Inhibition Of Protein Aggregation Research Articles

O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation without Affecting Membrane Binding.

The aggregation of neurodegenerative-disease associated proteins can be affected by many factors, including a variety of post-translational modifications. One such modification, O-GlcNAcylation, has been found on some of these aggregation prone proteins, including α-synuclein, the major protein that plays a causative role in synucleinopathies like Parkinson's disease. We previously used synthetic protein chemistry to prepare α-synuclein bearing a homogeneous O-GlcNAc modification at threonine 72 and showed that this modification inhibits protein aggregation. However, the effects of the other eight O-GlcNAcylation sites that have been identified were unknown. Here, we use a similar synthetic strategy to investigate the consequences of this modification at one of these sites, serine 87. We show that O-GlcNAcylation at this site also inhibits α-synuclein aggregation but to a lesser extent than that for the same modification at threonine 72. However, we also find that this modification does not affect the membrane-binding properties of α-synuclein, which differentiates it from phosphorylation at the same site. These results further support the development of therapies that can elevate O-GlcNAcylation of α-synuclein to slow the progression of Parkinson's disease.

Folic acid inhibits the amyloid fibrils formation of β-lactoglobulin

It is well known that amyloid fibrils are associated with a lot of neurodegenerative diseases. Thus, inhibition of protein aggregation and disruption of the formed amyloid fibrils have been attractive therapeutic strategies for amyloid-associated diseases. Here, we take the widely studied protein, β-lactoglobulin (β-LG), as a model protein to study the inhibition of amyloid fibrils. Using multiple biophysical and biochemical approaches, we successfully identify that folic acid can strongly inhibit amyloid fibrils formation of β-LG in 5 M urea solution. In the presence of folic acid, both the nucleation step and elongation step of β-LG fibrillation can be affected and the suppression efficiency followed a dose depended manner. Furthermore, the studies of protein intrinsic-fluorescence and anilinonaphthalene-8-sulfonic acid (ANS) fluorescence demonstrated that the folic acid decreased the hydrophobicity of the urea-denatured β-LG and changed the conformation of β-LG. Our study provides a convenient way to inhibit the amyloid fibrils formation, which has potential help to study the mechanism of amyloid-diseases.

Secondary structural alterations in glucoamylase as an influence of protein aggregation

Glucoamylase (EC 3.2.1.3) from Aspergillus niger possesses 31% α-helix, 36% β structure and rest aperiodic structure. A transition of glucoamylase structure in the presence of varying concentrations of glyoxal (GO) and trifluoroethanol (TFE) was studied by using multi-methodological approaches. At 20% GO, glucoamylase exists as molten globule state as evident by high tryptophan and ANS fluorescence, retention of secondary structure and loss of native tertiary structure. This state precedes the onset of the aggregation process and maximum is achieved at the highest concentration i.e. at 90% of GO. In parallel study TFE, on increasing concentration up to 25% induces secondary structure transformation leading to accumulation of intermolecular β sheets, altered tryptophan environment, high ANS and ThT fluorescence resulting in the formation of glucoamylase aggregates. Isothermal titration calorimetric curve is sigmoidal, indicating the weak binding of GO/TFE and glucoamylase. TEM studies showed that glucoamylase exists as globular and amorphous aggregates at 90% glyoxal and 25% TFE respectively. Further, TFE at 70% causes inhibition of enzyme aggregates; the majority of secondary structures observed at this concentration are α helices. Alpha helices being the main key player relocates glucoamylase native environment as evident by CD, FTIR and TEM. Hence induction of β sheet promotes protein aggregation and α helices inhibits protein aggregation.

Redox interactions of methylene blue with cysteine amino acid as a possible mechanizm of biological action of the dye

Design of the inhibitors of the pathogenic protein aggregation, associated with the neural cell damage, is of great importance for the therapy of the neurodegenerative diseases. In this work redox interactions of methylene blue as a potential drug against Alzheimer’s disease with its potential target – cysteine of the active cite of Tau proteins, have been studied in vitro, in order to establish the molecular mechanism of methylene blue biological action. First, a single product of direct oxidation of cysteine by methylene blue, viz. cystine, possessing a disulfide bridge between two cysteine molecules, has been detected by mass spectrometry. Such modification of cysteine residues was suggested to affect secondary structure of Tau-proteins, hampering amyloid fibril formation. Notably, we failed to detect any oxygen-containing products of cysteine oxidation. Finally, reduction of methylene blue in the above reaction resulted in the formation of its leuco form, which could be also considered as a potential drug in the therapy of neurodegenerative diseases.

Differential effects of silver and iron oxide nanoparticles on IAPP amyloid aggregation.

Recent studies have shown promise on the use of small molecules and nanoparticles (NPs) for the inhibition of protein aggregation, a hallmark of neurodegenerative diseases and type 2 diabetes (T2D). Towards this end here we show the differential effects of silver and iron oxide nanoparticles (AgNPs and IONPs) on the mesoscopic properties of human islet amyloid polypeptide (IAPP) aggregation associated with T2D. Both citrate- and branched polyethyleneimine-coated AgNPs (c-AgNPs, bPEI-AgNPs) inhibited IAPP aggregation at 500 μg mL-1, likely through electrostatic attraction and sequestering of IAPP monomers from fibrillation. In comparison, bare, brushed polyethylene glycol- and phosphorylcholine-grafted IONPs (bPEG-IONPs, bPC-IONPs) at 500 μg mL-1 elicited no major effect on IAPP fibril contour length, while bPC-IONPs induced significant fibril softening and looping likely mediated by dipolar interactions. While monovalent Ag+ up to 50 μg mL-1 showed no effect on the contour length or stiffness of IAPP fibrils, multivalent Fe3+ at 5 μg mL-1 halted IAPP fibrillation likely through ion-peptide crosslinking. Except bPEI-AgNPs, all three types of IONPs and c-AgNPs at 100 μg mL-1 alleviated IAPP toxicity in HEK293 cells indicating no clear correlation between protein aggregation and their induced cytotoxicity. This study demonstrates the complexity of protein aggregation intervened by NPs of different physicochemical properties and - together with existing literature - facilitates nanotechnological applications for mitigating amyloid-mediated pathologies.

Efficacy and safety of tau-aggregation inhibitor therapy in patients with mild or moderate Alzheimer's disease: a randomised, controlled, double-blind, parallel-arm, phase 3 trial

Leuco-methylthioninium bis(hydromethanesulfonate; LMTM), a stable reduced form of the methylthioninium moiety, acts as a selective inhibitor of tau protein aggregation both in vitro and in transgenic mouse models. Methylthioninium chloride has previously shown potential efficacy as monotherapy in patients with Alzheimer's disease. We aimed to determine whether LMTM was safe and effective in modifying disease progression in patients with mild to moderate Alzheimer's disease. We did a 15-month, randomised, controlled double-blind, parallel-group trial at 115 academic centres and private research clinics in 16 countries in Europe, North America, Asia, and Russia with patients younger than 90 years with mild to moderate Alzheimer's disease. Patients concomitantly using other medicines for Alzheimer's disease were permitted to be included because we considered it infeasible not to allow their inclusion; however, patients using medicines carrying warnings of methaemoglobinaemia were excluded because the oxidised form of methylthioninium in high doses has been shown to induce this condition. We randomly assigned participants (3:3:4) to 75 mg LMTM twice a day, 125 mg LMTM twice a day, or control (4 mg LMTM twice a day to maintain blinding with respect to urine or faecal discolouration) administered as oral tablets. We did the randomisation with an interactive web response system using 600 blocks of length ten, and stratified patients by severity of disease, global region, whether they were concomitantly using Alzheimer's disease-labelled medications, and site PET capability. Participants, their study partners (generally carers), and all assessors were masked to treatment assignment throughout the study. The coprimary outcomes were progression on the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) and the Alzheimer's Disease Co-operative Study-Activities of Daily Living Inventory (ADCS-ADL) scales from baseline assessed at week 65 in the modified intention-to-treat population. This trial is registered with Clinicaltrials.gov (NCT01689246) and the European Union Clinical Trials Registry (2012-002866-11). Between Jan 29, 2013, and June 26, 2014, we recruited and randomly assigned 891 participants to treatment (357 to control, 268 to 75 mg LMTM twice a day, and 266 to 125 mg LMTM twice a day). The prespecified primary analyses did not show any treatment benefit at either of the doses tested for the coprimary outcomes (change in ADAS-Cog score compared with control [n=354, 6·32, 95% CI 5·31-7·34]: 75 mg LMTM twice a day [n=257] -0·02, -1·60 to 1·56, p=0·9834, 125 mg LMTM twice a day [n=250] -0·43, -2·06 to 1·20, p=0·9323; change in ADCS-ADL score compared with control [-8·22, 95% CI -9·63 to -6·82]: 75 mg LMTM twice a day -0·93, -3·12 to 1·26, p=0·8659; 125 mg LMTM twice a day -0·34, -2·61 to 1·93, p=0·9479). Gastrointestinal and urinary effects were the most common adverse events with both high doses of LMTM, and the most common causes for discontinuation. Non-clinically significant dose-dependent reductions in haemoglobin concentrations were the most common laboratory abnormality. Amyloid-related imaging abnormalities were noted in less than 1% (8/885) of participants. The primary analysis for this study was negative, and the results do not suggest benefit of LMTM as an add-on treatment for patients with mild to moderate Alzheimer's disease. Findings from a recently completed 18-month trial of patients with mild Alzheimer's disease will be reported soon. TauRx Therapeutics.

Hsp70: Therapeutic Potential in Acute and Chronic Cardiac Disease Settings

Heat shock proteins are a family of proteins that are produced by cells in response to exposure to stressful conditions. The best studied heat shock protein is HSP70, which is known to act as a molecular chaperone to maintain cellular homeostasis and inhibit protein aggregation in response to stress. While early animal studies suggested that increasing HSP70 in the heart (using a transgenic, gene transfer or pharmacological approach) provided cardiac protection against acute cardiac stress, recent studies have found no benefit of increasing HSP70 in mouse models of chronic cardiac stress. As HSP70 has been considered a potential therapeutic target, it is important to comprehensively assess HSP70 therapies in preclinical models of acute and chronic cardiac disease.

Delaying aging in Caenorhabditis elegans with protein aggregation inhibitors

Recent evidence suggests that during aging there is widespread accumulation of aggregated insoluble proteins, even in the absence of pathological conditions. Pharmacological manipulation of protein aggregation might be helpful to unveil the involvement of protein aggregates during aging, as well as to develop novel strategies to delay aging. Here we investigated the effect of known protein aggregation inhibitors on the lifespan and health-span of Caenorhabditis elegans. For this purpose, we selected various structurally diverse anti-aggregation compounds and screened them in liquid and solid medium for their ability to alter the rate of aging in vivo. Our results show that treatment of C. elegans with diverse aggregation inhibitors significantly increases the animal lifespan and health-span. These findings indicate that protein misfolding and aggregation may play an important role in cellular dysfunction during aging, opening a novel approach to increase longevity and enhance the quality of life during aging.

Trehalose Inhibits Protein Aggregation Caused by Transient Ischemic Insults Through Preservation of Proteasome Activity, Not via Induction of Autophagy

Protein aggregation has been proved to be a pathological basis accounting for neuronal death caused by either transient global ischemia or oxygen glucose deprivation (OGD), and inhibition of protein aggregation is emerging as a potential strategy of preventing brain damage. Trehalose was found to inhibit protein aggregation caused by neurodegenerative diseases via induction of autophagy, whereas its effect is still elusive on ischemia-induced protein aggregation. In this study, we investigated this issue by using rat model of transient global ischemia and SH-SY5Y model of OGD. We found that pretreatment with trehalose inhibited transient global ischemia-induced neuronal death in the hippocampus CA1 neurons and OGD-induced death in SH-SY5Y cells, which was associated with inhibition of the formation of ubiquitin-labeled protein aggregates and preservation of proteasome activity. In vitro study showed that the protection of trehalose against OGD-induced cell death and protein aggregation in SH-SY5Y cells was reversed when proteasome activity was inhibited by MG-132. Further studies revealed that trehalose prevented OGD-induced reduction of proteasome activity via suppression of both oxidative stress and endoplasmic reticulum stress. Particularly, our results showed that trehalose inhibited OGD-induced autophagy. Therefore, we demonstrated that proteasome dysfunction contributed to protein aggregation caused by ischemic insults and trehalose prevented protein aggregation via preservation of proteasome activity, not via induction of autophagy.

The Fermentation Analogy: A Point of View for Understanding the Intriguing Role of Proline Accumulation in Stressed Plants.

The accumulation of proline under environmental stress is a conserved response of plants. Five decades have passed since the first report of proline accumulation in plants (Barnnet and Naylor, 1966). Many hypotheses have been put forward regarding assignment of a function to proline. These proposed roles include antioxidant capacity, osmoprotection, signaling, developmental function, and contributing in redox and cellular homeostasis (Smirnoff and Cumbes, 1989; Mattioli et al., 2008; Szabados and Savoure, 2010; Kishor et al., 2015). Among these, the osmotic adjustment, osmoprotectant, and antioxidant role have probably been the earliest and most widely accepted functions of proline. It is known that compatible osmolytes contribute in the retention of water, but also some of them force proteins to adopt a compactly folded structure, preventing the unfolding and reducing the exposed surface of the protein to damaging compounds (Attri et al., 2010). In case of proline this was demonstrated both in vitro and in single cell organisms. For example, it was observed that proline overproducing E. coli mutants had greater osmotolerance (Csonka et al., 1988) and also proline can inhibit protein aggregation in vivo (Ignatova and Gierasch, 2006). However, neither osmotic adjustment nor osmoprotection has been clearly confirmed in plants (Maggio et al., 2002; Kavi Kishor and Sreenivasulu, 2014). This is probably more complex in plants because plants have different osmoregulatory mechanisms. Controversy is present regarding its proposed antioxidant role because recent evidence demonstrates that proline cannot scavenge singlet oxygen, superoxide, nitric oxide, peroxynitrite nor nitrogen dioxide (Signorelli et al., 2013, 2016). In view of the lack of activity in previously suggested roles it has recently been proposed that proline acts exclusively as a hydroxyl radical scavenger (Signorelli et al., 2014, 2015), but more evidence for the physiological importance of this role is needed.

Cyclodextrin–protein interaction as inhibiting factor against aggregation

With the aim of better understanding how cyclodextrins can inhibit protein aggregation, a calorimetric study is reported concerning the determination of the thermodynamic properties characterizing the interaction process between natural or modified cyclodextrins and two model proteins: lysozyme and ovalbumin. The study will examine how the interaction depends on the dimensions of cyclodextrins, on the nature of their chemical modifications, and on the conformational state of the proteins. To that, the interaction has been studied in buffer, where proteins are in their native form, and in 8 mol kg−1 urea where they are in the denatured form. The technique employed is the isothermal calorimetry, which allows, in the case of the formation of a complex, to obtain the complete thermodynamic framework of the association process: enthalpy, association constant, Gibbs energy and entropy. The values and signs of these quantities will allow to investigate the forces acting in the interaction. The dimensions of the cavity and the presence of modifications on the cyclodextrin play a major role, determining the forces acting in the complexation process. The two proteins, in their native form, do not form complexes with natural cyclodextrins. Alkylated cyclodextrins, instead, form complexes with both proteins. In 8 mol kg−1 urea, where proteins are denatured, lysozyme binds to the α- and β-cyclodextrins examined, while ovalbumin forms complexes only with substituted β-cyclodextrins. The enthalpy–entropy compensation underlines that the association processes are determined by the changes experienced by water in the hydration shells of the interacting substances.

Efficient Inhibition of Protein Aggregation, Disintegration of Aggregates, and Lowering of Cytotoxicity by Green Tea Polyphenol-Based Self-Assembled Polymer Nanoparticles.

Green tea polyphenol epigallocatechin-3-gallate (EGCG) is known for its antiamyloidogenic property, and it is observed that molecular EGCG binds with amyloid structure, redirects fibrillation kinetics, remodels mature fibril, and lowers the amyloid-derived toxicity. However, this unique property of EGCG is difficult to utilize because of their poor chemical stability and substandard bioavailability. Here we report a nanoparticle form of EGCG of 25 nm size (nano-EGCG) which is 10-100 times more efficient than molecular EGCG in inhibiting protein aggregation, disintegrating mature protein aggregates, and lowering amyloidogenic cytotoxicity. The most attractive advantage of nano-EGCG is that it efficiently protects neuronal cells from the toxic effect of extracellular amyloid beta or intracellular mutant huntingtin protein aggregates by preventing their aggregation. We found that the better performance of nano-EGCG is due to the combined effect of increased chemical stability of EGCG against degradation, stronger binding with protein aggregates, and efficient entry into the cell for interaction with aggregated protein structure. This result indicates that the nanoparticle form of antiamyloidogenic molecules can be more powerful in prevention and curing of protein aggregation derived diseases.

Water‐Soluble Conjugated Polymers for the Detection and Inhibition of Protein Aggregation

Protein aggregation is etiologically associated with a wide range of devastating neurodegenerative diseases such as Alzheimer's and prion diseases, and therefore the new strategies for the detection and inhibition of protein aggregation are urgently desired. Herein, the capability of a cationic water‐soluble poly(phenylene vinylene) derivative (PPV‐NMe3+) to detect and inhibit the protein aggregation has been explored, using the hen egg white lysozyme (HEWL) as a model system whose aggregation is triggered by UV illumination. The PPV‐NMe3+ binds efficiently to the surface of the HEWL aggregates driven by the hydrophobic interactions, leading to the reduction of interchain contacts and therefore the enhancement of fluorescence intensity of the conjugated polymer. Importantly, the hydrophobic interactions between HEWL and PPV‐NMe3+ compete with the interaction that dominates the formation of the HEWL self‐assembly, and the electrostatic repulsion between the neighboring PPV‐NMe3+‐coated aggregates reduces and inhibits the further aggregation of HEWL. In this way, the water‐soluble conjugated polymer PPV‐NMe3+ serves as both the detector and inhibitor for the protein aggregation triggered by UV illumination, which promises advanced potential applications in the diagnose and prevention of diseases caused by protein aggregation.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Challenges with osmolytes as inhibitors of protein aggregation: Can nucleic acid aptamers provide an answer?

Protein aggregation follows some common motifs. Whether in the formation of inclusion bodies in heterologous overexpression systems or inclusions in protein conformational diseases, or aggregation during storage or transport of protein formulations, aggregates form cross beta-sheet structures and stain with amyloidophilic dyes like Thioflavin T and Congo Red, irrespective of the concerned protein. Traditionally, osmolytes are used to stabilize proteins against stress conditions. They are employed right from protein expression, through production and purification, to formulation and administration. As osmolytes interact with the solvent, the differential effect of the stress condition on the solvent mostly determines the effect of the osmolyte on protein stability. Nucleic acid aptamers, on the other hand, are highly specific for their targets. When selected against monomeric, natively folded proteins, they bind to them with very high affinity. This binding inhibits the unfolding of the protein and/or monomer–monomer interaction which are the initial common steps of protein aggregation. Thus, by changing the approach to a protein-centric model, aptamers are able to function as universal stabilizers of proteins. The review discusses cases where osmolytes were unable to provide stabilization to proteins against different stress conditions, a gap which the aptamers seem to be able to fill.

Aggregation of intrinsically disordered fibrinogen as the influence of backbone conformation

Fib having intrinsically disordered αC domains is involved in coagulation cascade and thrombosis. Fib molecules forms prefibrillar oligomers at 30%, and associate in 40 and 50% TFE to proceed α to β transition, suggesting the formation of an intermolecular β-structure. AFM images confirmed the nature of Fib aggregates at 40 and 50% TFE to be prefibrillar and fibrillar respectively. These aggregates possess high thioflavin T fluorescence with a shifted Congo red absorbance. Kinetics of Fib aggregation data at 50% TFE supports nucleation-dependent polymerization mechanism. At 60 and 70% TFE, no aggregation was observed. The inhibition of protein aggregation appears due to weakening of the hydrophobic interactions that were initially stabilizing the intermolecular β-sheet structure in the protein aggregation. The loss of hydrophobic contacts seems to favor the formation of intramolecular hydrogen bonds over intermolecular hydrogen bonds leading to helix formation. To conclude, protein aggregation is accompanied by the formation of β-sheet conformation, and induction of non-native helical segments in the protein inhibits aggregation. The discrepancy of the secondary structures on aggregation is proposed to stem from the disparity in the nature of the hydrogen bonds and packing of hydrophobic residues of the side chains in the β-sheet and α-helix conformation.

Potential applications of stress solutes from extremophiles in protein folding diseases and healthcare.

Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer's Aβ peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aβ and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.

Trehalose-Induced Structural Transition Accelerates Aggregation of α-Synuclein.

Fibrillation of α-synuclein proceeds through distinct stages, with oligomers combining to form the seed or the nucleus, followed by exponential and saturation phases. Osmolytes are considered to act as protein stabilizers by virtue of their ability to inhibit protein aggregation. Trehalose, a non-reducing disaccharide which is conventionally used as a stabilizer, was found to order α-synuclein, a natively disordered protein, into a non-native conformation such that the protein folding pathway is driven towards aggregation. Thus, by ordering the pathway intermediates, the osmolyte trehalose exerts variable effect on an intrinsically disordered protein when compared with its effect on natively folded proteins.

Screening: A platform for probing protein-aggregation inhibitors.

Screening: A platform for probing protein-aggregation inhibitors.

Bile Acids Protect Expanding Hematopoietic Stem Cells from Unfolded Protein Stress in Fetal Liver

During development, hematopoietic stem cells (HSCs) undergo a rapid expansion in the fetal liver (FL) before settling in the adult bone marrow. We recently reported that proliferating adult HSCs are vulnerable to ER stress caused by accumulation of mis-folded proteins. Here, we find that FL-HSCs, despite an increased protein synthesis rate and a requirement for protein folding, do not upregulate ER chaperones. Instead, bile acids (BAs), secreted from maternal and fetal liver, coordinate to serve as chemical chaperones. Taurocholic acid, the major BA in FL, supports growth of HSCs in vitro by inhibiting protein aggregation. In vivo, reducing BA levels leads to ER stress elevation and accumulation of aggregated proteins and significantly decreases the number of FL-HSCs. Taken together, these findings reveal that BA alleviation of ER stress is a mechanism required for HSC expansion during fetal hematopoiesis.