Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 281-328).

In order to study the behaviour of pathogenic viruses in groundwater, several tracer experiments were performed at chalk aquifer sites using three different bacteriophage. Laboratory experiments were performed in parallel to investigate the efficacy of the bacteriophage as models of pathogenic virus behaviour. The high titres per ml of bacteriophage available for the initial inoculum and the simplicity of the assay permitted the detection of a pattern of bacteriophage recovery from three different groundwater sites. The greatest percentage of original inoculum recovered was 1.9 percent. Bacteriophage were observed to migrate 1km over a five month period and 366m within just over 3.5 hours. The laboratory experiments explored: i) decay rates of viruses suspended within chalk groundwater; ii) adsorption of viruses to chalk in batch studies and; iii) virus behaviour in chalk columns representing natural aquifer conditions. Three human enteric viruses were used: poliovirus 1, echovirus 1 and coxsackievirus B5. In addition the simian rotavirus (SA-11), and a group of bacteriophage were tested. The laboratory experiments indicated that the bacteriophage included in this study survived longer than the animal viruses in groundwater and that all viruses adsorbed well to chalk. In the simulated aquifer conditions all viruses investigated percolated through a chalk column at a similar rate and with a similar pattern. No differences in behaviour between the viruses, whether in laboratory or field experiments, was found which could be directly attributed to their size or shape. A mathematical model was used which included coefficients for adsorption and decay observed in the laboratory experiments and certain other parameters found in one of the field sites under investigation. Those factors considered and included in the model were shown to be sufficient to produce the same behaviour pattern as that observed in the field. This confirmed that the main factors influencing viral migration in natural conditions have been considered in this investigation. Bacteriophage were found to be good tracers for determining certain properties of chalk aquifers and a potential model of the behaviour of human enteric viruses in groundwater.

Bovine coronavirus (BCV) has been previously detected in the enteric and respiratory tracts of cattle and is specifically associated with enteritis and diarrhoea in neonatal calves. Two diagnostic tests, an ELISA and an immunogold EM technique for the detection of BCV in faeces, were developed, optimised and compared with direct EM, immunosorbent EM and haemadsorption-elution-haemagglutination (HEHA). The immunogold EM technique was found to be the most sensitive test followed by the ELISA, HEHA, immunosorbent EM and direct EM. An IF test for detecting BCV in the respiratory tract and a neutralization test for quantifying anti-BCV antibody titres in serum and milk were also developed. Using the immunogold EM technique BCV was demonstrated in 39 of 123 field samples of bovine diarrheic faeces. From 25 of these samples 2 isolates were successfully adapted to grow in HRT 18 cells following initial isolation in bovine fetal tracheal organ culture. These, and three other strains of BCV and a human coronavirus (HCV) strain obtained from other laboratories, were compared in immunofluorescence (IF), haemagglutination inhibition (HAI) and neutralization tests. Polyclonal antisera against these 6 viruses were raised in rabbits. No significant differences between viruses were detected by IF incorporating homologous and heterologous antisera but HCV could be distinguished from the bovine coronaviruses in a cross neutralization test. In this test all BCV isolates were determined to be of one serotype. In the HAI test however, the HCV strain was distinguishable from the 5 BCV strains and differences between the BCV strains were shown. Two monoclonal antibodies prepared against one of the BCV strains distinguished the HCV from the BCV strains in all three tests. These monoclonal antibodies did not distinguish between the 5 BCV strains in the IF or HAI test but did so in the neutralization test. The various strains were also compared at the molecular level using the Western blotting technique. This technique showed no significant differences between the molecular weights or serological reactivity of the structural proteins of these strains. Experimental infection of a gnotobiotic calf with BCV resulted in diarrhoea and fever, but no clinical evidence of disease was seen when 4 conventionally reared colostrum fed calves and 4 gnotobiotic lambs were similarly infected. The oral infection of suckling mice with BCV produced diarrhoea in some animals but a full investigation is required to optimise this model. A prospective epidemiological survey on one farm was carried out and showed Cryptosporidium and BCV to be associated with diarrhoea. Additionally this survey showed that the detection of BCV in the respiratory tract was associated significantly with respiratory symptoms.

The proteins encoded by foot-and-mouth disease virus are expressed in the form of a polyprotein, which is processed by virus-encoded proteases to yield the mature viral proteins. The focus of this thesis is the 18 amino acid 2A region of foot-and-mouth disease virus (FMDV), which is (together with the first proline residue of 2B) capable of mediating a primary cleavage at its own carboxy-terminus, between the structural and replicative viral proteins. It was proposed that the 2A region performed this event via a novel proteolytic mechanism. Vectors encoding the FMDV 2A region, in frame, between two foreign gene sequences were constructed to allow the 2A region to be investigated in isolation from the rest of the virus proteins using in vitro translation systems. Detailed quantitative densitometric analyses of the translation products were carried out, which suggested non-stoichiometric expression of the two cleavage products. Single and multiple site-directed mutations were made to the 2A sequence and the activities of the resultant 2A regions determined to establish the identity of functional amino acid residues. The effect of the surrounding foreign protein sequence and the inclusion of progressively longer wild-type sequences prior to 2A was also examined. The carboxy-termini of cardiovirus 2A regions show significant sequence similarity to the FMDV 2A region and are known to mediate a similar primary cleavage. The cleavage activities of the carboxy-termini of cardiovirus 2A regions were compared to that of the FMDV 2A region and found to equally capable of mediating cleavage. The activity of FMDV 2A region was tested in prokaryotes and was found to be inactive. These studies indicated that the 2A cleavage was not a proteolytic cleavage, but more likely a translational effect. A new model for 2A-mediated activity is presented.

A series of P4 (Cbz and t-Boc) N-protected potential substrates and inhibitors, containing the P1 to P4 substrate recognition sequence of the type 2 adenovirus proteinase (Leu-Ala-Gly-Gly) were prepared by solution phase peptide coupling techniques and tested for activity against the proteinase. The potential substrates contained the amide and ester moieties at the P1 carbonyl position and the potential inhibitors contained the alcohol, acid, bromide, aldehyde, ketone, dimethylacetal, nitrile, alkenic, malonyl and epoxysuccinate moieties at the P1 carbonyl position. The esters, the t-Boc urethane and the p-nitroanilide moieties were substrates for the proteinase and the acid and the amides did not bind to the proteinase. Preliminary results show that the other inhibitors were mostly noncompetitive inhibitors for the adenovirus proteinase with approximate Ki's between 15 and 200 μmol dm-3. The test results indicate that the amides must contain a carbonyl group at P2' to bind to the proteinase; the loss of the P1' amine product is the rate limiting step for the hydrolysis of a substrate by the adenovirus proteinase; the P acid product leaves before the P' amine product, which is in complete contrast to classical cysteine proteinases such as papain; little protonation of the P1'amide nitrogen or the P1 carbonyl oxygen of the adenovirus proteinase-substrate complex occurs before the nucleophilic attack on the P1 carbonyl carbon of the adenovirus proteinase-substrate complex.

The development of a cell-free system in which adenovirus DNA synthesis can be initiated in vitro by using the viral genome or plasmids containing the origin of replication as template has led to the identification of the sequences important for origin function and the isolation and purification of the proteins required for viral DNA replication. In vitro studies on adenovirus types 2 and 5 have shown that their replication requires the formation of a large nucleoprotein complex. This is composed of three virally encoded proteins: adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, and two cellular proteins nuclear factor I and nuclear factor III. While the presence of DNA helicases in other eukaryotic DNA replication systems have been well characterised, this was not the case for adenovirus DNA replication. Initial attempts to identify DNA helicase activity associated with any of the adenovirus replication proteins were unsuccessful. However, a novel DNA unwinding activity was found associated with the DNA binding protein (Ad.DBP). We examined the interaction of DBP with partial DNA duplexes and demonstrated that it could displace oligonucleotides annealed to single-stranded M13 DNA. In addition, DBP could also unwind small fragments of fully duplex DNA. Unlike a DNA helicase, DBP promoted DNA unwinding was nucleoside-5'-triphosphate and Mg2+ independent and exhibited no directionality. The activity required saturating amounts of DBP and was both efficient and cooperative in nature. The helix-destabilising activity was shown to be situated in the C-terminal domain of the protein. These properties suggest a role for DBP in DNA replication in which DBP destabilises duplex DNA during origin unwinding and replication fork movement. The second part of the thesis dealt with the characterisation of the putative "active site" of the adenovirus DNA polymerase. This experimental approach was prompted by data from earlier studies which indicated that DBP could increase the processitivity of the polymerase as well as its sensitivity to nucleotide analogue inhibitors. The "active site" was labelled with pyridoxal-5'-phosphate (PLP), a substrate binding site directed reagent for DNA polymerases. Treatment of Ad.5 DNA polymerase with PLP followed by reduction of the enzyme-PLP adduct resulted in irreversible inactivation of the polymerase activity while the 3'-5' exonuclease associated with Ad.5 DNA polymerase was minimally affected. Substrate protection studies indicate that PLP inhibition is complex. Neither template-primer nor substrate dNTP alone showed any protective effect from PLP mediated inhibition. However, the presence of both template -primer and complementary dNTP significantly protected against PLP inhibition. Comparative tryptic mapping of labelled enzyme, modified in the presence and absence of substrates by PLP reaction, on a C-18 reverse phase column, indicated the protection of one peptide from pyridoxylation in the presence of substrates. Amino acid sequence analyses found no sequence to be present in this peak.

The development of systems in which adenovirus DNA can be replicated in vitro has led to the elucidation of the sequences essential for origin function and to the identification of the proteins required for viral DNA replication. Much of the information currently available has been derived from investigations carried out using adenovirus types 2 and 5 which in addition to the viral proteins, adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, require cellular proteins nuclear factor I and nuclear factor III for efficient initiation of DNA replication. In contrast adenovirus type 4 replicates Its DNA efficiently without these cellular proteins. Correspondingly its minimal origin of replication is remarkably simple in structure, consisting of only the terminal 18bp of the adenovirus genome ('the core sequence'). The effect of point mutations in the core region on adenovirus type 4 DNA replication in vitro was investigated and it was found that mutations within two discrete domains had a married deleterious effect on initiation of DNA replication. The crude Ad4 infected cell extracts initially used for in vitro DNA replication were fractionated and it was found that only four detectable proteins, three of which were identified as viral DNA polymerase, precursor terminal protein and DNA binding protein gave efficient DNA replication in vitro and furthermore behaved similarly to unfractionated infected cell extracts in the presence of template which contained point mutations. To examine a possible role of the core region of the origin as containing sites for specific interactions with viral replication proteins, purified adenovirus type 5 precursor terminal protein and DNA polymerase were assayed for their ability to recognise the terminal 1-18 sequence. It was found that both proteins independently and as a heterodimer bound specifically to a sequence corresponding to the core origin of replication, suggesting that sequences within this region are important for localisation of DNA replication proteins at the origin via a sequence specific DNA-protein interaction.

Objetivos – Descrever as características clínicas de crianças entre 1 e 12 meses hospitalizadas com diagnóstico de bronquiolite viral aguda (BVA), nos primeiros dias de internação, e verificar se o tempo de dessaturação de oxigênio (TD) tem valor prognóstico nesses pacientes. Metodologia – Estudo de coorte realizado de maio a outubro de 2001 com 111 pacientes entre 1 e 12 meses de idade internados no Hospital da Criança Santo Antônio, de Porto Alegre (RS), com diagnóstico de BVA na admissão, com saturação transcutânea de oxigênio da hemoglobina (SatHb) menor que 95% e em oxigenoterapia por cateter extranasal há menos de 24 horas. A gravidade foi verificada através do tempo de internação, tempo de oxigenoterapia e tempo para saturar 95% em ar ambiente (desfechos). Foram realizadas avaliações clínicas duas vezes ao dia (manhã e tarde), durante o período em que o paciente necessitou de oxigênio suplementar (até atingir saturação transcutânea de oxigênio de 95% em ar ambiente), com limite de dez avaliações. Os pacientes tiveram o oxigênio adicional retirado. Foi verificado, então, o tempo necessário para a saturação decrescer até 90% (TD90) e 85% (TD85), limitando-se a medida em no máximo cinco minutos. Foi constituído um escore de gravidade com os sinais clínicos anotados. Utilizou-se o teste do qui-quadrado ou teste exato de Fischer para comparar entre si os grupos de variáveis categóricas e o teste t ou MannWhitney para variáveis numéricas. Foi utilizada a correlação de Spearman para avaliar associações entre variáveis contínuas de distribuição assimétrica (escore de gravidade, tempo de internação, tempo de oxigenoterapia total e tempo para saturar acima ou igual a 95% em ar ambiente). Considerou-se alfa crítico de 5% em todas as comparações, exceto nas correlações em que foi utilizada a correção de Bonferroni para comparações múltiplas (30 correlações: p= 0,002; 10 correlações: p= 0,005). Os dados relativos ao peso e estatura para a idade foram digitados e analisados no programa específico do EpiInfo que utiliza o padrão NCHS (EpiNut). Resultados – Houve leve predomínio do sexo masculino (54%), predominância de idade inferior a quatro meses (61,3%), prevalência maior nos meses de junho e julho, freqüência elevada de história de prematuridade (23%) e de baixo peso de nascimento (14%). As manifestações clínicas prévias à hospitalização (falta de ar, chiado no peito, febre e parar de respirar) ocorreram, na sua maioria, nos três dias anteriores. Da população estudada, 45% tinha história de sibilância prévia, a maioria com um ou dois episódios relatados (31,5%). Esses pacientes foram analisados separadamente e tiveram resultados semelhantes ao grupo com BVA. A freqüência de desnutrição moderada e grave, excluídos os pacientes com história de prematuridade, foi de 26 pacientes (23%). Todos os pacientes utilizaram broncodilatador inalatório; 20% do grupo com BVA receberam corticosteróides sistêmicos e 47% de toda população, antibióticos. A mediana do uso de oxigênio em pacientes com BVA foi de 4,4 dias (IIQ 70,2-165,2) e o tempo de oxigenoterapia até saturar 95% em ar ambiente foi de 3,4 dias (IIQ 55-128). A mediana do tempo de internação hospitalar foi de 7 dias (IIQ 5-10,5) entre os pacientes com BVA; neste aspecto, apresentou diferença (p = 0,041) em relação ao grupo com sibilância prévia, que teve um tempo de internação mais longo (9 dias, IIQ 5-12). Observou-se pouca variabilidade clínica no período estudado, através da aplicação do escore clínico. Não se encontraram correlações estatisticamente significativas entre os escores clínicos e os TDs com os desfechos. Conclusões – Os TDs como elementos auxiliares na avaliação de pacientes em oxigenoterapia não foram clinicamente úteis neste estudo. É possível, no entanto, que, avaliando pacientes com maiores diferenças clínicas entre si, essas aferições possam mostrar-se importantes. / Objective – To describe the clinical characteristics of 1 to 12-month-old infants diagnosed with acute viral bronchiolitis (AVB) in the first days of hospitalization as well as to check if the oxygen desaturation time (DT) has prognostic value in such patients. Methodology – Cohort study done from May to October 2001 with 111 patients of 1 to 12 months of age at the Santo Antônio Children’s Hospital, Porto Alegre (RS), with AVB diagnosis upon admission, oxygen transcutaneous saturation of hemoglobin (HbSat) lower than 95% and in oxygen therapy through external nasal catheter for less than 24 hours. Severity was checked through length of stay , duration of oxygen therapy and time to reach 95% saturation in room air. Clinical checkups were done twice a day (morning and afternoon) during the time the patients needed additional oxygen (until oxygen transcutaneous saturation of 95% in room air was achieved), with a limit of ten checkups. The additional oxygen was removed from patients, and oxygen saturation checking was maintained. To check the desaturation time (DT90 and DT85), oxigen therapy was suspended for a maximum of five minutes. A severity score was developed with the recorded clinical signs. The χ2 Test - or Fisher Exact Test – was used in order to compare the groups of categorical variables, and Test t – or Mann-Whitney – for numerical variables. The Spearman correlation was used in order to evaluate associations in continuous variables of asymmetric distribution (severity score, lenght of stay (LOS), duration of oxygen therapy, and time to reach 95% saturation in room air. Results – There was a slight higher number of males (54%), the majority were under 4 months (61,3%), higher prevalency in June and July, high frequency of prematurity (23%) and of low birth weight (14%). Most clinical manifestations prior to hospitalization (shortness of breath, wheezing, fever and apnoea) had happened within three days of admission. Out of the population studied, 45% had had previous wheezing history, most of them with one or two reported episodes (31,5%). Such patients were analyzed separately and had similar results to the group with AVB. The frequency of moderate and severe malnutrition, except for the premature babies, was of 26 patients (23%). All patients had bronchodilators; 20% of the AVB group had given systemic corticorteroids, and 47% of the population were given antibiotics. The median of oxygen use in patients with AVB was 4,4 days (IQI 70,2–165,2) and duration of oxygen therapy until 95% saturation in room air was 3,4 days (IQI 55-128). The median lenght of stay was 7 days (IQI 5-10,5) in patients with AVB; in this aspect there was a difference (p=0,041) in relation to the group with previous history of wheezing, who had a longer LOS (9 days, IQI 5-12). Little clinical variability was observed in the period studied through the application of clinical score. There were no significant statistic correlations between the clinical scores and the DTs and the outcomes. Conclusion – DTs as an aid in the examination of AVB patients on oxygen therapy were not clinically useful in this study. It is possible, however, to achieve better results in the examination of patients with greater clinical differences.

Bronquiolite Viral Aguda (BVA) é uma doença de alta prevalência no Rio Grande do Sul e causa um importante número de hospitalizações em crianças menores de um ano de idade. Os pacientes com maior gravidade, nos três primeiros dias de internação, podem evoluir para insuficiência ventilatória e necessitarem oxigênio ou até ventilação mecânica, como forma de tratamento. Com o objetvo de identificar precocemente os episódios mais graves, investigaram-se fatores prognósticos através de sinais clínicos e laboratoriais, durante a hospitalização de crianças com BVA. Estudou-se uma coorte de 213 crianças menores de um ano com BVA admitidas no Hospital da Criança Santo Antônio de Porto Alegre. Esses pacientes foram seguidos desde a hospitalização, nos três dias subseqüentes, na alta hospitalar e até 30 e 60 dias após. Caracterizou-se a necessidade de oxigênio no terceiro dia ou de ventilação mecânica nos três primeiros dias de hospitalização, como critérios de gravidade. As hospitalizações ocorreram, predominantemente, nos meses de julho a setembro, e a maior parte das crianças (60%) apresentava idade inferior a quatro meses. Dentre as características sócio-econômicas estudadas, observou-se que 50% das famílias tinham renda mensal menor que três salários mínimos e 18% viviam em residências aglomeradas. Das 213 crianças acompanhadas até a alta hospitalar, 61(29%) necessitaram de oxigênio no terceiro dia, e 12(6%), ventilação mecânica nas primeiras 72 horas. Verificou-se que 17% dos pacientes apresentavam história de prematuridade, 24% estavam sendo amamentados quando hospitalizaram e 12% nunca haviam mamado no peito. Cerca de 35% das famílias referiram história de asma brônquica. As principais características clínicas na hospitalização foram: freqüência respiratória maior que 60(67%), presença de sibilos(76%) e estertores crepitantes(57%) à ausculta pulmonar e tiragem subcostal (56%). Entre os sinais investigados, assodaramse significativamente com nlaior gravidade: frequência respiratória maior que 70, enchimento capilar lento, cianose de extremidades e tiragem supraesternal. A saturação transcutânea de oxigênio da hemoglobina inferior a 91% e atelectasia ao exame radiológico de tórax, mostraram-se associadas significativamente com maior gravidade. Entre os fatores prognósticos, identificou-se o risco relativo associado a maior gravidade: idade inferior a 4 mes{~s (RRl~7), peso de nascimento inferior a 2500g (RR 2,3) e desnutrição grave(RR 2,0). Constituíram-se escalas de gravidade através de análise discriminante incluindo-se os seguintes itens: idade inferior a 3 meses, prostração, batimento de asas do nariz, freqüência respiratória maior ou igual a 70, tiragem (subcostal, intercostal e supraesternal), saturação de oxigênio menor ou igual a 90%, uso de oxigênio, internação em UTI e uso de ventilação mecânica. Através do somatório de cada um dos itens, resultaram os escores de gravidade. Os escores foram dicotomizados em menores ou iguaisl a 3 e maiores que 3 (mais graves). No seguimento após a alta hospitalar, observou-se que a Inaioria dos pacientes que compareceram apresentou episódios de sibilância e que foi elevado o número de reinternações (26% aos 60 dias). / Acute viral bronchiolitis(AVB) has a high prevalence in Rio Grande do Sul. It accounts for a high number of hospital admissions in infants. The patients with a more severe disease, in the first three days of hospitalization may develop respiratory failure and might need oxygen or mechanical ventilation . The aim of this study was to investigate prognostic factors (clinicaI signs and laboratory tests) in hospitalized infants with AVB. The research conducted was a cohort study of 213 infants with AVB who were admitted to the Hospital da Criança Santo Antônio, Porto Alegre. These patients were followed up from the admission, in the three first days, to their discharge and at 30 and 60 days after admission. Severity criteria were defined such as need of oxygen in the third day of admission or mechanical ventilation in the first three days. The admissions occurred predominantly from July to September and the majority (60%) were infants under four months of age. It was observed that 50% of the families had monthly wages below three minimum saIaries and 18% lived in crowded homes. Sixty one (29%) of the children needed oxygen in the third day of admission and 12 (6%) were put on mechanical ventilation. It was observed that 17% of the patients had a past history of prematurity, 24% were being breast fed at admission and 12% had never been breast fedo Around 35% of the families had a past history of bronchial asthma. The main clinicaI characteristics were: respiratory rate above 60 (67%), wheezes (76%), crepitations (57%) and subcostal retraction (56%). The following findings were significantly associated to severity : respiratory rate above 70 mpm, peripheric cyanosis, low capillary filling, supraesternal retraction, transcutaneous oxygen saturation below 91%, atelectasis at the chest X-ray. Among the prognostic features, some were identified as presenting high relative risk associated to severity: Age under 4 months (RR 1,7), birth weight below 2500 g (RR 2,3) and malnutrition (RR 2,0). Severity scales have been developed using discriminant analysis with the following items: age under 3 months, prostration, flaring of the alae nasi, respiratory rate above 70 mpm, retractions (subcostal, intercostal and supraesternal), transcutaneous oxygen saturation, need of oxygen, admission in intensive care unit and use of mechanical ventilation. Severity scores resulted from the addition of the value attributed to each of the items (O or 1). They were dichotomized in above (more severe) and below or equal to 3. At the follow up, after the discharge it was observed that the majority of the children who had been seen at thirty and sixty days had wheezing episodes and there was a high rate of re admissions (26% at 60 days).

In multicellular organisms, cell growth and differentiation is controlled in part by apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Resistance to Fas-mediated apoptosis is regulated through the production of a soluble Fas isoform (sFas), created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔ6) that can bind FasL and block apoptosis. Long noncoding RNAs (lncRNAs) are >200-nucleotide sequences that are important regulators of cellular programs. However, their role in erythropoiesis is just beginning to be appreciated with studies limited to murine systems. I studied the potential role of lncRNAs during human red blood cell development using RNA from cultured CD34+ purified from fetal liver (FL), cord blood (CB), or adult bone marrow (BM) to screen 82 documented lncRNAs. This screen revealed that the Fas Anti-sense (Saf) was consistently increased during maturation and levels high for BM compared to FL or CB. Next, I characterized the regulatory sequence of Saf and by In silico analysis identified canonical binding sites for the erythroid-specific transcription factors KLF1 and GATA1. Chromatin immunoprecipitation (ChIP) assays confirmed binding of both factors to their target sequences and luciferase reporter constructs revealed synergistic activity evidenced by increase in luciferase expression relative to controls. Genome wide expression analysis using cells with overexpression of Saf showed no effect on global gene transcription, suggesting Saf function at post transcriptional levels. Saf was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. Using a combination of biochemical assays, overexpression and knockdown studies, I showed how this occurs. Cell fractionation and RT-PCR demonstrated that Saf is localized in the nucleus. I found that Saf directly interacted with Fas pre-mRNA to form RNaseA-resistant double-stranded RNA intermediates at regions that flank Exon6. Post-transcriptional function of Saf was confirmed by qRT-PCR demonstrating significantly increased levels of sFas for Saf overexpressing cells. Enrichment for sFas RNA coincided with reduced Fas on the cell surface and increased sFas protein levels when conditioned supernatants were assayed by ELISA. Conversely, siRNA-mediated knockdown of Saf significantly reduced sFas production compared to non-targeting siRNA controls. Saf-interacting proteins were identified by mixing in vitro transcribed and biotin-labeled Saf RNA with nuclear lysates followed by mass spectrometry analysis. This screen identified human splicing factor 45 (SPF45) which has a known role in Fas pre-mRNA alternative splicing. Specific SPF45/Saf interaction was confirmed by RNA pulldown and western blot with SPF45-specific antibodies and the ability to detect sequences for Saf, Fas and sFas by RT-PCR of RNA that immunoprecipitated with SPF45. SPF45 knockdown decreased sFas transcripts and this reduction corresponded to limited production of sFas and increased sensitivity to Fas-mediated apoptosis when cells were exposed to the Fas-activating antibody CH11. Importantly, overexpression of Saf in SPF45 knockdown cells failed to rescue production of sFas supporting the hypothesis that Saf and SPF45 co-participate in modulating Fas pre-mRNA splicing. Protein phosphorylation modulates the interaction of the splicing factors with RNA. The effect of phosphorylation on the Saf-SPF45 interaction was evaluated using stable cell lines expressing a myc-tagged SPF45 protein or versions modified to introduce alanine in place of threonine 71 (T-71-A) or serine 222 (S-222-A) to prevent phosphorylation. Mutation in S-222-A reduced the interaction of SPF45 with Saf. I conclude that Saf interacts with Fas pre-mRNA at sequences that flank exon 6 and recruits phosphorylated SPF45 as mechanism to recognize exon 6 and allow for alternative splicing of Fas Pre-mRNA. Collectively, these studies reveal a novel mechanism to regulate apoptosis that may be responsible for cell proliferation and drug resistance.