No description available.

The AID/APOBEC protein family comprises a group of cytosine deaminases found in vertebrates that are capable of modifying cytosine to uracil in the context of RNA or singlestranded DNA. They exert diverse valuable physiological functions including antibody diversification and restriction of viral infection. However, off-target mutations have also been shown to contribute to cancer development, making it crucial to better understand the interactions and mechanisms that regulate AID/APOBEC activity and editing site fidelity. In this regard, a new focus on RNA as a putative regulator of AID/APOBECs has recently emerged. Regardless of whether it is used or not as a substrate for deamination, most members of the family have been shown to retain the ability to bind RNA, emphasizing a potential regulatory role for this interaction. However, little is known about AID/APOBECs RNA binding specificity. A promiscuous binding has been suggested in some cases while in vitro evidence for other members of the family indicate a certain level of specificity. Therefore, to thoroughly unravel the AID/APOBECs RNA binding specificity, in my doctoral research I applied cross-linking and immunoprecipitation (iCLIP), an unbiased technique that allows identification of protein-bound RNAs with nucleotide resolution in living cells. As a first approach, I adapted the technique for its use in yeast and probed the RNA binding of AID and APOBEC3G, revealing different degrees of preference for small structured RNAs and recognition of particular sites within them. I then expanded the analysis to mammalian cells (HEK293T) and evaluated an extended set of APOBECs finding that, even in the presence of a broader and more complex pool of RNAs, small RNAs were still significantly bound by some members of the family. Furthermore, the comparative analysis of AID, APOBEC1, APOBEC3G, APOBEC3A and APOBEC3B iCLIP data obtained in my research, revealed shared and individual preferences for certain RNAs, suggesting a degree of binding specificity among APOBECs. In summary, my thesis outlines for the first time a comprehensive analysis of the RNA binding specificity of different AID/APOBECs in vivo, including the description of novel interactions with nucleotide resolution. The results obtained are of great value and open the field for further investigation of the specific meaning and validation of each preferential binding, providing new insights into understanding the role of AID/APOBEC interaction with RNA.

Includes bibliographical references (43 leaves) This study shows that when LR3IGF-I is administered to animals in pharmacologically active doses, it may be present in either the free form or bound to IGF-binding protein(s) in the circulation. Age and nutrition which are factors that regulate synthesis of endogenous IGF-I and IGF-binding proteins, affect the in vivo formation of complexes between the analog and IGFBP(s). This study also suggests that IGFBP-1 inhibits the pharmacological activity of circulating LR3IGF-I on thymus whereas it appears to stimulate the pharmacological activity of LR3IGF-I in kidneys.

The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models. Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD. Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.

The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models. Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD. Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.

Understanding the temporal and spatial changes in calcium concentration has been a difficult endeavor for many years due to the relatively small changes in calcium concentration during messenging events, the rapid changes upon physiological messenging, and the unavailability of fast, efficient, and sensitive sensors to detect calcium changes. In addition, the key factors in calcium binding have yet to be determined due to the metal-metal interactions, cooperativity, and conformational change involved in calcium binding to natural calcium-binding proteins. To overcome these obstacles and to engineer calcium sensors for in vivo studies of calcium signaling events, calcium binding sites have been engineered into Green Fluorescent Protein. The engineered binding sites demonstrate terbium binding affinity from 2-30 ƒÝM and calcium binding affinity from 50-100 ƒÝM. Site 177 demonstrates green fluorescence when expressed in mammalian cells and produces a response to calcium concentration changes when expressed in the cytosol. Addition of the cycle 3 mutations (M153T, V163A, F99S) to Site 177 allowed for increased brightness in the emission of the chromophore but still exhibited calcium response. The second generation Site 1 demonstrates fluorescence response to calcium concentration changes when expressed both in the cytosol and in the endoplasmic reticulum. Addition of M153T and V163A to Site 1 allowed for expression of fluorescent protein at 37 ¢XC in HeLa cells and at 30 ¢XC in bacteria. Site 1-M153T/V163A exhibits chromophore fluorescence response to calcium with a Kd of 100 ƒÝM and competition with Rhodamine-5N produced a calcium Kd of 107 ƒÝM. This designed sensor, Site 1-M153T/V163A is the first demonstration of a designed calcium binding GFP with calcium response measured both in vivo and in vitro.

No description available.

Abstract Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Piscinodavirus (Betanodavirus) that causes vacuolating encephalopathy and retinopathy or viral nervous necrosis. MGNNV propagated in SSN-1 cells that were derived from fry tissue of striped snakehead fish, Channa striatus. SSN-1 was highly permissive to MGNNV infection and production, in which the TCID50 per ml increased from the magnitude of 10 4 to 10 8 after 5 passages. The thermal stability analysis unveiled that the optimal activity of MGNNV can be resumed at the temperature from frozen to less than 25 ¢J. Oreochromis mossambica was highly susceptible to MGNNV only when injecting the virus into the vitreous body of fish eye, via which the virus could propagate in the fish brain. Employing the technique of immobilizing virus on itrocellulose, several cellular MGNNV-binding proteins were detected. Two MGNNV-binding proteins of 100 and 56 kDa were found in SSN-1 cell lysate; four proteins of 60, 43, 36, and 30 kDa in rotifer lysate; four proteins of 62, 50, 20 and 25 kDa on the membrane of grouper fertilized eggs. In different organ lysates of Epinephelus malabaricus, two MGNNV-binding proteins of 80 and 43 kDa were found in brain; four MGNNV-binding proteins of 60, 45, 43 and 23 kDa were found in eye; two MGNNV-binding proteins of 80, 18 kDa were found in liver; four MGNNV-binding proteins of 60, 45, 43 and 30 kDa were found in pancreas; two MGNNV-binding proteins of 45 and 24 kDa were found in spleen; one MGNNV-binding proteins of 80 kDa were found in kidney; six MGNNV-binding proteins of 100, 95, 80, 48, 45 and 30 kDa were found in ovary.

The present study cloned successfully two lipid-metabolism genes, lipoprotein lipase (LPL) and fatty acid binding protein (FABPs) from cobia and studied the mRNA expressions of the two genes and their upstream gene PPARs when the cobia were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were supplemented by fish oil (FO, rich in n-3 HUFA), perilla oil (PE, rich in 18:2 n-6), safflower oil (SA, rich in 18:2 n-6), olive oil (OL, rich in 18:1 n-9) or palm oil (PA, rich in 16:0). The whole sequences of LPL, liver-FABP (L-FABP) and muscle-FABP (M-FABP) encode 520, 126 and 133 amino acids, respectively. RT-PCR and real time PCR analyses based on these gene sequences show that the mRNA expressions of L-FABP and M-FABP in the tissue of the cobia were diet-specific. The mRNA expression of LPL, on the other hand, did not respond to the treatments, except in visceral fat depot. Linear regression analysis shows that the mRNA expression of LPL in the liver and muscle was positively (P<0.05) related to dietary fatty acids and ther concentration, but that in the visceral fat depot was negatively related. The mRNA expression of FABPs was also positively correlated with dietary fatty acid levels. Among all fatty acids, the levels of C14:0, C20:1 n-9, EPA and DHA were positively correlated with the mRNA expression of PPAR£^and also with FABPs mRNA expression in the visceral fat depot and LPL mRNA expression in the muscle. Thus, LPL, L-FABP and M-FABP mRNA expression of the cobia were highly influenced by the kind and amount of dietary fatty acids. The role of PPARs was not clearly demonstrated.

Oskar (Osk) is a posterior body patterning determinant in Drosophila melanogaster oocytes. oskar (osk) mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein accumulates. Translational repression of osk prior to posterior localization is mediated by the RNA binding protein, Bruno (Bru). To better define Bru binding sites, I performed in vitro selections using full length Bru and the fragments containing either the first two RRMs (RRM1+2) or the third RRM (RRM3+). The aptamers from the final round from each of the selections produced a multitude of overrepresented primary sequence motifs. Examples of each of these motifs were found in the 3’UTRs of the mRNAs that Bru is known to regulate during oogenesis. GFP reporter transgenes under the control of the UAS-Gal4 expression system were constructed with each class of the binding sites within the reporter transgenes’ 3’UTRs to test the motifs’ ability to repress the reporters in vivo. In a wildtype background, the GFP reporters containing the binding sites were translationally repressed. In the aret mutant background, the GFP levels of the repressed GFP reporters increased with reduced Bru activity, suggesting the transgenes’ repression is mediated by Bru. Three of the motifs isolated in the in vitro selections reside in the AB and C regions of the osk 3’UTR, and the three classes of sites were mutated in the AB and C regions. The mutated AB and C regions were used to assay for a reduction of Bru binding affinity for the mutant RNAs. Additionally, the mutations were incorporated into an osk genomic transgene that was introduced into an osk RNA null as well as an Osk protein null background. The mutations reduced Bru binding to the AB and C regions. The transgenes containing the mutated Bru binding sites could not fully rescue the osk RNA null phenotype but can fully rescue the Osk protein null phenotype, suggesting an osk transcript can regulate other osk mRNAs in trans. / text