Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.

Ixodes ricinus est l’espèce de tique la plus abondante et ayant la plus vaste répartition géographique en Europe. Elle est le vecteur de nombreux agents pathogènes d’importance en santé publique et vétérinaire. Le remplacement des acaricides générant pollution environnementale et apparition croissante de résistances requiert le développement urgent de nouvelles stratégies de lutte efficaces contre les tiques et les agents pathogènes qu’elles transmettent. La découverte de telles stratégies passe nécessairement par une meilleure connaissance des interactions entre les tiques, leurs hôtes et les agents pathogènes transmis. La salive de tique, à l’interface de ces interactions, est un fluide essentiel pour ces arthropodes et possède notamment des propriétés protéolytiques, anticoagulantes, immunomodulatrices, analgésique, et anti-inflammatoires qui permettent à la tique de réaliser ses repas sanguins extrêmement longs. Afin de comprendre les mécanismes moléculaires impliqués dans la transmission des agents pathogènes et pour identifier de possibles candidats vaccinaux contre I. ricinus, une étude transcriptomique comparative entre des glandes salivaires infectées et non infectées par la bactérie Bartonella henselae a été antérieurement réalisée. Le transcrit le plus surexprimé suite à cette infection était IrSPI, un inhibiteur de sérine protéase de la famille des Kunitz. Les analyses fonctionnelles par ARN interférence ont montré l’implication de ce gène dans le gorgement et de l’infection des glandes salivaires par B. henselae. Ainsi, les travaux de thèse présentés ici ont concerné l’analyse structurelle, biochimique et fonctionnelle de IrSPI en tant que molécule impliquée dans les interactions tick-hôte-pathogène. Le premier objectif était de définir la structure et la séquence du gène IrSPI mais, malheureusement, bien que nos résultats aient permit des avancés sur cette question, nous n'avons pu obtenir la totalité de sa séquence. Dans un second temps, la dynamique d’expression d’IrSPI a été évaluée au cours du gorgement et de l’infection des tiques par différents agents pathogènes, montrant que son expression est induite par le repas sanguin, par des agents transmis par la tique mais pas par Escherichia coli, bactérie non transmise. De plus, nos résultats ont montré l’expression de IrSPI dans plusieurs organes de la tique, suggérant son implication dans diverses fonctions au sein de ce vecteur. Parmi elles, la mise en évidence d'une injection, par la salive, de la protéine à l'hôte vertébré nous a permis d'envisager un rôle sur les réponses de l'hôte à la piqûre de tique. Nos résultats n’ont montré aucune implication dans la voie extrinsèque de la coagulation ni dans la fibrinolyse, ni dans l’angiogenèse. En revanche, ils ont démontré que IrSPI inhibe la prolifération des lymphocytes TCD4+ sous stimulation monogénique quand chez des lymphocytes B non stimulés IRSPI, il induit une hausse de la prolifération. De plus IrSPI a montré une action négative significative sur la production de la majorité des cytokines et chimiokines pro-inflammatoires produites par les macrophages et les splénocytes. Ainsi, IrSPI, correspond à un des composants salivaires d’I. ricinus lui permettant de moduler la réponse immune de l’hôte pour lui permettre de prélever son repas sanguin tout en favorisant la transmission des agents pathogènes. Enfin, des résultats préliminaires dans l'identification des interactants de IrSPI à la fois chez la tique et l’hôte vertébré ouvre de nombreuses voies de recherche quant à la compréhension de ses fonctions. / Ixodes ricinus tick species, the most abundant and widespread tick in Europe, is an important vector of pathogens affecting both animal and human health. To replace the use of acaricides that generate environmental contamination and resistances, new environmentally sustainable approaches providing broad protection against ticks and tick-borne pathogens (TBP) are urgently needed. Such development requires improved understanding of the biology of ticks and more particularly of their interactions with vertebrate hosts and TBP. Tick saliva is an essential biofluid for ticks, as its proteolytic, anticoagulant, immunomodulatory, analgesic and anti-inflammatory activities allow ticks to acquire their blood meal under optimal conditions. Moreover, injection of saliva during blood feeding represents the principal route by which TBP are transmitted to the host. To understand the molecular mechanisms involved in TBP transmission, as well as to identify putative vaccine candidates against I. ricinus, salivary glands from bacteria infected and uninfected ticks were previously compared by high throughput transcriptomics. The most up-regulated transcript following infection was IrSPI, which belongs to the Kunitz/BPTI inhibitor family. Functional analyses via RNAi knockdown experiments revealed that IrSPI enhances both blood feeding and bacterial burden in the salivary glands. This present PhD work concerns then the structural, biochemical and functional characterization of IrSPI as a molecule involved in tick-host-pathogen interactions. Our aim was first to define the structure of IrSPI gene but, unfortunately, while our results have led to progress on this issue, we have not been able to get the full sequence. Then, the dynamic of IrSPI expression was evaluated during both tick feeding and colonization of ticks by pathogens, showing that its expression is induced by blood feeding and TBP but not by Escherichia coli that is not transmitted by I. ricinus. In addition, our results shown the expression of IrSPI in several tick organs, suggesting its implication in several functions in tick physiology. Among them, the discovery of the injection of IrSPI, through the saliva, to the vertebrate host allowed us to consider a role in host responses to tick bite. Evaluation of IrSPI effect on host showed no impact on coagulation through extrinsic pathway, as determined by analysis of thrombin generation time and by fibrinolysis, or in angiogenesis. However, it inhibited the proliferation of mitogen-stimulated CD4+ lymphocytes and increased unstimulated-B cell proliferation. In addition, IrSPI also modulated cytokine production from macrophages and splenocytes, repressing significantly most of proinflammatory cytokines and chemokines. Thus, we demonstrated that IrSPI plays a role in modulating the host immune response during blood feeding. Finally, preliminary results in the identification of the protein’s interactants open many research perspectives for understanding how IrSPI acts in tick physiology and counteracts host responses to tick injury and pathogen transmission.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

In this study, firstly the effects of aspartic acid group amino acids -which were reported to be the potential bottleneck in serine alkaline protease (SAP) synthesis- on SAP production were investigated by substituting at a concentration range of 0-15 mM by using recombinant Bacillus subtilis carrying pHV1434::subC gene. All aspartic acid group amino acids except threonine inhibited SAP activity when CAA&amp / #8805 / 2.5 mM. The highest SAP activities with asparagine, aspartic acid, lysine, threonine, isoleucine and methionine werefound to be 1.89-, 1.87-, 1.61-, 1.48-, 1.4-, and 1.4-fold higher than the reference medium activity, respectively, when the concentration of each amino acid was CAA=0.25 mM. The product and by-product distributions in defined and complex media in SAP production were also analyzed and compared in order to obtain a depth in-sight on functioning of the metabolic reaction network. The highest SAP activity in complex medium was found to be 3&ndash / fold higher than defined medium activity, while, specific SAP production rate was 1.2- fold higher. The highest cell concentration in complex medium (CX= 14.3 g/dm-3) was 8.1-fold higher than that obtained in defined medium (CX= 1.75 g/dm-3). In both media, oxaloacetic acid was observed extracellularly and intracellularly. In complex medium there was also succinic acid in the extracellular medium indicating that the operation of TCA cycle was insufficient. In both media serine, valine and glycine were observed neither in the extracellular nor in the intracellular media indicating that the synthesis of these amino acids can be a secondary rate limiting step. In defined medium asparagine was present neither in the cell nor in fermentation broth whereas, methionine was observed in the cell in high amounts, probably due to the lower flux values towards asparagine. Thus, in defined medium the synthesis of asparagine can also be a potential bottleneck in SAP production in defined medium.

Pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, is a cofactor for many enzymes involved in the metabolism of amino acids, amino acid derived metabolites and some amino sugars. PLP is one of the most versatile cofactors and the PLP-dependent enzymes catalyze a variety of reactions including transamination, decarboxylation, inter-conversion of L-and D-amino acids and removal or replacement of chemical groups bound at β or γ carbon of amino acids. The thesis describes the structural studies carried out on three PLP-dependent enzymes; N-acetylornithine aminotransferase (AcOAT), serine hydroxymethyltransferase (SHMT) and diaminopropionate ammonia lyase (DAPAL). Chapter 1 of the thesis begins with a brief introduction to PLP-dependent enzymes and their classification. This is followed by a review of structures of enzymes belonging to the subgroup II aminotransferases. The last section of chapter I contains a detailed description of the structures available till date for SHMT from various sources and the mutational studies carried out on SHMT. All the common experimental procedures and computational methods used for the current investigations are described in chapter II, as most of these are applicable to all structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization, and X-ray diffraction data collection. Computational methods include details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. AcOAT is one of the key enzymes in arginine and lysine metabolism. AcOAT belongs to the fold type I (αfamily) subgroup II family of PLP dependent enzymes. Both S. typhimurium and E. coli have two genes each, one involved in the biosynthesis of arginine and another in the biodegradation of arginine. Biosynthetic AcOAT catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate and the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate in lysine biosynthesis. Meso-DAP and lysine, the products of lysine biosynthesis pathway, are known to function as cross-linking moieties in the peptidoglycan component of bacterial cell wall. Therefore N-acetylornithine aminotransferase could serve as a target for designing antibacterials. Chapter III gives the details of the work carried out on AcOAT. Two genes each from S. typhimurium and E. coli coding for biosynthetic and biodegradative AcOAT were cloned in E. coli, overexpressed and purified by Ni-NTA affinity chromatography. Of the four enzymes, biosynthetic AcOAT from S. typhimurium (sArgD) crystallized in the unliganded form and in the presence of the inhibitor gabaculine or one of the substrates L-glutamate, diffracted to a maximum resolution of 1.90 Å and contained a dimer in the asymmetric unit. The structure was determined by the molecular replacement method using human ornithine aminotransferase (hOAT) as the starting model. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop Lαk-βm in only one subunit. The Km and kcat/Km values determined with the purified sArgD suggested that the enzyme could accept both acetylornithine (AcOrn) and ornithine (Orn) as the substrates and had much higher affinity for AcOrn than for Orn. However, OAT accepts only Orn as the substrate. Comparison of the structurte of sArgD with T. thermophilus AcOAT and hOAT suggested that the higher specificity of sArgD towards AcOrn may not be due to specific differences in the active site residues but could result from minor conformational changes in some of them. sArgD was inhibited by gabaculine with an inhibition constant (Ki) of 7 µM and a second order rate constant (k2) of 0.16 mM-1s-1. The crystal structure of sArgD obtained in the presence of gabaculine and the spectral studies of sArgD with gabaculine suggested that the enzyme might have a low affinity for the PLP-gabaculine complex. Biosynthetic AcOAT from E. coli (eArgD) crystallized in the presence of gabaculine in hanging drop vapor diffusion method and diffracted X-rays only to a resolution of 3.5 Å. Two data sets were collected for the eArgD crystals. One of the data sets belonged to P1 (data 1) and the other to P321 space group (data 2) with a solvent content of ~70%. Data 1 was twinned and the unit cell was unusually large and could accommodate ~24 molecules in the asymmetric unit where as data 2 had four molecules in the asymmetric unit. Biodegradataive AcOAT from E. coli also crystallized in presence of gabaculine in hanging drop vapor diffusion method and suffered from low diffraction quality, where as that from S. typhimurium did not yield crystals. In chapter IV, X-ray crystallographic studies on various site specific mutants of SHMT from Bacillus stereotherophilus (bs) and a detailed comparison of structural data with the biochemical results in relation to mechanism of catalysis are presented. SHMT is a member of the α-class of PLP-dependent enzymes and catalyzes the reversible conversion of L-Ser and THF to glycine and 5,10-methylene THF. 5,10-methylene THF serves as a major source of one-carbon units in the biosynthesis of nucleotides and a few amino acids. SHMT also catalyses the cleavage of β-hydroxy amino acids like L-allo-threonine, transamination, racemization and decarboxylation reactions. SHMT shows increased activity along with enhanced nucleotide synthesis and therefore is a potential target for cancer chemotherapy. The availability of structural and biochemical data on SHMT from different sources ranging from human to E. coli enabled the identification of active site residues and a more critical examination of the role of these residues in the different steps of catalysis. The important mutants studied in the present investigation are E53Q, Y51F, Y61F, Y61A, Y60A, N341A and F351G of bsSHMT. The crystal structures of all these mutants are solved in the presence of various ligands, which gave many interesting results. E53, one of the active residues, interacts with the side chain hydroxyl group of serine bound to PLP in the wild type serine complex and N10 and formyl oxygen in the wild type glycine-FTHF complex. In E53Q glycine and serine complexes, glycine carboxyl and serine side chain were in two conformations, respectively, the new conformation being stabilized by their interaction with the mutated residue Q53. The structure of E53Q-Gly complex obtained in the presence and absence of 5-formyl THF(FTHF) showed an interesting case of enzyme memory in which the final conformational state depends on the way it was obtained and suggested that E53 is crucial for FTHF/THF binding. Though the spectrum showed that FTHF binds to the mutant initially, no density was observed for FTHF in the final structure. FTHF is believed to dissociate from the active site with prolonged incubation leaving behind a few significant conformational changes. Y51, one of the highly conserved tyrosines in SHMT, has hydrogen bonding interactions with the phosphate group of PLP and the active site lysine (K226) in bsSHMT. Mutation of Y51 to F resulted in significant changes at the active site. In all the structures of Y51F complexes, the phosphate group is in two conformations and F51 has moved away from the phosphate and in turn changed the position of Y61, another tyrosine in the active site. The residue Y61 is hydrogen bonded to R357 in the internal aldimine complex of bsSHMT. Addition of glycine/serine to bsSHMT resulted in the conformational change of Y61 away from R357 and towards E53, allowing the added glycine/serine to interact with R357. Mutation of Y61 to A did not bring significant structural changes. Structures of Y51F and Y61A mutants complexed with L-allo-Thr (cleaved to Gly by the wild type enzyme) showed that L-allo-Thr was not cleaved to glycine and acetaldehyde and confirmed the biochemical observation that these two residues are essential even for the THF-independent reaction. Residues Y60 and N341 are also highly conserved residues among SHMTs. Y60 stacks over PABA ring of FTHF in the wild type glycine-FTHF ternary complex. N341 has strong hydrogen bonding interactions with N1 and N8 atoms of the pteridine ring of FTHF. Mutation of either Y60 or N341 to A destroys the binding ability of FTHF/THF to the enzyme according to the biochemical and structural observations. The residue F351 exhibits different conformations in the two subunits of wild type glycine-FTHF ternary complex and is thought to be an important residue in determining the asymmetric binding of FTHF. Mutation of F351 to G did not affect the catalytic activity. Surprisingly, in the crystal structure obtained in the presence of L-allo-Thr, the ligand did not get cleaved to glycine, though in solution, the mutant is as active as the wild type enzyme. Chapter V describes the preliminary structural studies carried out on DAPAL from E. coli and S. typhimurium. DAPAL catalyzes the α, βelimination of both L-and D-diaminopropionate (DAP). DAP is the immediate precursor of two neurotoxins 3oxalyl and 2,3-dioxalyl DAP present in Lathyrus sativus, a grain legume rich in proteins and capable of growing well in drought conditions. The presence of these two neurotoxins precludes its use as a source of protein rich food. This enzyme is present only in bacteria and few species of actinomycetes. Unlike many other PLP-dependent enzymes, DAPAL does not catalyze any side reaction and is the only enzyme known to remove an amino group from the βcarbon of the substrate. The enzymes from E. coli (eDAPAL) and S. typhimurium (sDAPAL) produced diffraction quality crystals. However, crystals of sDAPAL did not survive heavy atom soaking and eDAPAL crystals suffered from poor reproducibility and severe non-isomorphism making it difficult to obtain suitable heavy atom derivatives for structure determination. Production of selenomethionine labelled proteins for these enzymes was initiated and thin crystals were obtained for eDAPAL. Improvement of the quality of these crystals is necessary in order to solve the structure of DAPAL by MAD method.

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2009/11/30). Includes bibliographical references.

No description available.

The main focus of my PhD studies was aimed at understanding the general mechanism of nuclear translocation and isolating novel components of the nuclear translocation pathway in neurons. Using the Drosophila visual system as an in vivo model to study nuclear motility in developing photoreceptor cells (R-cells), I have identified a novel role for the Ser/Thr kinase Misshapen (Msn) and the endocytic trafficking pathway in regulating the nuclear translocation process. / The development of R-cells in the Drosophila eye disc is an excellent model system for the study of nuclear motility owing to its monolayer organization and the stereotypical translocation of its differentiating R-cell nuclei along the apical-basal plane. Prior to my thesis work, several laboratories had identified dynein and its associating proteins in R-cell nuclear translocation, however nothing was known about the signalling pathway that controlled their function in nuclear migration. Thus, one of my thesis goals was to elucidate the signalling mechanism controlling nuclear translocation in R-cells. / Using a combination of molecular and genetic approaches, I identified Msn as a key component of a novel signalling pathway regulating R-cell nuclear translocation. Loss of msn causes a failure of R-cell nuclei to migrate apically. Msn appears to control R-cell nuclear translocation by regulating the localization of dynein and Bicaudal-D (Bic-D). My results also show that Msn enhances Bic-D phosphorylation in cultured cells, suggesting that Msn regulates R-cell nuclear migration by modulating the phosphorylation state of Bic-D. Consistently, my results show that a Bic-D-phosphorylation-defective mutation disrupted the apical localization of both Bic-D and dynein. I propose a model in which Msn induces the phosphorylation of Bic-D, which in turn modulates the activity and/or subcellular localization of dynein leading to the apical migration of R-cell nuclei. / In addition to studying Msn, I have also searched for additional players in R-cell nuclear migration. From a gain-of-function approach, I found that the misexpression of the GTPase-activating-protein (GAP) RN-Tre caused a severe defect in R-cell nuclear migration. Since mammalian RN-Tre is involved in negatively regulating Rab protein activity, I speculated that the RN-Tre misexpression phenotype reflected a role for Rab-mediated vesicular transport in regulating R-cell nuclear migration. I systematically examined the potential role of Rab family proteins in R-cell nuclear migration and found that interfering with the function of Rab5, Rab11 or Shibire caused a similar nuclear migration phenotype. I propose that an endocytic pathway involving these GTPases is required for the targeting of determinants to specific subcellular locations, which in turn drive the apical migration of R-cell nuclei during development.

Myc transcriptionally regulates genes involved in processes such as cell proliferation, metabolism, differentiation, and angiogenesis.  MYC expression is deregulated in many types of human cancer; therefore discovering the mechanisms behind MYCs role in tumorigenesis is essential.  In this dissertation, I have focused on several Myc target genes, Spermidine synthase (Srm); Lactate dehydrogenase (Ldh); 3-phosphoglycerate dehydrogenase (Phgdh); Serine hydroxymethyltransferase (SHMT) 1 and 2; and Pim-3 (a member of the Pim family of serine/threonine kinases).  These enzymes play a role in various functions: Spermidine synthase (polyamine synthesis); Lactate dehydrogenase (glycolysis); Phgdh and Shmt (serine metabolism); and Pim-3 (cell signaling).  In order to elucidate the impact Myc over-expression has on metabolism in tumorigenesis, we use human cell lines, and transgenic mice as well as cell lines and tissues derived from these mice.  The impact of inhibition of these target genes on Myc-driven tumorigenesis was done by genetically inhibiting the target gene (using RNAi or mouse models) or inhibiting the protein with a chemical inhibitor.  Investigating these Myc target genes will help determine if inhibition of Myc target genes is a viable approach for chemotherapeutics, and under what conditions this inhibition may be the most valuable.  In paper I, we examine SRM; a highly expressed enzyme in the polyamine synthesis pathway that converts putrescine to spermidine, and is important for actively growing cells.  Genetic inhibition via RNAi against Srm, or chemical inhibition of Srm, resulted in decreased proliferation of B-cell tumor lines from transgenic mice in vitro.  In vivo treatment of λ-Myc transgenic mice with a chemical SRM inhibitor exhibited a significant chemopreventative effect on tumor formation. These results support previous findings that inhibition of polyamine synthesis pathway enzymes has a place in cancer therapy.  Many Myc target genes have been suggested as attractive targets in battling Myc-driven tumorigenesis.  Surprisingly in paper II, when we analyzed the inhibition of other Myc target genes, such as Ldh, Shmt, and Phgdh, we found that inhibition of these genes did not inhibit Myc-driven tumorigenesis to any significant degree. However, inhibition of Ldh, Phgdh and Shmt2 had a notable effect on in vitro Ras-driven transformation.  These findings suggest that chemotherapeutic inhibition of metabolic genes such as Ldh, Phgdh and Shmt2 may be effective in genetically defined settings, keeping in mind the oncogenic lesion behind the tumor.  The Pim kinase family consists of three serine/threonine kinases, Pim1-3.  In paper III, we found that Pim-3 is a direct Myc target gene and that Pim-3 expression is high in Burkitt Lymphoma samples taken from human patients, as well as spontaneously arising lymphomas from Myc transgenic mice. We also found that inhibition of Pim-3 using a pan-Pim kinase inhibitor, Pimi, in these spontaneously arising Myc lymphomas resulted in caspase independent cell death.  These results indicate that Pim kinase inhibition may be a potential chemotherapeutic strategy in human lymphomas that rely on Pim-3 kinase expression.