The groundbreaking use of transcription factors (Oct4, Sox2, Klf4, c-Myc) in reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides novel ways in regenerative medicine and disease modeling. The reprogramming process is a stepwise process involving global epigenetic remodeling. In recent years, small molecules like DNA methyltransferase inhibitor that alter the epigenetic status of cells were shown to enhance the reprogramming efficiency. It was postulated that chromatin modifying enzymes played an important role during the reprogramming process, and microRNAs (miRNAs) were the upstream regulators. The objectives of this study involve the identification of potential miRNAs regulating the expression of chromatin modifying enzymes and the study of their roles during reprogramming. Primary mouse embryonic fibroblasts (1o MEFs) were used for the establishment of a reprogramming system, where the delivery of transcription factors Oct4, Sox2, klf4 and cMyc was mediated by lentivirus. Another established secondary MEFs (2o MEFs) reprogramming system was also included in the study. Mouse iPSCs (miPSCs) derived from both systems were shown to express pluripotent markers. In-silico analysis predicted a set of miRNAs (miR-101, miR-135a, miR-148a and miR-148b) commonly targeted the chromatin modifying enzymes in mouse genome. Among them, miR-101 and miR-135a overexpression were found to inhibit the reprogramming efficiency significantly in both 1o and 2o MEFs. Conversely, the inhibition of miR-135a but not miR-101 expression significantly enhanced the reprogramming efficiency in both systems. In this study, it was postulated that miR-101 regulated enhancer of zeste homolog 2 (Ezh2) during reprogramming. Ezh2 was confirmed to be negatively regulated by miR-101 at protein level. The expression of Ezh2 was high in mouse embryonic stem cells (mESCs) but time dependently depressed during mESC differentiation, while its expression was increased during reprogramming of MEFs. Ezh2 expression was found to negatively correlate with miR-101 expression in these conditions. In addition, the knockdown of Ezh2 mimicked the inhibitory effect of miR-101 overexpression on reprogramming efficiency. The inhibitory role of miR-135a on reprogramming was linked to its potential target, Sirtuin 1 (Sirt1). Sirt1 was negatively regulated by miR-135a. The expression of miR-135a was upregulated upon mESC differentiation and decreased during reprogramming. Together with the previous finding in this laboratory, miR-135a expression was negatively correlated with Sirt1. Furthermore, miR-135a inhibition increased the proliferation rate of MEFs. More importantly, miPSCs reprogrammed from miR-135a knockdown MEFs maintained the pluripotent state. To further analyze the pluripotency of the miPSCs, the tetraploid complementation assay was established. Preliminary studies were performed to optimize the conditions for electrofusion. Although single electrofusion with a lower field strength (1000V/cm) resulted in lower fusion rate, the development of the mESC aggregated embryo was the best when compared to higher field strength and those with double electrofusion. Lastly, the mESCs aggregated into tetraploid embryo were mainly localize in the inner cell mass of the embryo. In conclusion, negative correlations were found between miR-101/Ezh2, and miR-135a/Sirt1 during somatic cell reprogramming. The identification of small molecules in reprogramming helps to understand the molecular mechanisms of reprogramming. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.

During tumor inception and progression, culprit gene variants confer selective advantage to progenitor cancer cells, allowing them to outcompete normal cells and proliferate uncontrollably. Both regions of somatic amplification as well as germline DNA sequence changes may be variants that are positively selected by the tumor. Traditionally, these two variant classes have been studied independently. While many discoveries have been made in such a manner, independent examination of these classes possesses certain limitations. Integrated examination of these two classes holds the potential to reveal specific nucleotide alleles that are amplified in the tumor, which in turn may reveal proximal genes. We present methods that focus on such integration. The first, the Amplification Distortion Test (ADT), aims to detect nucleotide alleles that are selectively amplified across tumor samples. Motivated to apply ADT on nascent next generation sequencing data, we developed a novel Hidden Markov Model-based method - Haplotype Amplification in Tumor Sequences (HATS) - that analyzes tumor and matched normal sequence data, along with training data for linkage information, to infer amplified alleles and haplotypes in regions of copy number gain. HATS is designed to handle biases in read data as well as accommodate rare variants. We demonstrate that HATS infers the amplified alleles more accurately on simulated and real tumor data than does an alternate naïve approach, especially at low to intermediate sequence coverage levels, and when allele-specific biases or stromal contamination is present. We present these methods with the motivation that they may aid the cancer community in identifying novel causal or associated putative variants.

Goal of work: evaluate and analyze the factors that have an impact on the quantity of the somatic cells in cow‘s milk. Objectives of work: 1. To select and collect the data of the milk tests of the cows under observance when the lactation was over, taken in 2003-2004. 2. To describe the collected data by setting the diversity of age and breeds of cows under observance and evaluating the greatness of farms. 3. To estimate statistically and describe the impact of such factors as breed, age, season, greatness of farm, type of sample collection and the person taking it, impact on the quantity of the somatic cells in the milk of the observed cows. Conclusion: Statistically, the breed and age of an animal influence upon the quantity of the somatic cells in milk. The least number of the somatic cells can be detected in the milk of the cows that are young or used to the local keeping and feeding conditions. 1. The greatest quality of milk from the perspective of the somatic cells is achieved in grand farms that keep over milking cows. 2. The influence of the person taking a sample on the quantity of the somatic cells in milk is confidential statistically. The right order of taking a sample has a crucial meaning to the further investigations on the productivity of cows. It is recommended to organize a periodical training of the control assistants and the owners of the observed cows, putting emphasis on the importance of the right way of taking a sample.

Padidėjęs somatinių ląstelių skaičius piene - svarbus mastito požymis. Perdirbamajai pramonei karvių mastitas buvo ir liks vienu iš svarbiausių trukdžių, gaminant aukštos kokybės pieno produktus. Klinikine forma serga 2 - 5 % lakuojančių ir užtrūkintų karvių, o slaptuoju uždegimu - iki 50 % karvių. / The increased number of somatic cells - an important feature of mastitis. Processing industry bovine mastitis has been and will remain one of the most important interference in the manufacture of high-quality dairy products. The clinical form of suffering 2-5% lakuojančių and užtrūkintų cows and subclinical inflammation - up to 50% of the cows.

Thesis (M.S.)--University of Michigan, 1964.

Thesis (M.S.)--University of Michigan, 1964.

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 223-251). Also available online through Digital Dissertations.

The aim of the study was to analyze the occurrence of individual genotypes in the CGIL4 locus in a given cattle population. The analysis was carried out in breeds of Holstein and Czech Pied Cattle. The relationship between the genotype in the given locus and the number of somatic cells in the milk was verified. An increased number of somatic cells is the main indicator of mastitis. The diploma thesis described the factors that provoked their origin. In the practical part, milk samples were taken in a selected cow population of both breeds. The DNA was isolated from the milk and samples for the CGIL4 locus were genotyped. Genetic and allelic frequencies were counted from genotyping results. Finally, the potential relationship between genotype in a given locus and the number of somatic cells in milk was evaluated statistically.

Submitted by VALENTINA ROLDAN MONTES null (valentiniya@hotmail.com) on 2016-11-14T17:33:47Z No. of bitstreams: 1 dissertação_Valentina_Roldan.pdf: 1303188 bytes, checksum: 91f87b135d433e48cad11782f8d4380d (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-11-21T13:30:26Z (GMT) No. of bitstreams: 1 roldanmonter_v_me_jabo.pdf: 1303188 bytes, checksum: 91f87b135d433e48cad11782f8d4380d (MD5) / Made available in DSpace on 2016-11-21T13:30:26Z (GMT). No. of bitstreams: 1 roldanmonter_v_me_jabo.pdf: 1303188 bytes, checksum: 91f87b135d433e48cad11782f8d4380d (MD5) Previous issue date: 2016-10-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Considerando a importância das doenças que afetam o desempenho produtivo dos animais na indústria leiteira em todo o mundo é necessário implementar ferramentas moleculares que auxiliem na identificação e controle destas doenças. Quando ocorre alguma infecção em um organismo superior, existe aumento do número de células de defesa e o sistema imune inato proporciona uma linha de defesa contra os patógenos. Os “Toll Like Receptors” (TLR) são proteínas da membrana que desempenham um papel chave na imunidade, reconhecendo patógenos e, posteriormente, ativando as respostas. O presente estudo foi realizado para identificar SNPs no gene TLR4 bubalino e analisar suas associações com características de importância produtiva, incluindo a contagem de células somáticas (CCS). Foram utilizadas amostras de DNA de 130 búfalas da raça Murrah. A região codificante do gene TLR4 foi amplificada através de reações de PCR e posteriormente sequenciada. Os polimorfismos encontrados tiveram suas frequências alélicas e genotípicas calculadas e verificadas quanto ao equilíbrio de Hardy-Weinberg, além de serem utilizados para os estudos de associação. Foram identificados 13 polimorfismos do tipo SNP para as regiões sequenciadas do gene TLR4, sendo que a maioria encontra-se em região codificante. Encontrou-se associação significativa, com porcentagem de gordura dos Snps g514>C/T (P=0,0040) e g536>A/T (P=0,0035). As associações para CCS demostrou-se altamente significantes (p=<0,001) para todos os Snps (g322>G/A, g514>C/T, g536>A/T, g8338>A/C, g8341>A/G, g8342>T/G, g8343>G/A, g8345>A/G, g8413>A/G, g8428>G/A, g8438>A/C, g8578>G/T e g8582>A/C). Sugere-se que os Snp do gene TLR4 possam ser utilizados como marcadores moleculares em búfalos, já que foram verificadas suas associações com características como porcentagem de gordura e proteína, e contagem de células somáticas. / Molecular markers might be developed to investigate genetic variants associated to the disease and assist selection process in order to identify resistant animals. When the mammary gland is infected, there is an increase in the defense cells, also called somatic cells. It is a defense line of the immune system against pathogens. The “tool like receptors” TLR are membrane proteins that have an important role in the immunity, recognizing pathogens and activating adequate responses. The present study aimed to investigate the association of TLR4 gene SNPs with productive characteristics and SCC in buffaloes. The DNA was extracted from hair follicules of 130 Murrah buffaloes. The fragments were amplified by Polymerase Chain Reaction (PCR) and sequenced. Thirteen SNPs were found. Allelic and genotypic frequencies were calculated as well as the adhesion to Hardy-Weinberg equilibrium and the linkage disequilibrium (r2) and the association to the characteristic. For SCC was tested methodology linear generalized mixed model, assuming Poisson distribution. Bonferroni correction was applied for the number of SNPs. Thirteen SNP polymorphisms were identified in coding region of the TLR4 (g322>G/A, g514>C/T, g536>A/T, g8338>A/C, g8341>A/G, g8342>T/G, g8343>G/A, g8345>A/G, g8413>A/G, g8428>G/A, g8438>A/C, g8578>G/T, g8582>A/C). The SNPs g514>C/T and g536>A/T had significant association whit %G. All the SNPs were associated (p=0.001) whit the CCS. Other authors also reported the association of TRL4 SNPs to the trait. The results show that the SNPs of TLR4 gene can be used as molecular markers in buffaloes.