Thesis (doctoral)--Swedish University of Agricultural Sciences, 2000. / Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 85-110.

Adenine nucleotides hydrolysis is an important step of the neuromodulation of CNS and some of its breakdown products are able to protect the nervous tissue against brain injuries. The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in cerebrospinal fluid (CSF) from patients without neural inflammatory process under different conditions and in the presence of several inhibitors. The samples were chose considering the low protein levels, normal glucose levels, low leukocyte count and CSF differential count. We chose use the supernatant 1 (S1) for enzyme assay due to the best enzymatic activities in this fraction. The best hydrolyze temperature was 37°C to ATP and ADP. This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in pH 8.0 in the presence of 5mM Ca+2. Sodum azide inhibited both nucleotide hydrolysis at concentrations higher than 10 mM. Sodium fluoride inhibited the ATP and ADP hydrolysis at concentrations of 15 mM and 20 mM. The Na+ K+ ATPase inhibitor ouabain, did not affect ATP or ADP hydrolysis. The inhibitor P-type ATPase lanthanum 5mM was ineffective on ATPDase hydrolysis. Suramin (30-300 μM) inhibited ATP and ADP hydrolysis and presented a maximal inhibitory effect of 50% at 300 μM. The results of the present study demonstrated that ATP and ADP hydrolysis from human CSF presented a similar response those obtained from rat synaptosomes. / A análise do líquor é de grande importância para a detecção de desordens neurológicas de diversas etiologias. O ATP junto a seus produtos de hidrólise (ADP, AMP e adenosina) desempenha importantes funções junto ao SNC, as quais envolvem ações neuroprotetoras em doenças de etiologias variadas. O presente estudo tem como objetivos verificar a ocorrência de hidrólise de nucleotídeos da adenina em líquor de pacientes sem doença inflamatória do sistema nervoso. A determinação da atividade enzimática da NTPDase foi feita em líquor humano em diferentes condições experimentais e na presença de inibidores. As amostras foram escolhidas de acordo com os baixos níveis protéicos, valores normais de glicose e contagem celular diminuída. Foi escolhido o sobrenadante 1 (S1) por apresentar melhores atividades enzimáticas. A melhor temperatura de hidrólise do ATP e ADP foi 37°C. Esta enzima é cátion dependente, sendo a atividade de hidrólise ótima do ATP em presença de 5 mM Ca+2 e o ADP 5-7 mM de Ca+2, ambos em pH 8.0. A azida sódica alterou a atividade enzimática do ATP e do ADP somente nas concentrações mais altas deste inibidor da ATPase mitocondrial. A ouabaína, um inibidor da Na+/K+ ATPase não afetou a hidrólise do ATP/ADP. O inibidor de ATPase tipo-P lantânio (5 mM) foi ineficaz na hidrólise dos nucleotídeos. O suramin (30-300 XM), inibidor específico de NTPDase, inibiu a hidrólise do ATP/ADP e apresentou máximo efeito inibitório na concentração de 300 XM. Os resultados deste estudo mostraram que a hidrólise de ATP/ADP em líquor humano apresentou uma resposta semelhante àquelas obtidas em sinaptossomas de ratos.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both the ade2-1 and the early nonsense pgk1 steady state mRNA populations. The half lives of the steady state mRNA populations for both ade2-1 and pgk1 (early nonsense) were shortened from >30 minutes to approximately 7 minutes. This was not observed for pgk1mRNAs that contained a late nonsense codon demonstrating that activation of NMD specifically targeted the proper substrates in these experiments. Therefore, in yeast, nonsense mRNAs residing in the cytoplasm are susceptible to NMD. While these findings are consistent with NMD occurring in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechanism. To investigate the involvement of the nucleus in NMD, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NMD) was characterized. Subcellular fractionation experiments demonstrated that the majority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NLS) of Nmd2p were found to have adverse effects on the protein's decay function. These effects on decay function, however, could not be attributed to a failure in nuclear localization. Therefore, the residues that comprise the putative NLS of Nmd2p are important for decay function but do not appear to be required for targeting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular compartment for NMD. This thesis then investigates the regulatory roles of the trans-acting factors involved in NMD (Upf1p, Nmd2p, and Upf3p) using a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100). Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy can1-100 expression in wild-type cells caused mRNA stabilization similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. These findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay.

The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation? The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro. This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation.

The XIST gene is implicated in X-chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST RNA does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2-D and 3-D space; hence the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its poly-adenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of non-chromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.

In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of Dicer, a key component of RNA silencing mechanisms. Our work demonstrates that adenovirus encodes two miRNAs within the loci of the virus-associated RNA I (VA RNA I). We find that one of these miRNAs, miR-VA “g”, enters into a functional, Argonaute-2 (Ago-2)-containing silencing complex during infection. Currently, the cellular or viral target genes for these miRNAs remain unidentified. Inhibition of the function of the miRNAs during infection did not affect viral growth in a highly cytopathic cell culture model. However, studies from other viruses implicate viral miRNAs in the establishment of latent or chronic infections. Additionally, we find that adenovirus infection leads to the reduced expression of Dicer. This downregulation does not appear to be dependent on the presence of VA RNA or its associated miRNAs. Rather, Dicer levels appear to inversely correlate with the level of viral replication, indicating that another viral gene product is responsible for this activity. Misregulation of Dicer expression does not appear to influence viral growth in a cell culture model of infection, and also does not lead to gross changes in the pool of cellular miRNAs. Taken together, our results demonstrate that RNA silencing pathways are active participants in the process of infection with human adenovirus. The production of viral miRNAs and the regulation of cellular Dicer levels during infection implicate RNA silencing mechanisms in both viral fitness as well as potential host defense strategies.

Large differences exist in the decay rates of individual mRNAs yet the molecular basis for such differences is substantially unknown. We have developed a procedure for the measurement of individual mRNAs in the yeast Saccharomyces cerevisiae which utilizes northern or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant strain (RY260). To assess the reliability of half-life measurements obtained in this manner, we have compared the results of this procedure to results obtained by three other procedures (pulse-chase analysis, approach to steady-state labeling, and inhibition of transcription with thiolutin) and also evaluated whether heat-shock alters mRNA decay rates. We find that: i) for most mRNAs, all four procedures yield comparable relative decay rates and ii) there are no significant differences in the mRNA decay rates measured in heat-shocked or non-heat-shocked cells. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MATα1, were unstable (t1/21/2> 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. The following parameters have been analyzed to determine their role in mRNA decay: i) mRNA size; ii) poly(A) tail metabolism; iii) translational status; iv) relative content of rare codons; and v) structures and sequences within the 3'-untranslated region (UTR). To identify the structural determinants responsible for the rapid decay of the unstable HIS3 and STE2 mRNAs, recombinants of their respective genes were constructed and transformed into strain RY260 on centromere-containing vectors, and the half-lives of the resulting chimeric mRNAs were measured in vivo. Chimeric genes were constructed in which the 3'-UTR of ACT1 was replaced with the corresponding region of the unstable HIS3 or STE2 mRNAs. The decay rate of the ACT1-5'-HIS3-3' mRNA was very similiar to that of the stable endogenous ACT1 mRNA, implying that the 3'-end of HIS3 is not sufficient to transfer the instability phenotype of the HIS3 mRNA. The HIS3-5'-ACT1-3' mRNA from the reciprocal construct was unstable, suggesting that HIS3 instability determinants are located within its 5'-UTR or coding sequence. A 411 nucleotide (nt) deletion within the HIS3 coding region (with either the HIS3 or ACT1 3'-UTR) was stabilized 3-fold suggesting this region is necessary for the rapid decay of HIS3 mRNA. Insertion of these 411 nts in-frame into the entire ACT1 gene had no significant effect on the stability of the hybrid mRNA implying that these HIS3 sequences are not sufficient to function on their own and that they may have to interact with HIS3 5'- sequences. The ACT1-5' -STE2-3' hybrid mRNA decayed with an intermediate half-life of 12 min. Furthermore, an 82% deletion of the STE2 coding region increased the half-life by nearly 2-fold. Both results suggest that instability determinants of STE2 mRNA are not restricted to the 3'-UTR. Our overall conclusion is that mRNA stability is not dictated by simple, transferable elements (sequences or structures), but may involve interactions between multiple determinants in the mRNA.

Les analogues de nucléosides représentent une famille d’agents thérapeutiques très largement utilisée en chimiothérapie anticancéreuse. Cependant des phénomènes de résistance d’origine multifactorielle apparaissent chez les patients atteints de leucémie et il semble que deux protéines appartenant à la famille des 5’-nucléotidases (la nucléotidase cytosolique de type II, cN-II et l’ectonucléotidase, CD73) puissent être impliquées. Nous nous sommes donc intéressés à la synthèse et à l’étude d’inhibiteurs potentiels de cN-II ou de CD73 du type analogue de nucléosides phosphonates. Ce manuscrit rapporte dans un premier temps le contexte biologique des travaux de thèse, et les découvertes ayant supposées l’implication des protéines cibles dans les mécanismes de résistance. Le deuxième chapitre décrit la synthèse d’une série d’analogues beta-hydroxyphosphonates incluant une nucléobase modifiée de type 1,2,3-triazolo, ainsi que leur évaluation biologique vis-à-vis de la cN-II purifiée. Enfin, des résultats préliminaires ont été obtenus concernant de nouvelles structures capables d’inhiber modestement l’activité enzymatique de CD73. / Nucleosidic analogs are widely used as therapeutic agents in antitumoral chemotherapy. However, cellular resistance appears in a multifactorial manner in leukemic patients and it seems that two proteins belonging to the 5’-nucleotidase family (the 5’-cytosolic nucleotidase, cN-II and the ectonucleotidase, CD73) may be involved in these phenomenon. We focused our interest on the synthesis and the study of potential inhibitors belonging to the family of phosphonate nucleoside analogs. First, we reviewed literature data about the biological context of our research, especially concerning the involvement of these two targeted proteins in resistance phenomenon. Then, we described the synthesis of a series of beta-hydroxyphosphonate nucleosides incorporating 1,2,3-triazolo moiety as nucleobase, as well as their evaluation as inhibitors against the purified enzyme. Finally, preliminary results concerning the design of novel inhibitors of CD73 were reported.

Made available in DSpace on 2014-06-11T19:27:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-04-29Bitstream added on 2014-06-13T19:35:47Z : No. of bitstreams: 1 pelicia_vc_me_botfmvz.pdf: 473034 bytes, checksum: 42ede2046acc80e2fb52f44e068b5c16 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Diante da crescente proibição da utilização dos antibióticos promotores de crescimento na produção animal, a busca por alternativas naturais é pauta atual entre os pesquisadores. Sobre estes aspectos, os nucleotídeos tornam-se um suplemento interessante na criação de frangos alternativos, pois vem demonstrando efeito benéfico sobre a integridade, desenvolvimento e turnover celular intestinal, entre outros. Com o presente trabalho, objetivou-se avaliar o efeito da suplementação com nucleotídeos na dieta sobre o desempenho, rendimento de carcaça e de partes e na morfologia da mucosa do intestino delgado de frangos de corte criados no sistema alternativo. Foram utilizados 600 pintos de corte machos da linhagem Ross distribuídos em delineamento em blocos casualizados com seis tratamentos (rações sem promotor de crescimento antimicrobiano Surmax® e com adição de 0; 0,04; 0,05; 0,06 e 0,07% de nucleotídeos e uma ração com promotor de crescimento antimicrobiano e sem adição de nucleotídeos), com quatro repetições de 25 aves cada. As rações foram formuladas a base de milho e farelo de soja sem ingredientes de origem animal e sem adição de anticoccidianos. Com três dias de idade as aves foram vacinadas contra coccidiose via água de bebida. As análises estatísticas dos dados de desempenho, rendimento de carcaça e partes e morfologia intestinal foram feitas pela análise de variância 5% de significância. Não foram encontradas diferenças significativas entre os tratamentos para as variáveis estudadas. Numericamente observou-se maior ganho de peso (GPD) e melhor conversão (CA) alimentar para o tratamento com 0,05% de inclusão de nucleotídeos aos 7 dias de idade e aos 21 e 42 dias de idade, maior GPD para o tratamento com 0,07% de inclusão. Diante desses resultados conclui-se que os nucleotídeos adicionados... / In view of increasing ban of antibiotics use as growth promoters in animal production, the demand for natural alternatives is the inquiry most current among researchers. Under these aspects, the nucleotides become an interesting supplement for the breeding of alternative broilers, because their demonstrated beneficial effect on the integrity, development and intestinal cell turnover, among others. The present study aimed to evaluate the effect of supplementation with nucleotides in broiler diet on raised performance, carcass and parts yield and on the morphology of intestinal mucosa of broilers in alternative breeding system. Six hundred 1-day-old male Cobb chicks were allotted in a randomized block design with six treatments (diet without antimicrobial growth promoter, and with addition of 0; 0.04; 0.05; 0.06 and 0.07% nucleotides and diet with antimicrobial growth promoter and without nucleotides addition), with four replicates of 25 chickens each. Diets were formulated utilizing corn and soybean meal without animal origin ingredients and without addition of anticoccidia drugs. Chicks were vaccinated against Coccidiosis by drinking water at three-day old. Statistical analyses of performance, carcass yield and parts and intestinal mucosa morphology were performed by analysis of variance and means compared by Tukey test was (P < 0.05). There were no significant differences between the treatments for these variables. Numerically there was higher weight gain and better conversion for treatments with 0.05% nucleotides inclusion to 7 days of age and 21 and 42 days, higher weight gain for treatments with 0.07% of inclusion. It was concluded that the nucleotides added to the diet did not influence carcass and parts yield and intestinal mucosa morphology, however in levels greater than or equal to 0.05%, nucleotides increase performance of alternative... (Complete abstract click electronic access below)