Ensaios clínicos observacionais e em animais sugerem que o estrógeno exerça proteção cardiovascular, entretanto existem controvérsias mesmo após estudos randomizados. Tem sido sugerido que existe uma janela de oportunidade terapêutica, teoria timing hypothesis, que analisa o tratamento com estrógeno, iniciado precoce ou tardiamente, em mulheres na pós-menopausa. Avaliamos a função de carótidas de camundongos fêmeas com senescência acelerada (SAMP8) ou resistentes (SAMR1) ovariectomizadas tratadas com estrógeno precoce e tardiamente. Em SAMR1 os tratamentos promoveram vasculoproteção e nas carótidas das SAMP8 o tratamento com início precoce não alterou o efeito da ovariectomia, porém o tratamento com início tardio aumentou a vasoconstrição à fenilefrina, a produção de tromboxano (TXA2) e a expressão proteica do ERα-36kDa, splicing alternativo do receptor clássico ERα-66kDa. Portanto, os efeitos do estrógeno não foram benéficos no envelhecimento, podendo contribuir para o desenvolvimento de doenças cerebrovasculares decorrentes de alteração na circulação cerebral. / Observational clinical trials and animal studies demonstrated that estrogen promote cardiovascular protection. However, there are controversies after randomized clinical trials. It has been suggested that there is a therapeutic window of opportunity timing hypothesis theory , which analyzes the early and late estrogen treatments in postmenopausal women. We evaluated the carotid function in ovariectomized senescence (SAMP8) and resistant (SAMR1) female mice treated with estrogen in mineral oil solution dose of 5 g/kg by subcutaneous injection every three days. In SAMR1 estrogen treatments promoted vasculoprotection. In SAMP8 early-onset estrogen treatment did not change the effect observed in the ovariectomy, however late-onset estrogen treatment increased the vasoconstriction to phenylephrine, the thromboxane production and the protein expression of ERα-36kDa, alternative splicing. Therefore, the effects of estrogen were not beneficial in aging, contributing to the development of cardio and cerebrovascular diseases due to alteration cerebral circulation.

<p>Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer.</p><p>The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition.</p><p>This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.</p>

Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition. This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.

Massively parallel whole transcriptome sequencing and its ability to generate full transcriptome data at the single transcript level provides a powerful tool with multiple interrelated applications, including transcriptome reconstruction, gene/isoform expression estimation, also known as transcriptome quantification. As a result, whole transcriptome sequencing has become the technology of choice for performing transcriptome analysis, rapidly replacing array-based technologies. The most commonly used transcriptome sequencing protocol, referred to as RNA-Seq, generates short (single or paired) sequencing tags from the ends of randomly generated cDNA fragments. RNA-Seq protocol reduces the sequencing cost and significantly increases data throughput, but is computationally challenging to reconstruct full-length transcripts and accurately estimate their abundances across all cell types. We focus on two main problems in transcriptome data analysis, namely, transcriptome reconstruction and quantification. Transcriptome reconstruction, also referred to as novel isoform discovery, is the problem of reconstructing the transcript sequences from the sequencing data. Reconstruction can be done de novo or it can be assisted by existing genome and transcriptome annotations. Transcriptome quantification refers to the problem of estimating the expression level of each transcript. We present a genome-guided and annotation-guided transcriptome reconstruction methods as well as methods for transcript and gene expression level estimation. Empirical results on both synthetic and real RNA-seq datasets show that the proposed methods improve transcriptome quantification and reconstruction accuracy compared to previous methods.

The presence of introns in all the eukaryotic genomes identified so far underscores the fundamental and ubiquitous role of pre-mRNA splicing. The spliceosomal machinery, comprised of five small nuclear RNAs and several protein factors, catalyzes the two-transesterification reactions of splicing with precision and consistency. Through a complex network of protein-protein and RNA-protein interactions it ensures the removal of the intron and ligation of the flanking exons to yield the mature mRNA. Prpl7 is a splicing factor that functions at the second-step of splicing (Vijayraghavan et all, 1989). Null alleles of prpl7 are viable at 23°C but die at temperatures above 33°C (Jones et al.9 1995). Besides its functions in pre-mRNA splicing, mutants in PRP17ICDC40 were independently shown to affect cell-cycle progression, particularly the Gl/S and G2/M transitions (Chawla et a/., 2003). In this study, we have attempted a further characterization of Prpl7 to analyze both its role in pre-mRNA splicing and in cell-cycle progression with an aim to decipher underlying reasons for the interlinking of these two cellular processes. Different experimental approaches were adopted to achieve this goal. Global gene-expression profiling provided an overview of all the transcripts affected in a prpl 7 mutant and allowed its comparison with mutants of other splicing factors. This exercise aided in identification of both pre-mRNA splicing and cell-cycle related effects of Prpl7. Biochemical analysis of the Prpl7 spliceosomal associations have provided further clarity on the part played by Prpl7 in pre-mRNA splicing. A genome-wide two-Hybrid screen for interacting partners of Prpl7 was undertaken and uncovered two Likely interacting partners of Prpl7. Global expression profiling of splicing mutants Pleiotropic phenotypes observed in mutants of prpl 7 and few other splicing factors have been speculated to arise from either the multi functionality of the factor or more likely due to a specific requirement of the factor in splicing of a select subset of transcripts, that encode proteins essential to the affected cellular pathway. These observations raise questions about the ubiquitous requirement of factors in pre-mRNA splicing. To understand these aspects of splicing, we studied the effects of splicing factor mutants on a genome-wide scale. Using splicing-sensitive DNA microarrays imprinted with all yeast ORFs and in addition, independent spots for a majority of the intron sequences, we analyzed the global expression changes triggered by the inactivation of temperature-sensitive mutations in PRP17 or PRP22. Experiments with prp2-l mutant strain detect, as expected, an increase in pre-mRNA levels at the intron spots and further demonstrated that the ORF spots detect a decrease in mRNA levels in these DNA microarrays. These results established the DNA micro arrays as tools for the analysis of splicing on a global scale. The temporal alterations in transcript profiles in prpl 7 and prp22 mutants, as compared to the wild type, revealed both shared and unique effects of these factors on clusters of intron-containing transcripts. Such differential effects, on intron-containing transcripts, amongst the splicing mutants implicate specialized roles for each of these factors. Through analysis of the set of intron-containing transcripts affected in prpl7Δ cells, we infer those attributes of these pre-mRNA substrates, which predispose a need for Prpl 7. We find that splicing of introns longer than 200nts has a stronger dependence on Prpl7. The distance between consensus intron elements- the branch-nucleotide and the 3'splice-site (B), also imposes a requirement for Prpl7. Introns with a 13nts or lesser distance between these elements are spliced even in the absence of Prpl 7, both in vivo and in vitro. The 5'splice-site to branch-nucleotide distance (A) also influences the need for Prpl7. Most introns with a A/B ratio of less than 2 undergo Prpl7 independent splicing in vivo. Intron-containing genes that could be responsible for the pleiotropic phenotypes of prpl7 were also identified through the global splicing analysis. These included splicing targets that act at the Gl-S phase such as ANC1/TAF14, TMD4, PHO85 and those at the G2-M phase of the cell-cycle; TUB], TUB3, GIM5, MOBl UBC9. Recently, a different study implicates ANC1ITAF14 as the intron-containing gene responsible for the cell-cycle phenotype associated with prpl7 (Dahan and Kupiec, 2004). Our global analysis of all intron-containing transcripts with compromised expression in prpl7A cells identify, in addition, PHO85 as a possible regulator underlying cell-cycle effects in this mutant. Pho85 is a cyclin-dependent kinase that functions at both the Gl/S and M/Gl phases of the division cycle (Moffate* al., 2000). Synergistic growth defects in double mutants of prpl7 and pho85 have uncovered a novel role for Prpl7 in bud morphogenesis. Our micro array data also reveals compromised expression levels for several key intronless cell-cycle rregulatory genes indicating a possible splicing-independent role for Prpl7 in the cell-cycle. Examples of such transcripts are: the Gl cyclins CLN1, CLN2 and CLN3; CDC6, required for assembly of the pre-replication complex at sites of replication origin; and the cell-cycle regulatory transcription factors: SWI5 and ACE2. The global analysis has therefore enabled, for the first time, a characterization of the splicing substrate specificity of Prpl7 and has also uncovered the effects of this protein on gene expression during cell-cycle progression (Fig. V.I A). Spliceosomal interactions of Prpl7 To understand the function and associations of Prpl7 in the spliceosome, we have examined its snRNP interactions and determined the time point of its coalescence on assembling spliceosomes. A functional epitope tagged-Prpl7 was created using the polyoma middle T-antigen and the poly-HIS tags (Stevens et aln 1999). Through immunoprecipitation analyses performed with splicing extracts, from this strain, we find Prpl7 to associate with three spliceosomal snRNPs- U2, U5 and U6, implicating an interaction with active spliceosomes or post-splicing complexes. Specific biochemical depletion of any one of these snRNAs, through oligo-directed RNaseH cleavage, did not have a drastic effect on the association of Prpl7 with the other two snRNAs. To decipher the point at which Prp 17 joins the assembling spliceosomes, we examined the presence of Prp 17 in in vitro assembled complexes generated under various conditions. The conditions adopted were designed to stall and enrich for •assembly intermediates. A co-immunoprecipitation of the input precursor RNA and reaction intermediates revealed an early association of Prp 17 with the assembling Spliceosome prior to its catalytic activation. This association occurred in the A2-1 complex, which contains the U4/U6.U5 tri-snRNP along with the Ul and U2snRNPs. Prpl7 was found to associate with all subsequent complexes until the completion of catalytic transesterification reactions and possibly continue with the spliced-out introns complex (Fig. V.1B). Identification of two novel interacting partners of Prpl7 from a genome-wide two-hybrid screen Although several genetic interacting partners of PRP17 are known, none display a direct physical association with Prpl7. Knowledge of the proteins that Prpl7 interacts with can further the functional characterization of this protein and aid in deciphering its link to cell-cycle progression. A genome-wide screen for interacting partners using Prpl7 as bait was carried out in a two-hybrid system with a yeast genomic DNA-B42 activation-domain library (Gyuris et al., 1993). Through this screen we identified two interacting partners of Prpl 7- YOL078W, an essential gene and SGML The domain in the 1176 amino acid YOL078W protein responsible for interaction with Prpl7 was mapped to a 225 amino acid segment in the C-terminai region of this protein. The N-terminal region of the protein appears to exert a negative effect on the interaction with Prpl7. While YOL078w does not have any apparent role in pre-mRNA splicing, a majority of the cells arrest with small buds indicating a late Gl or early S phase arrest upon transcriptional shut-down of YOL078W. YOL078W has been independently characterized as AVOl, a component of the TOR complex, involved in nutrient sensing and cell size regulation (Loewith et al, 2002). Other reports show it tto be a component of a complex that interacts with Ceglp, a nuclear protein involved in mRNA capping (Gavin et al, 2002). We hypothesize that Prpl7 and Avol may exist in a dynamic nucleocytoplasmic complex possibly functioning in either cell-cycle regulation, RNA processing or both. Through this study we have Established the use of splicing-sensitive microarrays as tools for the characterization of pre-mRNA splicing factors. Simultaneous assessment of the effects on other cellular pathways was accomplished through expression profiling of all the intron-containing and intronless genes. Deciphered the differential dependence of pre-mRNA substrates on spliceosome factors at a global scale. Predicted the substrate-specificity of the second-step splicing factor, Prpl7, and verified some of these predictions in vitro. Gathered evidence for a possible splicing-independent effect of Prpl7 on the cell division cycle. Uncovered a novel function of Prpl7 in bud morphogenesis, as deduced from its synergistic genetic interaction with PHO85. Identified U2, U5 and U6 snRNPs as interacting partners of Prpl7 in both xtracts and in in vitro splicing reactions. Determined the point of coalescence of Prpl7 during spliceosome assembly to be at an early assembly stage soon after the entry of U4/U6.U5 tri-snRNP and prior to catalytic activation. Demonstrated continued Prpl7 association with the spliceosome beyond the completion of the splicing reactions. Identified Avolp and Sgmlp as novel interacting partners of Prpl7 through a genome-wide two-hybrid screen.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

<p>\abstract</p><p>Transcription and translation describe the flow of genetic information from DNA to mRNA to protein. Recent studies show that at a single cell level, these processes are stochastic, which results in the variation of the number of mRNA and proteins even under identical environmental conditions. Because the number of mRNA and protein in each single cell are actually very small, these variations can be crucial for cellular function in diverse contexts, such as development, stress response, immunological and nervous system function. Most studies examine the origin and effects of stochastic gene expression using computer simulations. My goal is to develop a theoretical framework to study activity-dependent gene expression using simplified models that capture essential features. </p><p>I have examined the dynamics of stochastic gene regulation in three contexts. First, I examine how fluctuations in promoter accessibility lead to "bursty" transcription, during which genes are turned "on" or "off" stochastically. I describe a mathematical formalism to represent bursty gene expression in a coarse-grained manner as a Markov process and derive a master equation for the time evolution of the probability distribution of the number of mRNA molecules. This allows us to examine how transcript number responds to time varying stimuli. This model forms a basic building block for understanding the signal transmission and noise of the transcription process to time varying inputs as would be sensed by cells in dynamic environments. In addition to synthesis, gene expression is subject to additional modes of regulation. One such mechanism that controls transcript numbers is by microRNAs (miRNAs), which pair with target mRNAs to repress protein production following transcription. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. I explore the functional roles of feedback regulation by miRNAs and show that protein fluctuations strongly depend on the mode of miRNA-mediated repression. I discuss the functional implications of protein fluctuations arising from miRNA-mediated repression on gene regulatory networks. Finally, I examine the impact of fluctuations on alternative splicing, which is a major source for proteomic complexity in higher eukaryotes. Although the proteins regulating alternative splicing have been extensively studied, little is known about how noise arising from the stochastic nature of alternative splicing contributes to the entire gene expression process. I explore the functional roles and noise properties of alternative splicing, focusing on the case of exon skipping and intron retention. I show that while the overall counts of the mRNAs of the two isoforms are independent and Poisson distributed, diffusion and binding of the splicing factors contributes to the variance in the abundance of the isoforms. </p><p>Noise in gene expression may be of particular relevance in the nervous system. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify neuronal excitability and connectivity, ultimately influencing higher order brain function. Finally, I examine the implications of our studies for activity dependent gene expression in the nervous system.</p> / Dissertation

L’splicing és el mecanisme pel qual els introns són eliminats del pre-mRNA per generar un trànscrit madur. Aquest procés és dut a terme per un complex macromolecular anomenat spliceosoma i requereix el reconeixement dels senyals d’splicing al pre-mRNA. Aquests senyals no són sempre identificats correctament, el que permet la producció de trànscrits diferents a partir d’un únic pre-mRNA mitjançant un procés anomenat splicing alternatiu. Aquest procés pot ser regulat mitjançant factors proteics específics o per altres mecanismes que alteren el reconeixement dels senyals d’splicing com l’estructura secundària adoptada pels pre-mRNAs. En aquesta tesi hem investigat els mecanismes de regulació de l’splicing en eucariotes mitjançant tècniques computacionals. També hem estudiat la relació existent entre les proteïnes que intervenen en la regulació de l’splicing i els senyals d’splicing, i com han coevolucionat en diferents espècies. Finalment, i tenint en compte les possibilitats que l’splicing alternatiu ofereix des del punt de vista evolutiu, també hem analitzat l’impacte de l’splicing alternatiu en l’evolució gènica. / Splicing is the mechanism by which introns are removed from the pre-mRNA to create a mature transcript. This process is performed by a macromolecular complex, the spliceosome, and involves the recognition of the splicing signals in the premRNA. These signals are not always perfectly recognized, which allows the production of different mature transcripts from a single pre-mRNA through a process called alternative splicing. This process can be regulated by specific protein factors or by other mechanisms that affect the recognition of the splicing signals, such as the secondary structure adopted by the pre-mRNA. In this thesis we have investigated the mechanisms of splicing regulation in eukaryotes using computational approaches. Moreover, we have also studied the relationship that exists between protein factors involved in splicing regulation and splicing signals, and how they have co-evolved across species. Finally, and considering the possibilities that alternative splicing can offer from the evolutionary point of view, he have also analyzed the impact of alternative splicing in gene evolution.

Em eucariotos, a formação das subunidades ribossomais envolve múltiplos fatores, responsáveis pelas etapas de maturação dos rRNAs e por sua associação a proteínas ribossomais. A via de processamento de pré-rRNA é bastante complexa e inclui várias etapas de modificação de nucleotídeos e clivagens endo- e exonucleolíticas. As modificações de nucleotídeos são dirigidas por snoRNPs, formados por snoRNAs e proteínas, que são divididos em duas classes gerais, de box H/ACA (pseudouridilação) e de box C/D (metilação). Dentre os snoRNP de box C/D está o U3, que embora apresente as seqüências características e se associe a proteínas desse grupo de snoRNPs, não dirige metilações no rRNA, mas sim as clivagens iniciais no pré-rRNA 35S. O snoRNA U3 de Saccharomyces cerevisiae é codificado por dois genes que contêm introns, snR17A e snR17B. Embora a via de montagem do snoRNP U3 ainda não tenha sido determinada com precisão, sabe-se que algumas proteínas do core de box C/D ligam-se ao pré-snoRNA U3 co-transcricionalmente, afetando o splicing e o processamento da extremidade 3´ deste snoRNA. A proteína Cwc24p, cuja caracterização funcional foi o objetivo deste trabalho, foi isolada em nosso laboratório interagindo com Nop17p, um fator de montagem dos snoRNPs de box C/D. Cwc24p possui um domínio RING conservado e foi isolada previamente em um complexo contendo o fator de splicing Cef1p. Os resultados aqui obtidos mostram que, de maneira condizente com os dados de interação, Cwc24p é uma proteína nuclear e sua depleção leva ao acúmulo do pré-snoRNA U3, o que acarreta uma diminuição da velocidade de processamento do pré-rRNA 35S. O modelo aqui proposto prevê o recrutamento de Cwc24p para o pré-snoRNA U3 por Nop17p, onde atua como um fator de eficiência do splicing. Estes resultados levaram à conclusão de que Cwc24p está envolvida no splicing do pré-snoRNA U3, afetando indiretamente o processamento do pré-rRNA. / In eukaryotes, the ribosome biogenesis involves a large number of factors, that are responsible for the rRNAs maturation and for their association with ribosomal proteins. The pre-rRNA processing pathway is very complex and includes many steps of nucleotide modifications and endo- and exonucleolytic cleavage reactions. The nucleotide modifications are directed by snoRNPs that are formed by snoRNAs and proteins, divided in two major groups, of box H/ACA (which direct pseudouridilation), or of box C/D (methylation). Although the snoRNP U3 presents the snoRNA sequences and the proteins characteristics of box C/D class, it is not involved in methylation, but rather in the early cleavages of pre-rRNA 35S. U3 snoRNA is transcribed from two intron-containing genes in yeast, snR17A and snR17B. Although the assembly of the U3 snoRNP has not been precisely determined, at least some of the core box C/D proteins are known to bind pre-U3 cotranscriptionally, thereby affecting splicing and 3\'-end processing of this snoRNA. We identified the interaction between the box C/D assembly factor Nop17p and Cwc24p, a novel yeast RING-finger protein which had been previously isolated in a complex with the splicing factor Cef1p. Here we show that, consistently with the protein interaction data, Cwc24p localizes to the cell nucleus, and its depletion leads to the accumulation of both U3 pre-snoRNAs. U3 snoRNA is involved in the early cleavages of 35S pre-rRNA, and the defective splicing of pre-U3 detected in cells depleted of Cwc24p causes the accumulation of the 35S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre-U3 snoRNA splicing, indirectly affecting pre-rRNA processing.

Os fagócitos contêm uma nicotinamida adenina dinucleotídeo fosfato (NADPH) oxidase associada à membrana, que gera superóxido e outros reativos intermediários do oxigênio. Defeitos nesta oxidase em seres humanos resultam na doença granulomatosa crônica (DGC). Mutações próximas aos sítios de splicing que interferem com o processamento do RNA mensageiro, acarretando deleção de um ou mais exons, são cada vez mais freqüentes na literatura científica, nesses casos, os mecanismos moleculares que levam a DGC nem sempre são totalmente esclarecidos, assim como o efeito do IFN-g, seja sobre o processamento da mensagem ou estabilidade dos transcritos. Com base nessas informações o objetivo geral deste trabalho é investigar o efeito do IFN-g sobre a regulação do sistema NADPH oxidase fagocítico humano. Através dos resultados obtidos, não se pode constatar melhora na produção de ânions superóxido após o tratamento com IFN-g em pacientes com defeito de splicing, no entanto detectou-se aumento da expressão do gene CYBB através de PCR convencional e através de real-time PCR além de um aumento na marcação de proteínas do spliceossoma através do FAN. / Phagocytes have a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) associated to plasmatic membrane that generates superoxide and other oxygen reactive intermediates. Defects on this oxidase in humans result in a disorder called Chronic Granulomatous Disease (CGD). Mutations next to splicing sites that interfere with the mRNA processing leading to deletion of one or more exons, are even more frequent on scientific literature, in these cases, the molecular mechanisms causing CGD are not always completetly clear, as well as the effect of IFN-g on the mRNA processing or on the stability of transcripts. Based on this information our aim is to investigate the effect of IFN-g on the regulation of the human NADPH oxidase phagocyte system.. With the obtained results it wasnt possible to see an increase in anion superoxide production after the IFN-g treatment in patients with splicing defects, however it was detected an increase on the expression of CYBB gene by conventional and real-time PCR besides an increase in the marking of spliceossomal proteins by FAN.