<p> Fruit size is a polygenic trait controlled by multiple genomic regions each with small effect. The complex nature of fruit size regulation makes it challenging to dissect individual genes responsible for phenotypic variation. Though recent advances in high-throughput genome sequencing technology in conjunction with improved statistical and computational methods empowered science to explicitly understand the genetic basis underlying multiple fruit quality traits, much of the work that has been done through classical quantitative trait loci (QTL) approach resulted in reduced resolution and instability when evaluating in different genetic backgrounds and different environments. To increase the precision and improve the stability of QTL analyses and to identify genes controlling fruit size, we performed a set of multiple quantitative and molecular genetic analyses to elucidate the underlying genetic architecture of fruit mass. A total of nine genomic regions associated with fruit mass were identified, two of which are novel to this study; markers Md14_26050918 and Md14_26050904. Detected QTLs explained ~ 42% of the total genetic variation of which ~ 20% is explained by the two novel QTLs. Regions responsible for fruit mass variation appear to be under strong additive and epistatic genetic control. These regions exhibited high stability across-family as well as across-years and showed accurate genomic prediction across-family. Additionally, we identified the apple gene family of putative fw2.2 orthologs, naming them Cell Number Regulators (CNRs) genes (MdCNRs). Three CNRs (MdCNR1-3) showed increased expression at early fruit growth in small-fruited crabapple, associating with reduced relative cell production rate (RCPR), suggesting that alteration in cell number that leads to a subsequent reduction in fruit size is probably due to reduced cell division most likely due to changes in CNRs regulation. Furthermore, our study revealed that reduced fruit size is partially due to the shortened cell expansion period after which cell expansion ceases in the small-fruited crabapple species. Together, these data will advance our understanding of dissecting fruit mass genetic architecture and have high potential to be deployed for marker-assisted selection and further breeding approaches. </p>

Dietary soy isoflavones have been shown to ameliorate insulin resistance and Type 2 diabetes. However, many in vitro studies used supra-physiological concentrations of individual isoflavones that make it difficult to interpret the results as potential mechanisms in vivo. Since the insulin-sensitizing effects of thiazolidinediones, anti-diabetic drugs, have been shown to be mediated through activation of peroxisome proliferators-activated receptor gamma (PPARγ), the key transcription factor for adipocyte differentiation, we examined the effects of the two main soy isoflavones genistein and daidzein either as individual compound or combined on adipocyte differentiation and PPARγ expression, as well as whether the Wnt/β-catenin signaling pathway is the underlying molecular mechanism. In 3T3-L1 cells, genistein and daidzein significantly enhanced adipocyte differentiation. Similarly the expression of PPARγ increased particularly at 20 µmol/L. The stimulatory effect is greater when the two isoflavones are combined, indicating a synergistic effect. Genistein and daidzein also increased the relative abundance of insulin-responsive glucose transporter 4 (GLUT4) mRNA with a greater effect when combined. Wnt10b expression was not affected by soy isoflavones treatments, while Wnt5b expression was only increased by the combination of genistein and daidzein. Our results suggest, that the combination of soy isoflavones has a greater effect in increasing the newly formation of adipocytes that are highly insulin-sensitive via an increase in PPARγ expression as well as increasing the expression of GLUT4. However, genistein and daidzein actions on Wnt signaling remain unclear. These data further support the epidemiological findings for the beneficial effect of soy consumption on insulin sensitivity.

Author: Bc. Dagmar Bakerová Supervisor: MUDr. Vít Řeháček Charles University, Faculty of Pharmacy in Hradec Králové Title of diploma thesis: RhD antigen screening by molecular genetic methods This thesis deals with the genotyping of weak and partial antigens using molecular genetic methods. The main aim is to evaluate the rate of the representation of individual types of variant and weak RhD antigens in first-time blood donors, patients and pregnant women. Testing took place at the Transfusion Department of University Hospital Hradec Králové between October 2015 and February 2019. The PCR-SSP method was used for RHD genotyping using commercially supplied BAGene SSP kits from BAGene Health Care. The study includes 32 samples from first-time blood donors in the reference period to determine the specific type of RhD antigen, and 188 samples from patients and pregnant women, for whom serological methods could not be used to unequivocally identify the RhD antigen. For all pregnant women, moreover, the genotyping result was a factor in determining whether to administer anti-D immunoglobulin. This RHD genotyping for all serologically ambiguous samples has made it possible to determine a specific type of partial or weak RhD antigen. In the donor group, the weak RhD antigen was detected in 1.12 % of cases...

Este estudo foi realizado para detectar quantitative trait loci (QTL) afetando características e curva de crescimento numa população F2 Holandês x Gir. As características estudadas foram peso ao nascer (BW), peso à desmama (WW), peso aos 205 dias (W205), peso ao sobreano (YW), peso aos 720 dias (W720), ganho de peso diário do nascimento ao desmama (ADG0_60), ganho de peso diário da desmama ao peso aos 205 dias (ADG60_205), ganho de peso diário de peso ao 205 dias ao peso ao sobreano (ADG205_365), ganho de peso diário do sobreano ao peso aos 720 dias (ADG365_720), ganho de peso diário total (ADG), curva de crescimento de peso ao nascer ao peso aos 720 dias (GC). 180 marcadores microssatélites, abrangendo os 29 autossomos bovino e cobrindo 3,322 cM foram selecionados a partir do mapa genético bovino. A média de intervalo de marcador foi 22 cM. Vinte e oito QTLs foram detectados em dez cromossomos, quinze QTLs tem modo aditivo, dois QTLs tem modo de dominância e onze QTLs têm sobredominância como modo de ação. Um QTL sugestivo foi detectado para BW no cromossomo BTA 17 em 1 cM. Um QTL sugestivo foi detectado para W205 no BTA 3 a 20 cM. Quatro QTLs sugestivos foram encontrados para YW no BTA 3 a 7 cM, no BTA 6 a 134,9 cM, no BTA 12 a 1 cM, e no BTA 22 a 1 cM. Para W720, seis QTLs sugestivos foram identificados, no BTA 2 a 30 cM, no BTA 3 a 1 cM, no BTA 6 a 44 cM, no BTA 10 a 20 cM, no BTA 12 a 1cM e no BTA 22 a 1 cM. Um QTL sugestivo foi identificado para ADG0_60 no BTA 8 a 143 cM. Dois QTLs sugestivos foram relatados para ADG60_205 no BTA 3 a 19 cM e no BTA 23 19 cM. Um QTL sugestivo foi observado para ADG205_365 no BTA 12 a 7 cM. Para ADG365_720, três QTLs foram detectados. Dois QTLs têm efeitos sugestivos no BTA 1 a 12 cM e no BTA 10 a 22 cM, um QTL significativo no BTA 6 a 43 cM. Seis QTLs sugestivos para ADG foram encontrados no BTA 2 a 32 cM, no BTA 3 a 1 cM, no BTA 6 a 43 cM, no BTA 10 a 20 cM, no BTA 12 a 1 cM e no BTA 22 a 1 cM. Três QTLs sugestivos foram identificados por GC no BTA 2 a 34 cM, no BTA 12 a 2 cM e no BTA 22 a 3 cM. Nenhum QTL foi detectado para WW em todos os cromossomos. Existem vários efeitos pleiotrópcos nos cromossomos BTA 2, 3, 6, 10, 12 e 22, os quais influenciam as características de crescimento. Foram detectados 22 efeitos epistáticos complexos para as cinco características YW, W720, GC, ADG365_720 e ADG. / This study was conducted to detect quantitative trait loci (QTL) affecting growth traits and growth curve using F2 Holstein x Gyr population. Traits analyzed were birth weight (BW), weaning weights (WW), weight at 205 day (W205), yearling weight (YW), weight at 720 day (W720), average daily gain from birth to weaning weight (ADG0_60), average daily gain from weaning weight to weight at 205 days (ADG60_205), average daily gain from weight at 205 days to yearling weight (ADG205_365), average daily gain from yearling weight to weight at 720 days (ADG365_720), total average daily gain (ADG), and growth curve from birth weight to weight at 720 days (GC). 180 microsatellite markers covering the 29 bovine autosomes and covered 3322 cM were chosen from bovine genetic map. The average marker interval was 22 cM. Twenty eight QTLs detected span ten chromosomes, fifteen QTLs had additive mode, two QTLs had dominance mode and eleven QTLs had overdominance mode of gene action. Suggestive QTL was found for BW on BTA 17 at 1 cM. Also, Suggestive QTL was detected for W205 on BTA 3 at 20 cM. Four suggestive QTLs were found for YW on BTA 3 at 7 cM, on BTA 6 at 134.9 cM, on BTA 12 at 1 cM, and on BTA 22 at 1 cM. For W720, six suggestive QTLs were identified, on BTA 2 at 30 cM, on BTA 3 at 1 cM, on BTA 6 at 44 cM, on BTA 10 at 20 cM, on BTA 12 at 1cM and on BTA 22 at 1 cM. Suggestive QTL was observed for ADG0_60 on BTA 8 at 143 cM. Two suggestive QTLs were reported for ADG60_205 on BTA 3 at 19 cM and on BTA 23 at 19 cM. Suggestive QTL was observed for ADG205_365 on BTA 12 at 7 cM. For ADG365_720, three QTLs were detected. Two QTLs had suggestive effect on BTA 1 at 12 cM and BTA 10 at 22 cM, one significant QTL on BTA 6 at 43 cM. Six suggestive QTLs were found for ADG on BTA 2 at 32 cM, BTA 3 at 1 cM, BTA 6 at 43 cM, BTA 10 at 20 cM, BTA 12 at 1 cM and BTA 22 at 1 cM. Three suggestive QTLs were identified for GC on BTA 2 at 34 cM, BTA 12 at 2 cM and BTA 22 at 3 cM. No QTLs were detected for WW in all chromosomes. There were many pleiotropy effects on BTA 2, 3, 6, 10, 12 and 22 influencing growth traits. There were 22 complexes epistatic effects were detected for five traits YW, W720, GC, ADG365_720 and ADG.

Improvement of feeding regimens for production animals has been hindered by a lack of fundamental knowledge about how the capacity to regulate nutrient absorption across cell membranes affects the function of nutrient metabolizing enzymes. The objective is to determine if the activities and protein content of system X-AG glutamate transporter, its regulatory protein (GTRAP3-18 and ARL6IP1), glutamine synthetase (GS) and glutathione (GSH) content, changes in liver (Experiment 1), longissimus dorsi (LM) and subcutaneous adipose tissue (SF) (Experiment 2) as beef steers transitioned from predominantly-lean (growing) to -lipid (finished) tissue accretion phases. In liver (Experiment 1), system X-AG activity in canalicular membranes was abolished as steers developed from growing to finished stages but did not change in basolateral membranes. EAAC1 protein content in liver homogenates decreased in finished vs. growing steers, whereas GTRAP3-18 and ARL6IP1 content increased and GLT-1 content did not change. Concomitantly, hepatic GS activity decreased in finished steers whereas GS protein content did not differ. Hepatic GSH content did not differ in finished vs. growing steers. These results demonstrate a negative functional relationship between GTRAP3-18 and system X-AG activity with glutamine synthesis capacity in livers of fattened cattle. In addition to liver, skeletal muscle and adipose tissues play important roles in maintaining whole-body glutamate and nitrogen homeostasis. In Experiment 2, Western blot analysis of LM homogenates showed decreased EAAC1 and GS content, whereas GTRAP3-18 and ARL6IP1 did not differ in finished vs. growing steers. GSH content in LM was increased in finished vs. growing steers in concomitance with increased mRNA expression of GSH-synthesizing enzymes. In SF, GTRAP3-18 and ARL6IP1 content was increased, whereas EAAC1 and GS content did not differ. Concomitantly, GSH content in SF was decreased in finished vs. growing steers in parallel with decreased mRNA expression of GSH-metabolizing enzymes. These results demonstrate that the negative regulatory relationship between GTRAP3-18 and ARL6IP1 with EAAC1 and GS expression, which exists in liver, does not exist in LM and SF of fattened cattle; and antioxidant capacity in LM and SF changes and differs as steer compositional gain shifts from lean to lipid phenotype. To further explore the upstream regulatory machinery of EAAC1, transcriptome analysis (Experiment 3) was conducted to gain a greater understanding of hepatic metabolic shifts associated with the change in whole-body compositional gain of growing vs. finished beef steers. The expression of upstream regulators of EAAC1 was decreased in a manner consistent with the decreased EAAC1 activity in Experiment 1. Bioinformatic analysis found that, for amino acid metabolism, finished steers had increased capacities for ammonia, arginine, and urea production, and shunting of amino acid carbons into pyruvate. For carbohydrate metabolism, capacity for glycolysis was inhibited, whereas glycogen synthesis was stimulated in finished steers. For lipid metabolism, finished steers showed decreased capacity for fatty acid activation and desaturation, but increased capacity for fatty acid b-oxidation and lipid storage. In addition, redox capacity and inflammatory responses were decreased in finished steers. Collectively, these data describe novel regulatory relationships of system X-AG in liver and peripheral tissues, and the metabolic mechanisms that control nutrient use efficiency, as beef steers develop from lean to lipid phenotypes.

The ketogenic diet is commonly used to control epilepsy, especially in cases when medications cannot. The diet typically consists of high fat, low carb, and adequate protein and produces a metabolite called acetoacetate. Seizure activity is characterized by glutamate excitotoxicity and therefore glutamate regulation is a point of research for control of these disorders. Acetoacetate is heavily implicated as the primary molecule responsible for decreasing glutamate in the synapse; it is believed that acetoacetate interferes with the transport of glutamate into the synaptic vesicles. The effects on synaptic transmission at glutamatergic synapses was studied in relation to the ketogenic diet in Drosophila larvae for this thesis. Various measures of synaptic transmission were conducted. Acetoacetate decreased neurotransmission at the synapse. It was also found that acetoacetate has direct effects on the postsynaptic membrane, which indicates a novel role for the metabolite.

Type 2 Diabetes (T2D) disproportionally affects ethnic minorities in the United States. The development of T2D involves complex interaction between environmental factors and genetic predisposition. The genetic associations of six single nucleotide polymorphisms (SNPs) in TCF7L2 gene with the risk of T2D were evaluated in three high risk minority populations: Cuban Americans, Haitian Americans, and African Americans. For Cuban Americans, four SNPs (rs7901695, rs4506565, rs7903146 and rs11225537) were significantly associated with the risk of T2D after multivariable adjustment (p=0.018, p=0.016, p=0.014, and p=0.0008, respectively). Among controls, risk allele carriers of SNPs rs7901695, rs4506565 and rs7903146 had significantly higher fasting glucose level, compared to non-risk allele carriers. Additionally, a significant interaction between dietary fiber intake and SNP rs7903146 for the risk of T2D (p= 0.044) was found in Cuban Americans. Similarly, for SNP rs7901695, significant interaction was also found for fiber intake (p=0.014) as well as glycemic load (p=0.040). Subgroup analysis revealed that significant associations were only found within higher intake groups of dietary factors for both SNPs. For Haitian Americans, SNPs rs11196205 (p=0.059) and rs7895340 (p=0.069) showed marginal significance for the risk of T2D. After stratification by gender, SNPs with marginal significance from the gender-combined analysis became statistically significant with the same trend for the risk of T2D when analysis were done in males: rs11196205 (p=0.034) and rs7895340 (p=0.024). For African Americans, SNP rs7903146 (p=0.065) showed a marginal significance with the risk of T2D in gender-combined analysis and a statistical significance (p=0.013) in males. Two additional SNPs rs7901695 and rs4506565 were found to be significantly associated with the risk of T2D in males. Risk allele carriers of these two SNPs had significantly higher mean level of the fasting glucose level, compared to non-risk allele carriers in controls. T2D related quantitative trait analysis also demonstrated that in controls, compared to non-minor allele carriers of SNP rs12255372, minor allele carriers had significantly higher means of BMI, diastolic blood pressure, numbers of components of Metabolic Syndrome, significantly lower mean values of HDL-cholesterol and adiponectin. Taken together, our studies demonstrated ethno-specific genetic associations between TCF7L2 gene and the risk of T2D and related phenotypes.

Este estudo foi realizado para detectar quantitative trait loci (QTL) afetando características e curva de crescimento numa população F2 Holandês x Gir. As características estudadas foram peso ao nascer (BW), peso à desmama (WW), peso aos 205 dias (W205), peso ao sobreano (YW), peso aos 720 dias (W720), ganho de peso diário do nascimento ao desmama (ADG0_60), ganho de peso diário da desmama ao peso aos 205 dias (ADG60_205), ganho de peso diário de peso ao 205 dias ao peso ao sobreano (ADG205_365), ganho de peso diário do sobreano ao peso aos 720 dias (ADG365_720), ganho de peso diário total (ADG), curva de crescimento de peso ao nascer ao peso aos 720 dias (GC). 180 marcadores microssatélites, abrangendo os 29 autossomos bovino e cobrindo 3,322 cM foram selecionados a partir do mapa genético bovino. A média de intervalo de marcador foi 22 cM. Vinte e oito QTLs foram detectados em dez cromossomos, quinze QTLs tem modo aditivo, dois QTLs tem modo de dominância e onze QTLs têm sobredominância como modo de ação. Um QTL sugestivo foi detectado para BW no cromossomo BTA 17 em 1 cM. Um QTL sugestivo foi detectado para W205 no BTA 3 a 20 cM. Quatro QTLs sugestivos foram encontrados para YW no BTA 3 a 7 cM, no BTA 6 a 134,9 cM, no BTA 12 a 1 cM, e no BTA 22 a 1 cM. Para W720, seis QTLs sugestivos foram identificados, no BTA 2 a 30 cM, no BTA 3 a 1 cM, no BTA 6 a 44 cM, no BTA 10 a 20 cM, no BTA 12 a 1cM e no BTA 22 a 1 cM. Um QTL sugestivo foi identificado para ADG0_60 no BTA 8 a 143 cM. Dois QTLs sugestivos foram relatados para ADG60_205 no BTA 3 a 19 cM e no BTA 23 19 cM. Um QTL sugestivo foi observado para ADG205_365 no BTA 12 a 7 cM. Para ADG365_720, três QTLs foram detectados. Dois QTLs têm efeitos sugestivos no BTA 1 a 12 cM e no BTA 10 a 22 cM, um QTL significativo no BTA 6 a 43 cM. Seis QTLs sugestivos para ADG foram encontrados no BTA 2 a 32 cM, no BTA 3 a 1 cM, no BTA 6 a 43 cM, no BTA 10 a 20 cM, no BTA 12 a 1 cM e no BTA 22 a 1 cM. Três QTLs sugestivos foram identificados por GC no BTA 2 a 34 cM, no BTA 12 a 2 cM e no BTA 22 a 3 cM. Nenhum QTL foi detectado para WW em todos os cromossomos. Existem vários efeitos pleiotrópcos nos cromossomos BTA 2, 3, 6, 10, 12 e 22, os quais influenciam as características de crescimento. Foram detectados 22 efeitos epistáticos complexos para as cinco características YW, W720, GC, ADG365_720 e ADG. / This study was conducted to detect quantitative trait loci (QTL) affecting growth traits and growth curve using F2 Holstein x Gyr population. Traits analyzed were birth weight (BW), weaning weights (WW), weight at 205 day (W205), yearling weight (YW), weight at 720 day (W720), average daily gain from birth to weaning weight (ADG0_60), average daily gain from weaning weight to weight at 205 days (ADG60_205), average daily gain from weight at 205 days to yearling weight (ADG205_365), average daily gain from yearling weight to weight at 720 days (ADG365_720), total average daily gain (ADG), and growth curve from birth weight to weight at 720 days (GC). 180 microsatellite markers covering the 29 bovine autosomes and covered 3322 cM were chosen from bovine genetic map. The average marker interval was 22 cM. Twenty eight QTLs detected span ten chromosomes, fifteen QTLs had additive mode, two QTLs had dominance mode and eleven QTLs had overdominance mode of gene action. Suggestive QTL was found for BW on BTA 17 at 1 cM. Also, Suggestive QTL was detected for W205 on BTA 3 at 20 cM. Four suggestive QTLs were found for YW on BTA 3 at 7 cM, on BTA 6 at 134.9 cM, on BTA 12 at 1 cM, and on BTA 22 at 1 cM. For W720, six suggestive QTLs were identified, on BTA 2 at 30 cM, on BTA 3 at 1 cM, on BTA 6 at 44 cM, on BTA 10 at 20 cM, on BTA 12 at 1cM and on BTA 22 at 1 cM. Suggestive QTL was observed for ADG0_60 on BTA 8 at 143 cM. Two suggestive QTLs were reported for ADG60_205 on BTA 3 at 19 cM and on BTA 23 at 19 cM. Suggestive QTL was observed for ADG205_365 on BTA 12 at 7 cM. For ADG365_720, three QTLs were detected. Two QTLs had suggestive effect on BTA 1 at 12 cM and BTA 10 at 22 cM, one significant QTL on BTA 6 at 43 cM. Six suggestive QTLs were found for ADG on BTA 2 at 32 cM, BTA 3 at 1 cM, BTA 6 at 43 cM, BTA 10 at 20 cM, BTA 12 at 1 cM and BTA 22 at 1 cM. Three suggestive QTLs were identified for GC on BTA 2 at 34 cM, BTA 12 at 2 cM and BTA 22 at 3 cM. No QTLs were detected for WW in all chromosomes. There were many pleiotropy effects on BTA 2, 3, 6, 10, 12 and 22 influencing growth traits. There were 22 complexes epistatic effects were detected for five traits YW, W720, GC, ADG365_720 and ADG.

Breast cancer is one of the leading causes of death in women cancer cases worldwide. Cancer is the result of environmental and genetic factors that contribute to alterations in cellular control, proliferation, differentiation and apoptosis. Vitamin D is emerging as an important nutrient in the prevention and treatment of cancer due to its ability to modulate proliferation and apoptosis in vivo and in vitro. To accomplish this, Vitamin D exerts its biological activity by binding to a specific, high-affinity intracellular vitamin D receptor (VDR). VDR expression is identified in mammary cancer cell lines, but levels are reduced compared to non-cancerous cells, which limits vitamin D-induced gene expression. Our study investigated two compounds with histone deacetylase inhibitor (HDACI) activity, trichostatin A (TSA), and sulforaphane (SFN), and how they influence the expression of vitamin D-induced gene expression. By isolating mRNA to create cDNA, we were able to run RT-PCR to analyze the overall gene expression. The genes investigated were: CYP24A1, CYP27B1, VDR and TRPV6. We found that in MCF-7 breast cancer cells, 1,25(OH)2D3 treatment alone induced the expression of VDR, CYP24A1 and CYP27B1. TRPV6 mRNA expression was not evident. TSA alone increased expression of VDR and CYP24A1, but SFN alone had no effect. Co-treatments of 1,25(OH)2D3 and TSA raised CYP24A1, but not significantly. Co-treatments with SFN seemed to decrease CYP24A1 expression, not significantly. Our findings support further study of the effects of the HDACI TSA in breast cancer, and suggest that this HDACI may be beneficial in augmenting vitamin D cellular responsiveness.

Genetic variations in taste receptor genes play a notable role in human taste perception and food preferences and intake, which may affect nutritional and health status. Understanding how genetic variations in taste receptor genes influence food perception, preferences, and intake can play an important role in designing effective interventions to improve the quality of peoples' nutrition and minimize the risk of diet-related diseases such as obesity. The objective of this study was to investigate single nucleotide polymorphisms (SNPs) of umami taste receptor gene TAS1R1 and GRM4 and sweet taste receptor gene TAS1R3 and percentage of body fat mass (BF%) among young adults. 833 young adults aged 18-31 years old were enrolled in a cross-sectional study. Umami and sweet taste receptor genotypes were determined and analyzed. A strong association was observed between the allele frequencies of sweet taste receptor gene TAS1R3 for SNPs rs307355 and rs35744813 and BMI, and between the same SNPs rs307355 and rs35744813 and BF%. In addition, the allele frequencies of SNP rs2499729 were significantly related to the likelihood of having obesity based on BMI classification. However, there was no association between the allele frequencies of the SNPs of the umami taste receptor genes; TAS1R1 for rs34160967 and BMI or BF%. The results of this study also indicated association in total energy intake and the percentage of energy from carbohydrates, protein, and fat intake between the alleles of the sweet receptor gene TAS1R3 for rs307355 and 35744813. Furthermore, a notable association was also detected in the percentage of energy from fat intake among the alleles of the umami receptors gene TAS1R1 rs34160967, and a significant relation in the percentage of energy from carbohydrates and protein intake between the different genotype polymorphisms of the umami receptor GRM4 gene for rs2499729.