Improved NYVAC-based vaccine vectors.

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.

Rationale and design of a randomized, double-blind, parallel-group study of terutroban 30 mg/day versus aspirin 100 mg/day in stroke patients: the prevention of cerebrovascular and cardiovascular events of ischemic origin with terutroban in patients with a history of ischemic stroke or transient ischemic attack (PERFORM) study.

BACKGROUND: Ischemic stroke is the leading cause of mortality worldwide and a major contributor to neurological disability and dementia. Terutroban is a specific TP receptor antagonist with antithrombotic, antivasoconstrictive, and antiatherosclerotic properties, which may be of interest for the secondary prevention of ischemic stroke. This article describes the rationale and design of the Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient ischeMic Attack (PERFORM) Study, which aims to demonstrate the superiority of the efficacy of terutroban versus aspirin in secondary prevention of cerebrovascular and cardiovascular events. METHODS AND RESULTS: The PERFORM Study is a multicenter, randomized, double-blind, parallel-group study being carried out in 802 centers in 46 countries. The study population includes patients aged > or =55 years, having suffered an ischemic stroke (< or =3 months) or a transient ischemic attack (< or =8 days). Participants are randomly allocated to terutroban (30 mg/day) or aspirin (100 mg/day). The primary efficacy endpoint is a composite of ischemic stroke (fatal or nonfatal), myocardial infarction (fatal or nonfatal), or other vascular death (excluding hemorrhagic death of any origin). Safety is being evaluated by assessing hemorrhagic events. Follow-up is expected to last for 2-4 years. Assuming a relative risk reduction of 13%, the expected number of primary events is 2,340. To obtain statistical power of 90%, this requires inclusion of at least 18,000 patients in this event-driven trial. The first patient was randomized in February 2006. CONCLUSIONS: The PERFORM Study will explore the benefits and safety of terutroban in secondary cardiovascular prevention after a cerebral ischemic event.

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer.

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.

Improved SNR efficiency in gradient echo coronary MRA with high temporal resolution using parallel imaging.

Contemporary coronary magnetic resonance angiography techniques suffer from signal-to-noise ratio (SNR) constraints. We propose a method to enhance SNR in gradient echo coronary magnetic resonance angiography by using sensitivity encoding (SENSE). While the use of sensitivity encoding to improve SNR seems counterintuitive, it can be exploited by reducing the number of radiofrequency excitations during the acquisition window while lowering the signal readout bandwidth, therefore improving the radiofrequency receive to radiofrequency transmit duty cycle. Under certain conditions, this leads to improved SNR. The use of sensitivity encoding for improved SNR in three-dimensional coronary magnetic resonance angiography is investigated using numerical simulations and an in vitro and an in vivo study. A maximum 55% SNR enhancement for coronary magnetic resonance angiography was found both in vitro and in vivo, which is well consistent with the numerical simulations. This method is most suitable for spoiled gradient echo coronary magnetic resonance angiography in which a high temporal and spatial resolution is required.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

The role of vectors in the epidemiology of avian malaria

Studies on host-parasite relationships have commonly reported that parasitized hosts undergo changes in their behavioural and life history traits. How do these changes affect the fitness of the hosts? What are the ecological and evolutionary drivers of these changes? These open questions are crucial to predict the parasite spread amongst hosts. Surprisingly, mosquito vectors of diseases to humans and animals have long been seen as passive parasite transporters, being unaffected by the infection though they also function as hosts. Natural parasite-vector interactions are therefore poorly documented in the literature. In this thesis, we seek to address the role of wild vectors in the epidemiology of avian Plasmodium, the etiological agents of malaria in birds. We first conducted avian malaria surveys in field-caught mosquitoes to identify the natural vectors in our temperate study area. We report that ornithophilic Culex pipiens primarily act as a vector for Plasmodium vaughani in spring, this parasite species being progressively replaced by P. relictum along with the season. Season-related factors may thus shape the mosquitoes' vectorial capacity. We then used experimental approaches to determine the effect of avian malaria on wild, naturally infected C. pipiens. We show that infected mosquitoes incur unavoidable physiological costs associated with parasite exploitation, these costs being expressed as a reduced survival under nutritionally stressed conditions only. These results are of significant importance for the epidemiology of avian malaria since seasonal changes in climate may likely influence food quality and quantity available to the mosquitoes. The host-selection preferences of the vectors with respect to the malaria-infection status of their bird hosts largely determine the disease spreading. In a second laboratory experiment, we thus offered wild C. pipiens the opportunity to choose between uninfected and naturally infected great tits, Parus major. We show that host-seeking mosquitoes have innate orientation preferences for uninfected birds. This suggests that avian malaria parasites exert strong selective pressures on their vectors, pushing them to evolve anti-parasite behaviours. We lastly investigated the links between malaria-associated symptoms in birds and resulting attractiveness to the mosquitoes. We show that experimentally malaria-infected canaries, Serinus canaria, suffer severe haematocrit reduction at peak parasitaemia and reduced basal metabolic rate later in the course of the infection. However, no links between infection and bird attractiveness to the mosquitoes were shown in an experiment using canaries as live bait for mosquito trap in the field. These links may have been masked by confounding environmental factors. Using a system where the vectors, parasites and hosts co-occur in sympatry, this thesis illustrates that vectors are not always Plasmodium permissive, which opposes to the traditional view that malaria parasites should have little effect on their vectors. The way that the vectors respond to the parasite threat is largely determined by the environmental conditions. This may have major implications for the epidemiology of avian malaria. - Les études portant sur les relations hôtes-parasites mentionnent souvent que les hôtes parasités subissent des modifications de leurs traits d'histoire de vie ou bien comportementaux. Comment ces changements affectent-ils la valeur sélective des hôtes et celle de leurs parasites ? Quels sont les déterminants de ces modifications ? Ces questions sont d'un grand intérêt en épidémiologie. Pour autant, les moustiques vecteurs de maladies infectieuses ont longtemps été perçus comme de simples transporteurs de parasites, n'étant pas affectés par ces derniers. Cette thèse porte sur le rôle des vecteurs dans l'épidémiologie des Plasmodium aviaires, agents étiologiques de la malaria chez les oiseaux. Dans le but d'identifier les vecteurs naturels de malaria aviaire dans notre zone d'étude, nous avons tout d'abord collecté des moustiques sur le terrain, puis déterminé leur statut infectieux. Nous rapportons que les moustiques Culex pipiens sont principalement impliqués dans la transmission de Plasmodium vaughani au printemps, cette espèce de parasite étant progressivement remplacée par P. relictum au fil de la saison de transmission. Nous avons ensuite conduit une expérience visant à déterminer l'effet de la malaria aviaire sur des C. pipiens sauvages, naturellement infectés. Nous montrons que des coûts sont associés à l'infection pour les moustiques. Ces coûts occasionnent une diminution de la survie des vecteurs seulement lorsque ceux-ci sont privés de ressources nutritionnelles. Des changements saisonniers de climats pourraient affecter la quantité et la qualité des ressources disponibles pour les vecteurs et donc, leur aptitude à transmettre l'infection. Les traits comportementaux des moustiques vecteurs, tels que la recherche et le choix d'un hôte pour se nourrir, sont d'une importance majeure pour la dispersion de la malaria. Pour cela, nous avons offert à des C. pipiens sauvages l'opportunité de choisir simultanément entre une mésange charbonnière (Parus major) saine et une autre naturellement infectée. Nous montrons que les moustiques s'orientent préférentiellement vers des mésanges saines. Les Plasmodium aviaires exerceraient donc de fortes pressions de sélection sur leurs vecteurs, favorisant ainsi l'évolution de comportements d'évitement des parasites. Enfin nous avons cherché à identifier de potentiels liens entre symptômes de l'infection malarique chez les oiseaux et attractivité de ces derniers pour les moustiques. Nous montrons que des canaris (Serinus canaria) expérimentalement infectés sont fortement anémiés au moment du pic infectieux et que leur métabolisme basai diminue plus tard au cours de l'infection. Toutefois, aucun lien entre le statut infectieux et l'attractivité des canaris pour les moustiques n'a pu être montré lors d'une expérience réalisée en nature. Il se peut que ces liens aient été masqués par des facteurs environnementaux confondants. Dans son ensemble, cette thèse illustre que, contrairement aux idées reçues, les vecteurs de malaria aviaire ne sont pas toujours permissifs avec leurs parasites. L'environnement apparaît aussi comme un facteur déterminant dans la réponse des vecteurs face à la menace d'infection malarique. Cela pourrait fortement affecter l'épidémiologie de la malaria aviaire.

C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes.

Phenotypic convergence is a widespread and well-recognized evolutionary phenomenon. However, the responsible molecular mechanisms remain often unknown mainly because the genes involved are not identified. A well-known example of physiological convergence is the C4 photosynthetic pathway, which evolved independently more than 45 times [1]. Here, we address the question of the molecular bases of the C4 convergent phenotypes in grasses (Poaceae) by reconstructing the evolutionary history of genes encoding a C4 key enzyme, the phosphoenolpyruvate carboxylase (PEPC). PEPC genes belong to a multigene family encoding distinct isoforms of which only one is involved in C4 photosynthesis [2]. By using phylogenetic analyses, we showed that grass C4 PEPCs appeared at least eight times independently from the same non-C4 PEPC. Twenty-one amino acids evolved under positive selection and converged to similar or identical amino acids in most of the grass C4 PEPC lineages. This is the first record of such a high level of molecular convergent evolution, illustrating the repeatability of evolution. These amino acids were responsible for a strong phylogenetic bias grouping all C4 PEPCs together. The C4-specific amino acids detected must be essential for C4 PEPC enzymatic characteristics, and their identification opens new avenues for the engineering of the C4 pathway in crops.

Verkkovaihtosuuntaajan vektorisäädön toteutus FPGA-piirillä

Verkkovaihtosuuntaajalla pystytään muuntamaan tasajännite vaihtojännitteeksi ja päinvastoin. Verkkovaihtosuuntaajan toiminta perustuu tehokytkinten ohjaukseen ja sopivan modulointimenetelmän käyttöön. Vektorisäädössä vaihtosuuntaajanvirrat ja jännitteet esitetään kompleksitasossa, jolloin virta- ja jännitekomponentit voidaan esittää vektoreina. Vektorisäädössä verkkovaihtosuuntaajan ohjaustoteutetaan laskemalla kompleksitasossa vektoreille arvot, jotka tuottavat vaihtosuuntaajan lähtöön halutun vektorin. Koska FPGA-piirit mahdollistavat nopean rinnakkaisen laskennan, soveltuvat ne hyvin vektorisäädön toteuttamiseen. FPGA-piirien rakenteesta johtuen on säätöjärjestelmän suunnittelussa huomioitava kiinteän pilkun lukujen riittävä bittileveys ja järjestelmän diskretointiaika. Työssä suunnitellaan verkkovaihtosuuntaajan vektorisäätö ja tutkitaan bittileveyden vaikutusta säädön toteuttamiseen FPGA-piirillä. Bittileveyden tarkasteluun esitetään käytettäväksi tilastollisia menetelmiä. Työssä tarkastellaan kiinteän pilkun järjestelmän ja liukulukujärjestelmän erosuureen tilastollisia tunnusmerkkejä sekä histogrammia. Tarkasteluissa huomattiin, että maksimivirhe itsessään ei tarjoa riittävästi tietoa erosuureen jakautumisesta. Näin ollen maksimivirhe ei ole kaikissa tilanteissa sovelias menetelmä riittävän bittitarkkuuden määrittämiseen. Työssä esitetään riittävän bittitarkkuuden määrittelemiseen käytettäväksi otossuureista otosvarianssia, keskipoikkeamaa ja vaihteluväliä.

Novel insulated gamma and lentis retroviral vectors towards safer genetic modification of stem cells

In otherwise successful gene therapy trials insertional mutagenesis has resulted in leukemia. The identification of new short synthetic genetic insulator elements (GIE) which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts with e.g. b-globin HS4, have met with poor efficacy and genetic instability. We have investigated potential improvement with two new candidate synthetic GIEs in SIN-gamma and lentiviral vectors. With each constructs two internal promoters have been tested: either the strong Fr- MuLV-U3 or the housekeeping hPGK.We could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro and lentivectors. In target cells a dramatic shift of expression is observed with an homogenous profile the level of which strictly depends on the promoter strength. These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI). In comparison, control native and SIN vectors expression levels show heterogeneous, depend on the MOI and prove unstable. We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing. Data will be presented. Further genotoxicity assessment will include in vivo studies. We have established insulated vectors which harbour both boundary and enhancer-blocking effect and show stable in prolonged in vitro culture conditions. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006-018933

Lentiviral vectors efficiently transduce the EGFP gene into cardiomyocytes in vivo : immunohistology and Elisa quantification of the transgene

RESUME Les maladies cardio-vasculaires représentent la cause la plus importante de mortalité et de morbidité dans les pays occidentaux. La thérapie génique offre une nouvelle approche au traitement de ces maladies. L'expression de gènes protecteurs dans le myocarde par des technologies de transfert génique peut améliorer la fonction ventriculaire lors de l'insuffisance cardiaque ou stimuler la formation de nouveaux vaisseaux dans la maladie coronarienne. Etant donné qu'une majorité des maladies cardiaques sont des maladies chroniques, l'expression durable du gène thérapeutique introduit dans le coeur est souhaitable dans de nombreux cas. Malheureusement, l'utilité des vecteurs de transfert génique les plus utilisés en thérapie génique cardiovasculaire est limitée par une performance faible (ADN plasmidique) et une courte durée d'expression (adénovirus). Récemment, des vecteurs de transfert génique dérivés des lentivirus, une sous-famille des rétrovirus, ont retenu l'attention de la communauté scientifique en raison de leur capacité à exprimer des gènes à long terme. Contrairement aux vecteurs rétroviraux traditionnels, les vecteurs lentiviraux transduisent des gènes même dans des cellules qui ne se divisent pas, ce qui est le cas des cardiomyocytes adultes. Ces vecteurs présentent un profil de biosécurité comparable à celui des vecteurs rétroviraux traditionnels. Nous avons donc décidé de tester l'utilité des vecteurs lentiviraux pour le transfert génique dans des cardiomyocytes de rat adulte in vitro et in vivo. Plusieurs versions de vecteurs lentiviraux contenant différent promoteurs ont été construites. Ces vecteurs contenant le gène marqueur EGFP (enhanced green fluorescent protein) ont été testés dans des cardiomyocytes de rat in vitro, ainsi que dans des coeurs de rat in vivo. Le but de ces expériences était de déterminer la durée de l'expression du transgène après injection intramyocardique chez le rat. Pour ce faire, nous avons développé une technique ELISA pour détecter la protéine EGFP dans des extraits de tissu cardiaque. Les résultats ont montré que la protéine EGFP était encore présente à des niveaux significatifs jusqu'à dix semaines après l'injection de vecteurs lentiviraux, alors que l'expression transgénique obtenue avec un vecteur adénoviral traditionnel a été plus limitée dans le temps. Ces résultats démontrent la capacité des vecteurs lentiviraux à exprimer des gènes d'intérêt de manière performante et stable dans le cœur de rat adulte in vivo. SUMMARY Cardiovascular diseases are the first cause of morbidity and mortality in Western countries. Gene therapy offers a new approach to these diseases. Expression of therapeutic genes in the myocardium by gene transfer technologies can improve ventricular function in heart failure and stimulate neovascularization in coronary disease. Chronic heart diseases likely require sustained expression of the therapeutic gene within the heart itself. Unfortunately, the most commonly used vectors in cardiovascular gene therapy, i.e. plasmid DNA and recombinant adenovirus vectors, are limited by poor DNA uptake and transient transgene expression, respectively. Recently, lentivirus-derived vectors have attracted much interest because of their ability to achieve long-term transgene expression. In contrast to traditional retroviral vectors, lentiviral vectors are also able to transduce non- dividing cells, while presenting a comparable biosafety profile. Adult cardiomyocytes are terminally differentiated cells that do not divide under normal conditions. For these reasons, we have decided to evaluate the efficiency of lentiviral vectors for gene-transduction of adult cardiomyocytes both in vitro and in vivo. We constructed various types of lentiviral vectors containing various promoters. Vectors encoding EGFP as a reporter gene were tested in rat cardiomyocytes in vitro and in rat hearts in vivo. The aim of the experiments involved in this thesis work was to determine the duration of the expression of the transgene after rat intramyocardial injection using a quantitative assay. Therefore, an ELISA technique was set up to measure the EGFP protein in rat heart tissue extracts. Our results showed that the EGFP protein was still present at significant levels at ten weeks after lentiviral vector injection, whereas the duration of expression with adenoviral vectors was shorter. These results demonstrate that lentiviral vectors efficiently deliver genes and achieve sustained transgene expression in adult rat cardiomyocytes in vivo.

Data-parallel computation in parallel and distributed environments

The past few decades have seen a considerable increase in the number of parallel and distributed systems. With the development of more complex applications, the need for more powerful systems has emerged and various parallel and distributed environments have been designed and implemented. Each of the environments, including hardware and software, has unique strengths and weaknesses. There is no single parallel environment that can be identified as the best environment for all applications with respect to hardware and software properties. The main goal of this thesis is to provide a novel way of performing data-parallel computation in parallel and distributed environments by utilizing the best characteristics of difference aspects of parallel computing. For the purpose of this thesis, three aspects of parallel computing were identified and studied. First, three parallel environments (shared memory, distributed memory, and a network of workstations) are evaluated to quantify theirsuitability for different parallel applications. Due to the parallel and distributed nature of the environments, networks connecting the processors in these environments were investigated with respect to their performance characteristics. Second, scheduling algorithms are studied in order to make them more efficient and effective. A concept of application-specific information scheduling is introduced. The application- specific information is data about the workload extractedfrom an application, which is provided to a scheduling algorithm. Three scheduling algorithms are enhanced to utilize the application-specific information to further refine their scheduling properties. A more accurate description of the workload is especially important in cases where the workunits are heterogeneous and the parallel environment is heterogeneous and/or non-dedicated. The results obtained show that the additional information regarding the workload has a positive impact on the performance of applications. Third, a programming paradigm for networks of symmetric multiprocessor (SMP) workstations is introduced. The MPIT programming paradigm incorporates the Message Passing Interface (MPI) with threads to provide a methodology to write parallel applications that efficiently utilize the available resources and minimize the overhead. The MPIT allows for communication and computation to overlap by deploying a dedicated thread for communication. Furthermore, the programming paradigm implements an application-specific scheduling algorithm. The scheduling algorithm is executed by the communication thread. Thus, the scheduling does not affect the execution of the parallel application. Performance results achieved from the MPIT show that considerable improvements over conventional MPI applications are achieved.

Introducing massively parallel computers into operational weather forecasting

Numerical weather prediction and climate simulation have been among the computationally most demanding applications of high performance computing eversince they were started in the 1950's. Since the 1980's, the most powerful computers have featured an ever larger number of processors. By the early 2000's, this number is often several thousand. An operational weather model must use all these processors in a highly coordinated fashion. The critical resource in running such models is not computation, but the amount of necessary communication between the processors. The communication capacity of parallel computers often fallsfar short of their computational power. The articles in this thesis cover fourteen years of research into how to harness thousands of processors on a single weather forecast or climate simulation, so that the application can benefit as much as possible from the power of parallel high performance computers. The resultsattained in these articles have already been widely applied, so that currently most of the organizations that carry out global weather forecasting or climate simulation anywhere in the world use methods introduced in them. Some further studies extend parallelization opportunities into other parts of the weather forecasting environment, in particular to data assimilation of satellite observations.

A survey of potential vectors of apricot chlorotic leaf roll

A comparative survey was done in leafhopper populations captured in apricot orchards in two areas of Valencia, one with considerable natural spread of apricot chlorotic leaf roll (ACLR), and the other where such natural spread is virtually nonexistent. An identification of the leafhopper species found in the first and in the second area suggest that Neoaliturus haematoceps and/or Neoaliturus fertestratus are the potential vectors of ACLR, at least under the conditions of Valencia province. Psammotettix striatus and Austroagallia sinuata are potential secondary vextors of ACLR.

Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade.

Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation. Indeed, we found that Hog1 phosphorylates its activating kinase Ssk2 on several sites, and cells expressing a non-phosphorylatable Ssk2 mutant are partially defective for feedback regulation and proper control of basal Hog1 activity. Together, our data suggest that Hog1 activity is controlled by intertwined regulatory mechanisms operating with varying kinetics, which together tune the Hog1 response to balance basal Hog1 activity and its steady-state level after adaptation to high osmolarity.