. METHODS: 383 cows from commercial dairy farms were included in this study. Uterine size and secretion were evaluated at treatment 21â35 days post partum and 14 days later. Cows were randomly allocated to one of three treatment groups: PGF2alpha and PGE2, PGF2alpha or placebo. For every animal participating in the study, the following reproduction variables were recorded: Interval from calving to first insemination, days open, number of artificial inseminations (AI) to conception; subsequent treatment of uterus, subsequent treatment of ovaries. Plasma progesterone level at time of treatment was used as a covariable. For continuous measurements, analysis of variance was performed. Fisher's exact test for categorical non-ordered data and exact Kruskal-Wallis test for ordered data were used; pairwise group comparisons with Bonferroni adjustment of significance level were performed. RESULTS: There was no significant difference among treatment groups in uterine size. Furthermore, there was no significant difference among treatments concerning days open, number of AI, and subsequent treatment of uterus and ovaries. Days from calving to first insemination tended to be shorter for cows with low progesterone level given PGF2alpha and PGE2 in combination than for the placebo-group (P = 0.024). CONCLUSION: The results of this study indicate that the administration of PGF2alpha or a combination of PGF2alpha and PGE2 21 to 35 days post partum had no beneficial effect upon measured fertility variables. The exception was a tendency for a shorter interval from calving to first insemination after administration of the combination of PGF2alpha and PGE2, as compared to the placebo group. Further research should be done in herds with reduced fertility and/or an increased incidence of postpartum vaginal discharge
Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil
Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30Â years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars