JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.

PV-1: Leukocyte trafficking molecule and vascular marker

The distinction between lymphatic vessels and blood vessels is a crucial factor in many studies in immunology, vascular biology and cancer biology. They both share several characteristics and perform related, though different functions. They are equally important for the performance of the human immune system with the continuous recirculation of leukocytes from the tissues via lymphatics to the blood vessels and back into the tissue presenting the link between both systems. This study was undertaken to elucidate the differences in the gene expression between primary blood- and lymphatic endothelial cells as well as the two immortalized cell lines HMEC-1 (human microvascular endothelial cell line 1) and TIME (telomerase immortalized microvascular endothelial cell line). Furthermore, we wanted to investigate the mystery surrounding the identity of the antigen recognized by the prototype blood vascular marker PAL-E. In the last step we wanted to study whether the PAL-E antigen would be involved in the process of leukocyte migration from the bloodstream into the surrounding tissue. Our results clearly show that the gene expression in primary blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and the cell lines HMEC-1 and TIME is plastic. Comparison of a large set of BEC- and LEC datasets allowed us to assemble a catalog of new, stable BEC- or LEC specific markers, which we verified in independent experiments. Additionally, several lines of evidence demonstrated that PAL-E recognizes plasmalemma vesicle associated protein 1 (PV-1), which can form complexes with vimentin and neuropilin-1. Finally, numerous in vitro and in vivo experiments identify the first function of the protein PV-1 during leukocyte trafficking, where it acts as regulatory molecule.

Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis

The aim of this thesis was to develop new herpes simplex virus (HSV) vectors for gene therapy of experimental autoimmune encephalomyelitis (EAE), the principal model of multiple sclerosis (MS), and to study the pathogenesis of wild-type HSV-1 and HSV-1 vectors in vivo. By introducing potential immunomodulatory factors into mice with EAE we strived to develop therapies and possibly find molecules improving recovery from EAE. We aimed at altering the immune response by inducing favorable Th2-type cytokines, thus shifting the immune response from a Th1- or a Th17-response. Our HSV vector expressing interleukin (IL)-5 modulated the cytokine responses, decreased inflammation and alleviated EAE. The use of a novel method, bacterial artificial chromosome (BAC), for engineering recombinant HSV facilitated the construction of a new vector expressing leukemia inhibitory factor (LIF). LIF is a neurotropic cytokine with broad functions in the central nervous system (CNS). LIF promotes oligodendrocyte maturation and decreases demyelination and oligodendrocyte loss. The BAC-derived HSV-LIF vector alleviated the clinical symptoms, induced a higher number of oligodendrocytes and modulated T cell responses. By administering HSV via different infection routes, e.g. peripherally via the nose or eye, or intracranially to the brain, the effect of the immune response on HSV spread at different points of the natural infection route was studied. The intranasal infection was an effective delivery route of HSV to the trigeminal ganglion and CNS, whereas corneal infection displayed limited spread. The corneal and intranasal infections induced different peripheral immune responses, which might explain the observed differences in viral spread.

Long term dietary deficiency in steers: vital functions and T3 and IGF-1 relationships

To evaluate the influence of diets with different degrees of energy deficiency on the hormonal profile and vital functions, 12 steers were randomly distributed into 3 groups of 4 animals. For 140 days, each group received (G1) a diet to promote a weight gain of 900gr/day (17.7 Mcal/d DE and 13% CP), (G2) 80% of the maintenance requirements (5.8 Mcal/d DE and 7% CP), or (G3) 60% of the maintenance requirements (4.7 Mcal/d DE and 5% CP). In G2 and G3, the energy deficit caused a marked decrease in the heart rate and respiratory rate and a reduction in the blood levels of Insulin like growth factor-1 (IGF-1) and triiodothyronine (T3). The decrease in heart rate, respiratory movement and, to a lesser extent, reduction of the rectal temperature, reflected the low status of energy and was negatively impacted by the low levels of T3. There was a strong correlation between the hormones T3 and IGF-1 (r=0.833). There were also strong correlations between T3 and HR (r=0.701), T3 and RR (r=0.632), IGF-1 and HR (r=0.731), and IGF-1 and RR (r=0.679). There were intermediate correlations between T3 and TºC (r=0.484), T3 and insulin (r=0.506), IGF-1 and insulin (r=0.517), and IGF-1 and TºC (r=0.548). This study showed the influence of a long period of providing an energy-deficient diet on animal performance, correlating hormonal status and vital functions in growing cattle. The results indicated that the evaluated parameters represent an important tool for the early detection of dietary deficiency.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

Blockade of NK-1 receptors in the lateral commissural nucleus tractus solitarii of awake rats had no effect on the cardiovascular responses to chemoreflex activation

The neurotransmission of the chemoreflex in the nucleus tractus solitarii (NTS), particularly of the sympatho-excitatory component, is not completely understood. There is evidence that substance P may play a role in the neurotransmission of the chemoreflex in the NTS. Microinjection of substance P (50 pmol/50 nl, N = 12, and 5 nmol/50 nl, N = 8) into the commissural NTS of unanesthetized rats produced a significant increase in mean arterial pressure (101 ± 1 vs 108 ± 2 and 107 ± 3 vs 115 ± 4 mmHg, respectively) and no significant changes in heart rate (328 ± 11 vs 347 ± 15 and 332 ± 7 vs 349 ± 13 bpm, respectively) 2 min after microinjection. Previous treatment with WIN, an NK-1 receptor antagonist (2.5 nmol/50 nl), microinjected into the NTS of a specific group of rats, blocked the pressor (11 ± 5 vs 1 ± 2 mmHg) and tachycardic (31 ± 6 vs 4 ± 3 bpm) responses to substance P (50 pmol/50 nl, N = 5) observed 10 min after microinjection. Bilateral microinjection of WIN into the lateral commissural NTS (N = 8) had no significant effect on the pressor (50 ± 4 vs 42 ± 6 mmHg) or bradycardic (-230 ± 16 vs -220 ± 36 bpm) responses to chemoreflex activation with potassium cyanide (iv). These data indicate that the activation of NK-1 receptors by substance P in the NTS produces an increase in baseline mean arterial pressure and heart rate. However, the data obtained with WIN suggest that substance P and NK-1 receptors do not play a major role in the neurotransmission of the chemoreflex in the lateral commissural NTS.

Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity

The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100% of subtype B (sensitivity = 1.0; specificity = 0.95), 100% of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95% of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50% of subtype A (sensitivity = 0.5; specificity = 0.95), 60% of subtype D (sensitivity = 0.6; specificity = 1.0), and 28% of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 × 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 × 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Pentoxifylline decreases tumor necrosis factor and interleukin-1 during high tidal volume

Tumor necrosis factor-alpha (TNF-alpha) is one of the most important proinflammatory cytokines which plays a central role in host defense and in the acute inflammatory response related to tissue injury. The major source of TNF-alpha are immune cells such as neutrophils and macrophages. We tested the hypothesis that pentoxifylline, a methylxanthine derivative, down-regulates proinflammatory cytokine expression during acute lung injury in rats. Male Wistar rats weighing 250 to 450 g were anesthetized ip with 50 mg/kg sodium thiopental and randomly divided into three groups: group 1 (N = 7): tidal volume (V T) = 7 ml/kg, respiratory rate (RR) = 50 breaths/min and normal saline infusion; group 2 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and normal saline infusion; group 3 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and pentoxifylline infusion. The animals were ventilated with an inspired oxygen fraction of 1.0, a positive end-expiratory pressure of 3 cmH2O, and normal saline or pentoxifylline injected into the left femoral vein. The mRNA of TNF-alpha rapidly increased in the lung tissue within 180 min of ventilation with a higher V T with normal saline infusion. The concentrations of inflammatory mediators were decreased in plasma and bronchoalveolar lavage (BAL) in the presence of higher V T with pentoxifylline infusion (TNF-alpha: plasma, 102.2 ± 90.9 and BAL, 118.2 ± 82.1; IL-1ß: plasma, 45.2 ± 42.7 and BAL, 50.2 ± 34.9, P < 0.05). We conclude that TNF-alpha produced by neutrophil influx may function as an alert signal in host defense to induce production of other inflammatory mediators.

Enhanced expression of Ang-(1-7) during pregnancy

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

The reliability of the Brazilian version of the Composite International Diagnostic Interview (CIDI 2.1)

The objective of the present study was to determine the reliability of the Brazilian version of the Composite International Diagnostic Interview 2.1 (CIDI 2.1) in clinical psychiatry. The CIDI 2.1 was translated into Portuguese using WHO guidelines and reliability was studied using the inter-rater reliability method. The study sample consisted of 186 subjects from psychiatric hospitals and clinics, primary care centers and community services. The interviewers consisted of a group of 13 lay and three non-lay interviewers submitted to the CIDI training. The average interview time was 2 h and 30 min. General reliability ranged from kappa 0.50 to 1. For lifetime diagnoses the reliability ranged from kappa 0.77 (Bipolar Affective Disorder) to 1 (Substance-Related Disorder, Alcohol-Related Disorder, Eating Disorders). Previous year reliability ranged from kappa 0.66 (Obsessive-Compulsive Disorder) to 1 (Dissociative Disorders, Maniac Disorders, Eating Disorders). The poorest reliability rate was found for Mild Depressive Episode (kappa = 0.50) during the previous year. Training proved to be a fundamental factor for maintaining good reliability. Technical knowledge of the questionnaire compensated for the lack of psychiatric knowledge of the lay personnel. Inter-rater reliability was good to excellent for persons in psychiatric practice.

Cultural adaptation and validation of the "Kidney Disease and Quality of Life - Short Form (KDQOL-SF™ 1.3)" in Brazil

The objective of the present study was to translate the Kidney Disease Quality of Life - Short Form (KDQOL-SF™1.3) questionnaire into Portuguese to adapt it culturally and validate it for the Brazilian population. The KDQOL-SF was translated into Portuguese and back-translated twice into English. Patient difficulties in understanding the questionnaire were evaluated by a panel of experts and solved. Measurement properties such as reliability and validity were determined by applying the questionnaire to 94 end-stage renal disease patients on chronic dialysis. The Nottingham Health Profile Questionnaire, the Karnofsky Performance Scale and the Kidney Disease Questionnaire were administered to test validity. Some activities included in the original instrument were considered to be incompatible with the activities usually performed by the Brazilian population and were replaced. The mean scores for the 19 components of the KDQOL-SF questionnaire in Portuguese ranged from 22 to 91. The components "Social support" and "Dialysis staff encouragement" had the highest scores (86.7 and 90.8, respectively). The test-retest reliability and the inter-observer reliability of the instrument were evaluated by the intraclass correlation coefficient. The coefficients for both reliability tests were statistically significant for all scales of the KDQOL-SF (P < 0.001), ranging from 0.492 to 0.936 for test-retest reliability and from 0.337 to 0.994 for inter-observer reliability. The Cronbach's alpha coefficient was higher than 0.80 for most of components. The Portuguese version of the KDQOL-SF questionnaire proved to be valid and reliable for the evaluation of quality of life of Brazilian patients with end-stage renal disease on chronic dialysis.

Cardiovascular actions of angiotensin-(1-7)

Angiotensin-(1-7) (Ang-(1-7)) is now considered to be a biologically active member of the renin-angiotensin system. The functions of Ang-(1-7) are often opposite to those attributed to the main effector component of the renin-angiotensin system, Ang II. Chronic administration of angiotensin-converting enzyme inhibitors (ACEI) increases 10- to 25-fold the plasma levels of this peptide, suggesting that part of the beneficial effects of ACEI could be mediated by Ang-(1-7). Ang-(1-7) can be formed from Ang II or directly from Ang I. Other enzymatic pathways for Ang-(1-7) generation have been recently described involving the novel ACE homologue ACE2. This enzyme can form Ang-(1-7) from Ang II or less efficiently by the hydrolysis of Ang I to Ang-(1-9) with subsequent Ang-(1-7) formation. The biological relevance of Ang-(1-7) has been recently reinforced by the identification of its receptor, the G-protein-coupled receptor Mas. Heart and blood vessels are important targets for the formation and actions of Ang-(1-7). In this review we will discuss recent findings concerning the biological role of Ang-(1-7) in the heart and blood vessels, taking into account aspects related to its formation and effects on these tissues. In addition, we will discuss the potential of Ang-(1-7) and its receptor as a target for the development of new cardiovascular drugs.

A multiplex PCR assay able to simultaneously detect Torque teno virus isolates from phylogenetic groups 1 to 5

Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. TTV has an extreme genetic heterogeneity which is reflected in its current classification into five main phylogenetic groups (1-5). Using specific PCR assays, it has been shown that many individuals are co-infected with TTV isolates belonging to different phylogenetic groups. Here, a multiplex PCR assay was developed, using five recombinant plasmids. Each plasmid carried an insert of different size issued from a TTV isolate belonging to a different group. The assay was able to simultaneously amplify DNAs of TTV isolates belonging to all five phylogenetic groups. Multiplex PCR was then tested satisfactorily on DNAs extracted from 55 serum samples (47 health care workers and 8 AIDS patients). All individuals but nine were infected with at least one TTV isolate. Co-infection with multiple isolates was found in 29/47 (62%) health care workers and in 8/8 (100%) AIDS patients. A number of discrepancies were observed when results obtained with three thermostable DNA polymerases were compared. For example, four TTV phylogenetic groups were detected in a particular serum sample by using one of the three DNA polymerases, whereas the other two enzymes were able to detect only three TTV groups. However, none of the three enzymes used could be broadly considered to be more efficient than the others. Despite its limitations, the assay described here constitutes a suitable tool to visualize the degree of co-infection of a given population, avoiding time-consuming experiments.