An important task in brain modeling is that of estimating model parameters and quantifying their uncertainty. In this thesis we tackle this problem from a Bayesian perspective: we use experimental data to update the prior information about model parameters, in order to obtain their posterior distribution. Uncertainty quantification via a direct computation of the posterior has a prohibitive computational cost in high dimensions. An alternative to a direct computation is offered by Markov chain Monte Carlo (MCMC) methods. The aim of this project is to analyse some of the methods within this class and improve their convergence. In this thesis we describe the following MCMC methods: Metropolis-Hastings (MH) algorithm, Metropolis adjusted Langevin algorithm (MALA), simplified manifold MALA (smMALA) and Approximate Bayesian Computation MCMC (ABCMCMC). SmMALA is further analysed in Paper A, where we propose an algorithm to approximate a key component of this algorithm (the Fisher Information) when applied to ODE models, with the purpose of reducing the computational cost of the method. A theoretical analysis of MCMC methods is carried out in Paper B and relies on tools from the theory of large deviations. In particular, we analyse the convergence of the MH algorithm by stating and proving a large deviation principle (LDP) for the empirical measures produced by the algorithm. Some of the methods analysed in this thesis are implemented in an R package, available on GitHub as “icpm-kth/uqsa” and presented in Paper C, and are applied to subcellular pathway models within neurons in the context of uncertainty quantification of the model parameters. / En viktig uppgift inom hjärnmodellering är att uppskatta parametrar i modellen och kvantifiera deras osäkerhet. I denna avhandling hanterar vi detta problem från ett Bayesianskt perspektiv: vi använder experimentell data för att uppdatera a priori kunskap av modellparametrar, för att erhålla deras posteriori-fördelning. Osäkerhetskvantifiering (UQ) via direkt beräkning av posteriorfördelningen har en hög beräkningskostnad vid höga dimensioner. Ett alternativ till direkt beräkning ges av Markov chain Monte Carlo (MCMC) metoder. Syftet med det här projektet är att analysera några MCMC metoder och förbättra deras konvergens. I denna avhandling beskriver vi följande MCMC algoritmer: “Metropolis-Hastings” (MH), “Metropolis adjusted Langevin” (MALA), “Simplified Manifold MALA” (smMALA) och “Approximate Bayesian Computation MCMC” (ABCMCMC). SmMALA analyseras i artikel A. Där presenterar vi en algoritm för att approximera en nyckelkomponent av denna algoritm (Fisher informationen) när den tillämpas på ODE modeller i syfte att minska metodens beräkningskostnad. En teoretisk analys av MCMC metoder behandlas i artikel B och bygger på verktyg från teorin av stora avvikelser. Mer specifikt, vi analyserar MH algoritmens konvergens genom att formulera och bevisa en stora avvikelser princip (LDP) för de empiriska mått som produceras av algoritmen. Några av metoderna analyserade i den här avhandlingen har implementerats i ett R paket som finns på GitHub som “icpm-kth/uqsa” och presenteras i artikel C. Metoderna tillämpas på subcellulära vägmodeller inom neuroner i sammanhanget av osäkerhetskvantifieringen av modellparametrar. / <p>QC 2023-08-21</p>

Conversion of aspartate and asparagine residues to isoaspartate is a prevalent covalent protein modification in cells. The accumulation of these altered residues can lead to the loss of protein function and the consequent loss of cellular function. The L-ISOASPARTATE METHYLTRANSFERASE (EC 2.1.1.77) (PIMT) iteratively methylates abnormal isoaspartyl residues leading to conversion to L-aspartate, thereby mitigating the injurious effects of aging. Arabidopsis thaliana is unique among eukaryotes studied to date in that it possesses two genes (At3g48330 (PIMT1) and At5g50240 (PIMT2)) encoding PIMT. The PIMT2 gene exhibits a complex transcriptional control involving different transcriptional initiation sites and 5'- and 3'- alternative splice site selection in the first intron. Varying the transcriptional initiation site results in alternative targeting of the PIMT2 proteins thus produced to: 1) the nucleus, or 2) the cytoplasm, while PIMT1 is cytosolic. Inclusion of a 51 nucleotide 5 alternatively spliced sequence with or without a nine nucleotide 3 alternatively spliced sequence dramatically alteres the subcellular protein localization from the cytoplasm and around the chloroplast to inside the chloroplast. All recombinant PIMT2 isoform tested exhibit PIMT activity, although solubility varied among them. Multiplex RT-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to -TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous ABA. Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA application or applied stress presumably through an ABA-dependent pathway. Furthermore, Cleaved Amplified Polymorphic Sequence analysis of the PIMT2 amplicons has shown that the ratio among the splicing variants alters upon ABA application, implicating a role for the spliceosome or differential RNA stability in orchestrating the plant's response to stress. T-DNA insertional mutants of both genes were isolated but no obvious phenotype has been identified. The double mutant has been generated and will be evaluated.

No description available.

N-acylation of phosphatidylethanolamine (PE) with free fatty acids catalyzed by N-acyl phosphatidylethanolamine (NAPE) synthase was reported in cotyledons of 24-h-old cotton seedlings. Here I report subcellular localization of this enzyme. Differential centrifugation, sucrose density gradient fractionation,aqueous two-phase partitioning and electron microscopy techniques were utilized to elucidate subcellular site(s) of NAPE synthase. Marker enzymes were used to locate organelles in subcellular fractions. Differential centrifugation indicated that NAPE synthase is present in more than one organelle and it is a membrane bound enzyme. Sucrose density gradient fractionations indicated that NAPE synthase is present in membranes derived from endoplasmic reticulum (ER),Golgi and possibly plasma membrane (PM) but not mitochondria, glyoxysomes or plastids. Aqueous two-phase partitioning experiments with cotton and spinach tissues supported these results but Goigi appeared to be the major site of NAPE synthesis. Electron microscopy of subcellular fractions was used to examine isolated fractions to provide visual confirmation of our biochemical results. Collectively, these results indicate that NAPE is synthesized in plant ER, Golgi and possibly PM.

Maspina é uma proteína supressora de tumor e metástase e sua localização subcelular está relacionada ao prognóstico do câncer de mama. Nosso grupo mostrou em MCF-10A que quando fosforilada maspina se acumula no citoplasma. Porém, esta correlação ainda não foi relatada in vivo. Aqui investigamos a expressão, fosforilação e localização subcelular de maspina ao longo do desenvolvimento da glândula mamária murina. Maspina foi detectada no estágio mais tardio da gestação, na lactação e na involução. Os níveis de fosforilação de maspina são maiores no período de lactação do que na involução. Interessantemente, a porcentagem de células que apresenta maspina no núcleo é maior na fase de involução do que na fase de lactação Estes dados mostram que a correlação entre níveis de fosforilação de maspina e localização subcelular também é observada in vivo e que esses processos são reguladas ao longo do desenvolvimento na glândula mamária murina. / Maspin is a protein with tumor and metastasis suppressing activity and its subcellular localization is related to breast cancer prognosis. Using MCF-10A cells as a model system, our group demonstrated a correlation between maspin phosphorylation and cytoplasmic accumulation. Here we investigated maspin expression, phosphorylation levels and subcellular localization in vivo during the murine mammary gland development. Maspin was detected in late pregnancy, during lactation and involution. Maspin phosphorylation levels is higher during lactation than during involution. Interestingly, the percentage of cells which present nuclear maspin is higher in the involution than in lactation. These data show that the correlation between maspin phosphorylation and subcellular localization is also observed in vivo and these processes are regulated during murine mammary gland development.

Maspina, uma proteína supressora de tumor de 42 kDa, pertence à superfamília das serpinas (inibidores de serina protease), no entanto, seu mecanismo de ação independe da inibição de proteases. Maspina é expressa por epitélio de diferentes órgãos e apresenta regulação diferencial durante a progressão tumoral. Entre suas atividades biológicas, foram descritas a regulação da adesão celular, migração, morte, expressão gênica e resposta ao estresse oxidativo. A localização subcelular de maspina está relacionada a regulação de suas funções biológicas. Estudos clínicos recentes indicam que é a localização nuclear de maspina, ao invés de seus níveis de expressão, correlaciona-se com bons fatores prognósticos e sobrevida global em alguns tipos de câncer, incluindo câncer de mama. No entanto, pouco se sabe sobre como a localização subcelular de maspina é regulada. A maioria dos estudos sobre maspina é conduzido em linhagens de células tumorais, que apresentam uma grande variabilidade no contexto celular. Portanto, é importante usar uma linhagem celular não transformada para esta abordagem. Nesse trabalho investigamos fatores solúveis, interação célula-célula e interação célula-substrato como possíveis reguladores da localização de maspina na célula e as vias de sinalização envolvidas nesta regulação. Usando a linhagem celular epitelial mamária não transformada MCF10A como modelo, observamos por experimentos de imunofluorescência que o Fator de Crescimento Epidermal (EGF) promove a localização nuclear de maspina. EGF também altera a fosforilação da proteína conforme mostram nossos experimentos de 2D-SDS-PAGE e gel contendo Phostag. A fosforilação ocorre em resíduos de serina e a desfosforilação em resíduos de tirosina. À procura por vias de sinalização a jusante de do receptor de EGF envolvidas na regulação da localização subcelular de maspina, identificamos as vias de PI3K e Stat3 . Além disso, a adesão célula-célula parece bloquear a localização nuclear de maspina. Na tentativa de investigar funções celulares relacionadas à regulação de maspina por EGFR, identificamos proteínas que co-imunoprecipitam com maspina após o tratamento com EGF. O agrupamento funcional dessas proteínas sugere que a maspina pode estar envolvida em metabolismo de RNA, adesão celular e citoesqueleto. Desta forma, este trabalho identificou diferentes mecanismos que regulam a localização subcelular de maspina, que dependem da ativação das vias de PI3K e AKT pela via de EGFR. / Maspin, a 42 kDa tumor suppressor protein, belongs to the serpin (serine protease inhibitors) superfamily, however, its mechanism of action does not depend on protease inhibition. Maspin is expressed by epithelium of different organs and presents differential regulation during tumor progression. Among its biological activities, regulation of cell adhesion, migration, death, gene expression and oxidative stress response were described. Subcellular localization of maspin is related to the regulation of its biological functions. Recent clinical studies indicate that nuclear localization of maspin, not its expression levels, correlates with good prognostic factors and overall survival in some types of cancer, including breast cancer. However, little is known about how maspin subcellular localization is regulated. Most studies on maspin are conducted in tumor cell lines, which show great variability in cell context. Therefore, it is important to use a nontransformed cell line for this approach. Here we investigated soluble factors, cell-cell interaction and cell-substrate interaction as possible regulators of maspin subcellular localization and the signaling pathways involved. Using the MCF10A untransformed mammary epithelial cell line as a model system, we observed by immunofluorescence experiments that the Epidermal Growth Factor (EGF) promotes nuclear localization of maspin. EGF also alters the phosphorylation of the protein as observed by 2D-SDSPAGE and gel containing Phos-tag. Phosphorylation occurs on serine residues and dephosphorylation, on tyrosine residues. Looking for signaling pathways downstream of EGF receptor involved in regulation of maspin subcelular localization, we identified the PI3K and STAT3 pathways. On the other hand, cell-cell adhesion seems to block nuclear localization of maspin. In an attempt to investigate cellular functions related to regulation of maspin by EGFR, we identified proteins that co-immunoprecipitate with maspin in EGF-treated cells. The functional grouping of these proteins suggests that maspin may be involved with RNA metabolism, cell adhesion and cytoskeleton. Thus, this study identified different mechanisms that regulate subcellular localization of maspin, which depends on PI3K and STAT3 activation downstream of EGFR pathway.

Orientador: Henrique Ferreira / Banca: Marco Aurélio Takita / Banca: Fernando Carlos Pagnocca / Resumo: O cancro cítrico é uma doença causada pela bactéria Xanthomonas axonopodis pv. citri (Xac), e que afeta plantas de citros por todo o mundo. O genoma de Xac foi completamente seqüenciado, o que revelou grandes quantidades de ORFs (~30%) codificando para produtos com função desconhecida (proteínas hipotéticas). Baseando-se no princípio de que muitos eventos bioquímicos acontecem em sítios específicos no interior celular, a localização de proteínas em fusão com GFP tem sido amplamente utilizada para a obtenção de informações valiosas a respeito de suas funções. Para iniciarmos estudos de localização de proteínas hipotéticas em Xac, construímos um vetor integrativo capaz de expressá-las em fusão com o polipeptídio GFP, pPM2a. O vetor de expressão para Xac carrega um cassete promotor/repressor de xilose (xylR/pxyl), o gene gfp, um RBS sintético e um fragmento do gene de α-amilase de Xac, para direcionar a integração do sistema de expressão no lócus amy do cromossomo bacteriano. Mostramos aqui a integração estável do vetor no lócus amy de Xac. Além disso, mutantes de Xac expressando o polipeptídio GFP não apresentam nenhuma alteração em seu fenótipo de patogenicidade para o hospedeiro (laranja doce). Mutantes de Xac expressando versões marcadas com GFP para as proteínas ParB e ZapA, ambas codificadas por Xac, foram utilizadas para a padronização dos estudos de localização subcelular. GFP-ZapAXac apresentou um padrão de localização análogo ao de seu ortólogo presente em Bacillus subtilis: uma estrutura semelhante a uma barra, posicionada no meio do bacilo, onde o septo se desenvolve, orientado perpendicularmente com relação ao eixo longitudinal da célula. Este é o primeiro relato de um estudo de localização realizado em Xac. Ao contrário de GFP-ZapAXac, ParBXac-GFP não mostrou nenhum padrão de localização, apesar de a fusão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Citrus canker is a disease caused by the bacterium Xanthomonas axonopodis pv. citri (Xac), which affects citrus plants worldwide. The genome of Xac was completely sequenced, which unveiled an expressive amount of ORFs (~30%) coding for products of unknown function (hypothetical proteins). Based on the principle that many biochemical events happen at specific sites within the cells, protein localization studies have been extensively used to gather valuable information about function. In order to start subcellular localization studies of hypothetical proteins encoded by Xac using fluorescent microscopy, we constructed an integrative expression vector for GFP-tagging of proteins in this bacterium, pPM2a. The expression vector for Xac carries a xylose repressor/promoter cassette (xylR/pxyl), the gfp gene, a synthetic Ribosome Binding Site (RBS), and a fragment of the α-amylase gene of Xac, to drive the integration of the whole expression system into the amy locus of the bacterial chromosome. We show here stable integration of the expression vector into the amy locus of Xac. Furthermore, Xac mutants expressing the polypeptide GFP do not exhibit any alteration in pathogenicity to the host plant sweet orange. Mutants of Xac expressing GFPtagged versions of ParB and ZapA proteins, both encoded by Xac, were used to standardize the subcellular localization studies. GFP-ZapAXac showed a localization pattern analogous to its ortholog encoded by Bacillus subtilis: a bar-like structure positioned in the middle of the rods, where the septum develops, oriented perpendicularly to the longitudinal axis of the cell. This is the first report of a protein localization study performed in Xac Unlike GFP-ZapAXac, ParBXac-GFP did not display any detectable localization pattern, despite the fact that we were able to detect the production of the fusion ParBXac-GFP in Western blot experiments... (Complete abstract click electronic access below) / Mestre

Maspina (mammary serpin) foi identificada em 1994 como uma serpina (serine protease inhibitor) que apresenta atividade de supressão tumoral. Foi classificada como uma serpina devido à homologia na sequência de aminoácidos, porém, maspina não apresenta atividade de inibição de serina proteases. Entre os efeitos biológicos de maspina estão a modulação da adesão, a inibição do crescimento e a invasão tumoral, a inibição da angiogênese, o efeito pró-apoptótico e o controle da resposta ao stress oxidativo, propriedades que contribuem para supressão tumoral. Esta diversidade de funções se reflete nos inúmeros ligantes de maspina e na sua localização subcelular, já que é encontrada na membrana plasmática, no citoplasma, núcleo e mitocôndrias. A localização subcelular de maspina guarda importante relação com sua função, já que foi demonstrado que sua localização nuclear está correlacionada com bom prognóstico em diversos tumores e seu efeito supressor de tumor foi observado somente quando maspina está localizada no núcleo. Entre os ligantes de maspina estão a HDAC1, IRF6, GST, HSP90 e HSP70, &#946;1 integrina, uPAR e colágeno tipo I e III. O mecanismo molecular envolvido na regulação dessas atividades não foi elucidado, e até o momento, somente um gene e uma proteína de maspina foram descritos, desta forma alterações pós-traducionais devem estar envolvidas na regulação dessas atividades. Com objetivo de verificar se há modificações pós-traducionais em maspina, utilizamos células MCF10A, que expressam grande quantidade dessa proteína, e submetemos seu extrato proteico à separação por gel bidimensional seguido de western blot. Identificamos quatro formas de maspina com a mesma massa molecular (42kDa), mas pontos isoelétricos distintos. Três destas formas são sensíveis ao tratamento com fosfatase ácida, o que sugere que estas sejam fosforiladas. Utilizamos ainda peroxidovanadato de sódio, um potente inibidor de tirosina fosfatase para investigar o papel da fosforilação de maspina em resíduos de tirosina. Através de western blot e imunofluorescência, observamos que o tratamento das células com o inibidor resultou no aumento dos níveis celulares de maspina assim como no seu acúmulo no citoplasma. Deste modo, concluímos que existem três diferentes fosfoformas de maspina em células MCF10A e ainda a inibição de tirosinas fosfatases aumentam os níveis de maspina e resultam no acúmulo da proteína no citoplasma. Esses resultados sugerem que a fosforilação pode estar envolvida na localização subcelular de maspina e na regulação dos seus níveis proteicos na célula. / Maspin (mammary serpin) was identified in 1994 as a serpin (serine protease inhibitor) which presents tumor suppressor activity. It was classified as a serpin due to its homology in amino acids sequence; however, maspin doesn\'t exhibit serine protease inhibition activity. Among maspin biological effects are modulation of cell adhesion, inhibition of tumor growth, invasion and angiogenesis, a pro-apoptotic effect and control of oxidative stress response, properties which contribute to tumor suppression. This functional diversity reflects maspin numerous ligands and its subcellular localization, since it is found on the plasma membrane, in the cytoplasm, nucleus and in mitochondria. Maspin subcellular localization is closely related to its function, as its nuclear localization correlates with good prognostic in several tumors and maspin tumor suppressor activity is only observed when it is located in the nucleus. Among maspin ligands are histone H1 deacetylase, IRF6, GST, HSP90 e HSP70, &#946;1 integrin, uPAR and type I and III collagen. The molecular mechanisms involved in the regulation of maspin biological activities are poorly understood. So far, only one gene and one protein have been assigned to maspin, so posttranslational modification should be involved. In order to verify posttranslational modification in maspin, we utilized MCF10A cells, which express great amount of this protein, and we submitted its proteic extract to 2D-SDS-PAGE followed by western blot. We identified four maspin forms with the same molecular mass (42kDa), but different isoelectric point. Three of these forms are sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated maspin forms. We also utilized sodium peroxovanadate, a potent tyrosine phosphatase inhibitor to investigate the role of maspin tyrosine phosphorylation. Through western blot and immunofluorescence analyses, we observed that cell treatment resulted in increase in maspin cellular levels as well as its cytoplasmic accumulation. Thus, we concluded that there are three diferente maspin phosphoforms in MCF10A cells and yet tyrosine phosphatase inhibition increases maspin levels and results in accumulation of the protein in the cytoplasm. These data suggest that phosphorylation may be involved in maspin subcellular localization and regulation of its protein levels in the cell.

Les travaux présentés dans cette thèse de doctorat portent sur létude de la distribution cellulaire dynamique des facteurs essentiels dépissage de la famille SR dArabidopsis thaliana. Le processus dexcision/épissage du pré-mRNA consiste en la reconnaissance précise des introns au niveau des sites dépissage, en leur excision et en la ligature des exons. Les facteurs d'épissage SR possèdent un domaine particulier riche en dipeptides sérines et arginines répétés et également un ou deux domaines hautement conservés de liaison au RNA. Ils sont impliqués dans la reconnaissance et le choix des sites dépissage ainsi que dans lassemblage du spliceosome. Au cours de cette thèse de doctorat, nous avons montré la distribution subcellulaire dynamique des protéines SR d'Arabidopsis (RSp31, RSZp22, RSp34 et RSZ33) fusionnée à la GFP, et ce dans divers systèmes expérimentaux (cellules foliaires de tabac et dArabidopsis, cellules BY-2). Celles-ci se localisent au sein du noyau et se concentrent en certains sites nucléaires précis dénommés speckles. Nous avons aussi observé que lune dentre-elles, RSZp22, peut se localiser au sein du nucléole suivant les conditions cellulaires, ce qui suggérait un rôle possible de cette protéine dans le transport du mRNA. Nous avons étudié le rôle des différents domaines structuraux des protéines SR dans leur distribution cellulaire en réalisant des délétions partielles des protéines RSp31 et RSZp22 et en analysant la localisation des protéines mutantes. Par co-expression de différents couples de protéines SR fusionnées à deux variantes de protéines fluorescentes (la GFP et la mRFP1 ou monomeric Red Fluorescent Protein 1), nous avons également montré une co-localisation générale des protéines SR végétales, à lexception de RSZp22 qui est la seule à présenter cette localisation nucléolaire. Nous avons aussi analysé la redistribution des protéines SR après traitement par divers inhibiteurs de la phosphorylation et déphosphorylation et également de la transcription. Aussi, l'utilisation de diverses méthodes de microscopie confocale (comme le FRAP ou Fluorescence Recovery After Photobleaching ou encore le FLIP ou Fluorescence Loss In Photobleaching) nous a permis de montrer que les protéines SR dArabidopsis sont hautement dynamiques au sein du noyau. Enfin, nous avons observé grâce à la technique du FLIP que RSZp22 est capable de faire la navette entre le noyau et le cytoplasme et que ce transport nucléo-cytoplasmique dépend de la voie dexportation via le récepteur CRM1/exportine1 [1-3]. 1. Docquier, S., et al., Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants. Chromosoma, 2004. 112(5): p. 255-66. 2. Tillemans, V., et al., Functional distribution and dynamics of Arabidopsis SR splicing factors in living plant cells. Plant J, 2005. 41(4): p. 567-82. 3. Tillemans, V., et al., Insights into Nuclear Organization in Plants as Revealed by the Dynamic Distribution of Arabidopsis SR Splicing Factors. Plant Cell, 2006. 18(11): p. 3218-34.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.