Stable hybrid containing haploid genomes of two obligate diploid Candida species

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

Identification of heat shock factor binding protein in Plasmodium falciparum

Background: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum. Methods: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum. Results: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite. Conclusions: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

Rad51-Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A(CaCse4) levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A(CaCse4) in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A(CaCse4) levels at the programmed stall sites of early replicating centromeres.

Genomic mapping of cAMP receptor protein (CRPMt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRPMt) at endogenous expression levels using a specific alpha-CRPMt antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRPMt binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRPMt during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRPMt-binding site represented only a minor portion of this transcriptional reprogramming with similar to 19% of those representing transcriptional regulators potentially controlled by CRPMt. The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRPMt can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRPMt-binding sites.

Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

Introduction: Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed `mammospheres'. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression. Methods: Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. Results: In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser(116) phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser(116) phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser(116) residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser(116) phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo. Conclusions: Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.

CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth. 10.1111/(ISSN)1469-8137

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90 degrees light scattering and FtsZ polymer pelleting assays. The gamma 32P-GTP synthesised by NDK from GDP and gamma 32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound P-32-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knock-down studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase

We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment.

DNA methylation analysis of phenotype specific stratified Indian population

Background: DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. Methods: Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. Results: Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). Conclusion: Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.

Functional genomic studies of the structure and regulation of eukaryotic transcriptomes

The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.

The first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.

The second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.

The third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.

The fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).

The last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.

Análise de uma heme oxigenase funcional em Trypanosoma cruzi

O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação no hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglobina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito na tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação por PCR do gene da HO-1 utilizando oligonucleotídeos desenhados no genoma de T. cruzi. Os dois pares de oligonucleotídeos utilizados nao foram capazes de amplificar uma sequência equivalente a uma HO. Em seguida, utilizamos a técnica de imunoprecipitação, seguida de immunoblotting, com anticorpo anti-HO-1, com objetivo de concentrar a proteína alvo, e observamos um aumento significativo do imunocomplexo nas células tratadas com heme 300 mM, cerca de 2 vezes em relação ao controle. Dando seguimento à tentativa de identificação da HO-1 de T. cruzi, utilizamos a técnica de espectrometria de massa a partir de eletroforese unidimensional, que mostrou uma grande alteração do perfil protéico na presença de heme, mas futuros experimentos são necessários, como eletroforese 2D, para a identificação da proteína alvo