This dissertation consists of four different topics in the areas of proteomics, genomics, and cardiology. First, a data-based method was developed to assign the subcellular localization of proteins. We applied the method to data on the bacteria Rhodobacter sphaeroides 2.4.1 and compared the results to PSORTb v.3.0. We found that the method compares well to PSORTb and a simulation study revealed that the method is sound and produces accurate results. Next, we investigated genomic features involved in the lethality of the knockout mouse using the random forest technique. We achieved an accuracy rate of 0.725 and found that among other features, the evolutionary age of the gene was a good predictor of lethality. Third, we analyzed DNA breakpoints across eight different cancer types to determine if common hotspots or cancer-type specific hotspots can be well-predicted by various genomic features and investigated which of the genomic features best predict the number of breakpoints. Using the random forest technique, we found that cancer- type specific hotspots are poorly predicted by genomic features but common hotspots can be predicted using the relevant genomic features. Additionally, we found that among the genomic features analyzed, indel rate and substitution rate were consistently chosen as the top predictors of breakpoint frequency. Lastly, we developed a method to predict the hypothetical heart age of a subject based on the subject’s electrocardiogram (ECG). The heart age predictions are consistent with current ECG science and knowledge of cardiac health.

Subcellular localization is crucial for determining the functions of proteins. A system called prediction of protein subcellular localization (P2SL) that predicts the subcellular localization of proteins in eukaryotic organisms based on the amino acid content of primary sequences using amino acid order is designed. The approach for prediction is to nd the most frequent motifs for each protein in a given class based on clustering via self organizing maps and then to use these most frequent motifs as features for classication by the help of multi layer perceptrons. This approach allows a classication independent of the length of the sequence. In addition to these, the use of a new encoding scheme is described for the amino acids that conserves biological function based on point of accepted mutations (PAM) substitution matrix. The statistical test results of the system is presented on a four class problem. P2SL achieves slightly higher prediction accuracy than the similar studies.

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Infection of a host cell by the Herpes Simplex Virus Type 1 leads to the efficient reprogramming of the cells' synthetic machinery to replicate the viral genome ultimately producing progeny virions. Two proteins introduced upon viral fusion are thought to initiate this effect. The potent transactivator of immediate early genes (VP16) and the mRNA destabilizing virion host shutoff protein (vhs), work in concert with one another to invoke the cascade of viral gene expression, and to destroy pre-existing cellular mRNA. Due to the non-specific nature of vhs induced mRNA degradation, its activity is downregulated at later times during infection to spare virally encoded mRNA. Recent evidence has shown that VP16 is responsible for this vhs downregulation, a process thought to occur by mutual interactions between the two proteins and a potential compartmentalization of vhs within the nucleus (Lam 𝘦𝘵 𝘢𝘭., 1996; Smibert 𝘦𝘵 𝘢𝘭., 1994). Furthermore, such an event is also thought to position vhs so it can be efficiently packaged, a supposition supported by the observation that vhs lacking the ability to bind VP16 is not incorporated into new virions (Read 𝘦𝘵 𝘢𝘭. , 1993). To ascertain if VP16 was indeed capable of relocalizing vhs to the nucleus of a cell in the absence of any other viral factors, we created multiple constructs consisting of various portions of vhs fused in frame to the fluorescent marker protein EGFP. In addition, various truncated forms of VP16 were also fused to EGFP for the purpose of delineating the region of VP16 that is responsible for VP16 and possibly vhs nuclear localization. Co-transfection experiments utilizing EGFP-vhs fusions demonstrated that vhs relocalizes to perinuclear regions in the presence of VP16, an effect absolutely dependent upon its ability to interact with VP16. In addition, deletion mapping of VP16 implicated the region spanning amino acids 335 to 355 as being necessary for this localization, with a stretch of 15 amino acids (330 to 344) appearing to constitute a putative bipartite nuclear localization signal. Interestingly, our observation that the vhs/VP16 complex localizes to a region of the cell thought to ultimately encompass the tegument of new virions gives credence to the notion that this interaction and subsequent localization may indeed function to package vhs into new virions. Furthermore, it is also suggested that vhs may in fact be downregulated at intermediate times during infection through VP16 mediated compartmentalization within the nucleus. For these reasons we propose that the disruption of the vhs/VP16 interaction could severely abrogate the infectivity of HSV and as such could present a novel target for antiviral intervention. / Thesis / Master of Science (MS)

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Listeria monocytogenes est l'un des meilleurs organismes modèles pour l'étude des interactions bactérie-hôte. Ce pathogène intracellulaire facultatif peut infecter, survivre et se répliquer dans le cytoplasme des cellules eucaryotes, démontrant la co-évolution étroite de Listeria avec son hôte. Le style de vie intracellulaire de ce pathogène implique la manipulation de divers composants de la cellule hôte, dont l'un est la chromatine. En induisant des modifications de la chromatine au niveau des histones, Listeria peut influencer le programme transcriptionnel de l'hôte. Ce projet de thèse porte sur une modification spécifique des histones, la désacétylation de la lysine 18 de l'histone H3, induite par la désacétylase de l'hôte Sirtuin 2 (SIRT2) lors de sa relocalisation du cytoplasme vers le noyau pendant l'infection. Le détournement de SIRT2 par Listeria fournit un système idéal pour étudier les mécanismes de la localisation subcellulaire de SIRT2, qui est mal comprise, et c'est le but de cette thèse. En utilisant la spectrométrie de masse, nous avons identifié une nouvelle modification posttraductionnelle de SIRT2, la phosphorylation de la sérine 25 (S25), ciblée spécifiquement par l'infection, et essentielle pour l'association de SIRT2 à la chromatine. Nous avons caractérisé le complexe moléculaire impliqué dans la déphosphorylation de SIRT2-S25 et nous montrons que cette modification est essentielle pour contrôler la fonction de SIRT2 en tant que répresseur transcriptionnel, et est nécessaire pour une infection efficace. Notre approche protéomique a aussi permis la caractérisation d'un interactome de SIRT2. De nombreuses protéines ont été identifiées et quelques-unes ont été confirmées et étudiées pour leur rôle dans le transport nucléo-cytoplasmique de SIRT2. De plus, une collaboration au laboratoire a mis au jour un mécanisme de subversion de la réponse aux dommages de l'ADN de l'hôte par Listeria. Dans son ensemble, ce travail a contribué à la compréhension de mécanismes originaux de l’interaction entre les bactéries et la chromatine et a révélé un processus cellulaire contrôlant la localisation subcellulaire et la fonction de la protéine de l’hôte SIRT2. / One of the best model organisms for the study of bacterial-host interactions is Listeria monocytogenes. This facultative intracellular pathogen can infect, survive, and replicate in the cytoplasm of eukaryotic cells, demonstrating the close co-evolution of Listeria with itshost. The intracellular life style of this pathogen involves manipulation of various host cellcomponents, one of which is chromatin. By inducing chromatin modifications at the level of histones, Listeria can influence the transcriptional program of the host. This thesis focuses on one specific histone modification, deacetylation of histone H3 of lysine 18, which is induced by the host deacetylase Sirtuin 2 (SIRT2) upon its relocalization from the cytoplasmto the nucleus during infection. Hijacking of SIRT2 by Listeria provides an ideal system tostudy the mechanisms of SIRT2 subcellular localization, which is poorly understood, and is the purpose of this thesis. By using mass spectrometry we have identified a novel posttranslational modification of SIRT2, Serine 25 (S25) phosphorylation, specifically targeted byinfection, and essential for SIRT2 chromatin association. We have characterized themolecular complex involved in dephosphorylating SIRT2-S25 and we show that this modification is essential for controlling SIRT2 function as a transcriptional repressor andnecessary for productive infection. Our proteomic approach further allowed the characterization of a SIRT2 interactome. Many proteins were identified and a few wereconfirmed and studied for their role in nucleo-cytoplasmic shuttling of SIRT2. In addition, a laboratory collaboration uncovered a mechanism for subversion of the host DNA DamageResponse by Listeria. As a whole, this work has contributed to the understanding of original mechanisms of chromatin-bacteria cross talk, and has revealed a cellular process controlling subcellular localization and function of the host protein SIRT2.

Phototransduction is a well characterized system for study of G protein coupled receptor (GPCR) signaling. The GPCR rhodopsin couples to the heterotrimeric G protein transducin. Light-stimulated activation of transducin in turn activates phosphodiesterase (PDE), leading to closure to cGMP-gated channels and inhibition of glutamate release. Rod and cone photoreceptors are highly polarized neurons consisting of the outer segment (OS) where phototransduction biochemistry occurs, the inner segment containing mitochondria and other organelles, the nuclear layer, an axon, and a glutamatergic synapse. Upon illumination, activated G protein transducin redistributes from the rod OS (where it is localized in the dark) to the inner compartments of the cell. Interestingly, cone transducin does not translocate in light. Opposite to this, visual arrestin migrates from the inner compartments to the OS, where it binds to rhodopsin. Previous reports from other groups and our lab argue for either an active or passive mechanism for transducin and arrestin redistribution. Our lab has shown that arrestin migration occurs by diffusion which is restricted by molecular sinks (Nair et al, 2005b). The focus of my dissertation was to unravel the molecular mechanism of rod transducin translocation. Specifically, I found energy (ATP) was not required for transducin movement within photoreceptors. Also, I found that the disc membranes of the rod outer segments as well as protein-protein interactions with retinal guanylate cyclase serve to restrict transducin diffusion through the cell. In addition, I used the insights gained from these studies of transducin to re-examine the relationship of other G proteins' subcellular localization and signal transduction. Ultimately, I found that most G proteins do not undergo subunit dissociation under physiological activating conditions.