Author details

Sushil Bhunia1 , Ayan Mahalanobis2 \*, Pralhad Shinde2 and Anupam Singh2

1 IISER Mohali, India

2 IISER Pune, Pune, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The MOR Cryptosystem in Classical Groups with a Gaussian Elimination Algorithm… DOI: http://dx.doi.org/10.5772/intechopen.84663


**7. References**

346 Congenital Heart Disease – Selected Aspects

possible to observe the appearance of skeletal myosin in the cardiomyocytes. This testifies that, during hypertrophy development, the synthesis changes over from cardiac myosin to a skeletal one. Plain fluorescent microscopy of preparations stained with ethidium bromide revealed a drastic decrease in intensity of the fluorescent marker in infants aged under 6 months, as compared to the control group, and a rapid growth of ethidium bromide incorporation in infants aged 6 months and upward. It indicates a prevalence of the population of cardiomyocytes with diploid nuclei in the hearts of infants aged up to 6 months. In patients aged above 6 months, the heart remodelling process proceeds, with the processes associated with polyploidization of nuclear material and subsequent development of cell hypertrophy dominating. It should be emphasized that the level of polyploidization in LV cardiomyocytes is essentially higher than that in RV. Hence, the hyperplastic processes associated with intensive polyploidization of the genetic material and an increase in the quantity of desoxyribonucleic acid play an important role in the remodelling of the

This study enabled us to come to the following conclusion on the development of a pathological mechanism causing the deaths of TGA patients at an early age. As a result of aorta and pulmonary artery transposition, low-oxygen venous blood flows into the systemic circulation system, limits the growth of newborns. The need for a sufficient volume of oxygen can be met only by increased load on the myocardium, with the development of heart hypertrophy uniform for LV and RV in patients with IVS and more pronounced in RV in patients with VSD. In this case the growth of TGA infants conforms to the age norm and even slightly exceeds it, while the body mass falls far short of the norm by 25-30 %. For hypertrophy and hyperplasia to develop dramatically, there should be an increased supply of nutritional and caloric substances, including CE. As the delivery of CE turns out to be insufficient, or their consumption increases, a 50 % deficiency of such microelements as Cl, Cr, Sr, Zn, Br, Rb and especially Se, which, as an active antioxidant, protects cardiomyocytes from lipid peroxidation, can be seen. As a consequence, structural disorders of the myocardium occur on the morphological/molecular level. Also observed are the following abnormalities: a decrease in the diameter of muscle fibres and the average area of nuclei, a drop in the level of the total calcium ions against the background of intensive polyploidization of genetic material, an increase in the content of the quantity of desoxyribonucleic acid, and change from the cardiac myosin synthesis over to a skeletal one. All these changes lead to alteration of the myocardium, occurrence of cardiosclerosis, development of cardiac insufficiency and reduction of arterial blood saturation down to 32 % and below, which is fatal, and results in death of the organism. This is a picture of TGA development pathogenesis of infants from neonatal age up to 1 year old. In this case, only definitive repair of the disease can break the pathogenetic chain of TGA in an early period, and the best time when effective cardiac surgery could be performed for TGA infants is in

We suppose, that to prevent the development of congenital heart diseases including TGA, pregnant women and nursing mothers should get the optimum quantity of microelements Cr, Zn, Sr, Ni, Rb, Br and most of all Se, protecting the myocardium from lipid

heart in patients aged above 6 months.

**6. Conclusion**

the neonatal period.

peroxidation.

Trounova, V.A.; Zolotarev, K.V. & Baryshev, V.B. et al. (1998). Analytical possibilities of

Trunova, V. A. & Zvereva, V. V. (2008). Investigation of the distribution macro - and

(1998), pp. 532-536

*Chemistry*, Vol. 49, (2008), pp. 211-216

SRXRF station at VEPP-3 SR source. *Nuclear Instruments and Methods A*, Vol. 405,

traceelements in human myocardium and vessels by SRXRF. *Journal of Structural*


**2. Legume Lectins as a Factor in an Effective Symbiosis**

The full establishment and functioning of the legume-rhizobial symbiosis depends signifi‐ cantly on a number of abiotic, biotic and anthropogenic factors. In particular, the effective‐ ness of symbiotic nitrogen-fixing is affected by temperature, aeration, pH level and moisture content of the substrate, the presence of pesticides, the content of nitrogen and other macroand micronutrients. Of further importance is the virulence and activity of root nodule bacte‐ ria. The study of the influence and the establishment of the degree of importance of different factors to the effectiveness of symbiosis, will determine the conditions, and help to develop methods of optimizing the functioning of the symbiotic systems.

Wide dissemination of lectins in a variety of plants and the presence of these proteins in vir‐ tually all organs and tissues of plant organisms demonstrate the importance of their role in life processes. Initially it was assumed that the presence of lectins was a distinctive feature of seeds from the legume family. However, the number of organisms in which lectins have been found is increasing every day and there is reason to believe that all the above men‐ tioned plants as well as algae, lichens and fungi contain lectins [9-11]. Nevertheless, in the present day researchers focus much of their attention on lectins of leguminous plants, the increased study of which leads us to understand the activity of these proteins in relation to the structure of the latter.

Legume lectins are a large group of carbohydrate binding proteins derived mainly from seeds. Lectins contained in the seeds of legumes, are localized in the proteins and make up approximately 10% of soluble protein extract. In recent years it has been found that these proteins are present in other parts of plants, including stems, leaves, bark, roots, and root nodules. Lectins accumulate in the vacuoles of cells and can come to the surface of the plants. They can also be associated with membranes and cell walls [12-15].

The symbiotic and associative systems of plants and diazotrophs are an example of the evo‐ lution of the interaction of living organisms. Their study is particularly relevant with the im‐ plementation of highly productive and environmentally friendly farming. The biological fixation of molecular nitrogen from the air is a process of fixation and assimilation of nitro‐ gen by microorganisms. It is of great practical importance, since the industrial production of chemical nitrogen fertilizer requires significant use of costly energy resources, which by themselves can be harmful for the environment. A comprehensive study of this problem is necessary due to the need to create new and effective biological preparations. The creation and use of biological agents on the basis of nitrogen-fixing microorganisms is the most justi‐ fiable method of increasing the productivity of plants and the quality of their harvest, which allows maintenance of the natural fertility of soils and the ecological balance of the environ‐ ment. Their use makes it possible to regulate the number and activity of beneficial micro‐ flora in the rhizosphere of crops, and to provide plants with nitrogen fixed from the atmosphere. For example, in addressing the shortage of high-grade protein the key role be‐ longs to the soybean. However, the soils on which the crop is grown for the first time [8] usually do not have nodule bacteria compatible with soybean or bacteria number is small

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

(up to 20 per gram of soil).

Relationships

62

the structure of the latter.

**2. Legume Lectins as a Factor in an Effective Symbiosis**

methods of optimizing the functioning of the symbiotic systems.

The full establishment and functioning of the legume-rhizobial symbiosis depends signifi‐ cantly on a number of abiotic, biotic and anthropogenic factors. In particular, the effective‐ ness of symbiotic nitrogen-fixing is affected by temperature, aeration, pH level and moisture content of the substrate, the presence of pesticides, the content of nitrogen and other macroand micronutrients. Of further importance is the virulence and activity of root nodule bacte‐ ria. The study of the influence and the establishment of the degree of importance of different factors to the effectiveness of symbiosis, will determine the conditions, and help to develop

Wide dissemination of lectins in a variety of plants and the presence of these proteins in vir‐ tually all organs and tissues of plant organisms demonstrate the importance of their role in life processes. Initially it was assumed that the presence of lectins was a distinctive feature of seeds from the legume family. However, the number of organisms in which lectins have been found is increasing every day and there is reason to believe that all the above men‐ tioned plants as well as algae, lichens and fungi contain lectins [9-11]. Nevertheless, in the present day researchers focus much of their attention on lectins of leguminous plants, the increased study of which leads us to understand the activity of these proteins in relation to

Legume lectins are a large group of carbohydrate binding proteins derived mainly from seeds. Lectins contained in the seeds of legumes, are localized in the proteins and make up approximately 10% of soluble protein extract. In recent years it has been found that these The basis of the biological activity of lectins is a reversible reaction with carbohydrates, which defines the different types of biological reactions [16] and their physiological signifi‐ cance. Lectins do not have uniform structural characteristics. In legumes, these proteins are generally composed of two or four subunits with a molecular mass of 25 000 - 30 000 and each having one carbohydrate binding site [15, 17]. Lectins can interact with both mono-and oligosaccharides, as well as the remnants of carbohydrates present in the complex organic substances - glycoproteins, polysaccharides, and glycosides. In the early 1980's there was es‐ tablished a carbohydrate specificity for many legume seed lectins. However indisputable physiological carbohydrate ligands for any of these proteins have not been identified [14].

Over the past half-century many hypotheses were developed regarding the role of lectins in the life of legumes, but so far none of them have been fully confirmed. These proteins were detected due to their ability to bind carbohydrates, and most hypotheses about their func‐ tions are based solely on this wonderful property. Since it is known that the plant may con‐ tain a significant number of lectins, which are localized in different tissues and may form a gene duplication, while carrying out a variety of functions, it is believed that any attempt to dedicate a specific role to these proteins is doomed to failure [14].

An important feature of most of the representatives of the family *Fabaceae* (Legumes) is the ability to enter into symbiotic relationships with rhizobia. They belong to the family of *Rhizo‐ biaceae* (bacteria genera *Rhizobium, Bradyrhizobium, Mezorhizobium, Sinorhizobium, Azorhizobi‐ um*), and form special structures on the roots called nodules. Lectins are considered as a component of the molecular and chemical interactions underlying the formation of symbiot‐ ic structures [6, 9, 11]. The symbiosis between rhizobia and legumes is based on a complex sequence of morphophysiological changes in the cells of both partners. Stages in the develop‐ ment of symbiosis are: preinfection (chemotaxis of bacteria, the exchange of signals, the ad‐ sorption of microsymbionts on the root surface), infection of the roots and development of nodules (penetration, formation of infection threads, the transformation of rhizobia into bac‐ teroids), as well as the functioning of the nodules of nitrogen fixation [9, 18, 19].

Plants throughout their lifespan release various matter into the environment. The presence of lectins has been found in the soil [20], which are secreted during the germination of seeds together with other biologically active substances [21]. It is believed that in the first stages of interaction between rhizobia and legumes an important role is played by the chemotactic re‐ sponse to bacteria. Nodule bacteria and other soil microorganisms, in response to plant exu‐ dates, stimulate the reproduction and active movement of bacteria towards the roots, colonizing the rhizosphere and rhizoplane. A positive chemotaxis of rhizobia on the root exudates and germinating seeds can be either nonspecific (due to the excretion of organic acids, carbohydrates, vitamins), or specific (induced by flavonoids and lectins) [9, 18, 22].

The initial stage of many plant-microbe interactions is recognition of partners, which is largely due to the exchange of molecular signals [23, 24]. This process begins with the exu‐

dation by the host plant of chemicals such as flavonoids and betaines that induce the gene expression of nodulation in rhizobia [25]. Flavonoids induce the transition of bacteria from free-living to those in a symbiotic state [26]. The first chemically-characterized inducer of the expression of nod-gene was luteolin, drawn from the extract of lucern seeds [27]. For each type of rhizobia, plants produce individual flavonoid signals that can stimulate or inhibit the nod-genes of rhizobia [28]. The identification of factors by microorganisms which are re‐ leased by plants initiates physiological processes required for the infection of the host plant. In turn, the microbial signals induce the plant to express the genes required for the forma‐ tion of responses [5, 18, 25, 29]. Amongst the signals produced by the bacteria, the most studied are the lipoсhitooligosaссharide Nod-factors [14, 24]. During the formation of sym‐ biotic relationships lectins on the plant recognize the nod-factor signal released by the bacte‐ ria. It is believed that lectins binding to the bacteria whith the surface of root hairs are able to identify the rhizobial signaling molecules, through surface polysaccharides on the rhizo‐ bia [14, 30]. It has been shown that the lectin of dolichos roots, which is localized on the sur‐ face of root hairs, is able to connect with some of the Nod-factors [14]. Furthermore, from the soybean root tissue there has been found and characterized a chitin-binding protein of the plasma membranes of cells. The presence of this protein and the specificity of the induc‐ tion of the biological response to the binding of ligand *Bradyrhizobium japonicum* indicates its importance in the initiation of response to the binding of chitin in soybean [31].

Therefore Nod-factors play the role of a trigger mechanism for initiating bacterial invasion and nodulation of the plant [24]. Lipopolysaccharides inform the plant about the transition to a symbiotic interaction and the formation of the so called functional areas [32]. Legume plants are unique in their ability to form a response to Nod-factors, which consist of a prepa‐ ration for the penetration of bacteria into the roots. Unfortunately, we do not know how the representatives of this family developed the ability to recognize such signals and how they improved upon these mechanisms [26].

An important stage of preinfection is the adsorption of bacterial cells on the surface of root hairs [33, 34]. The process of attachment of the bacteria from the *Rhizobiaceae* family consists of several stages with the participation of bacterial surface proteins in the first stage (rickad‐ hesin, porin) and polysaccharide fibrils in the second stage. The first stage, involving pro‐ teins, is more decisive for the success of infection than the second stage, mediated by specific cellulose fibrils. The latter promote the retention of bacterial cells on the surface of the plant, but are not necessary for the infection [35].

The adsorption of rhizobia has also been associated with the ability of legumes to synthesize specific glycoproteins called lectins, which bind to polysaccharides on the surface of rhizo‐ bia cells [36, 37]. It is believed [33, 34] that the ability of rhizobia to be adsorbed on the sur‐ face of the roots of the host plant (like the chemotaxis) may be either specific or nonspecific. The initial binding of rhizobial cells to the surface of root hairs occurs by the means of exo‐ polysaccharides (EPS). The degree of affinity between EPS microsymbionts and host plant lectin determines the degree of homology between the symbiotic partners and provides an advantage over other homologous strains in the process of plant infection.

Dazo and his co-writer [38] proposed a model of the attachment of rhizobia to the surface of the roots of dicotyledonous plants. The first phase is non-specific attachment which is char‐ acterized by the fact that the polyvalent host plant lectin binds carbohydrate receptors on the cell surface of nodule bacteria. This results in an intercellular "bridge" between lectin and polysaccharide. The second phase is the "anchoring" of bacteria on the surface of plant cells. This is the phase of specific attachment. The bacterial cells attach to the plant cell, which is the signal for further stages of infection. At this point microfibrils are formed be‐ tween the contacting surfaces which consist of cellulose [35].

dation by the host plant of chemicals such as flavonoids and betaines that induce the gene expression of nodulation in rhizobia [25]. Flavonoids induce the transition of bacteria from free-living to those in a symbiotic state [26]. The first chemically-characterized inducer of the expression of nod-gene was luteolin, drawn from the extract of lucern seeds [27]. For each type of rhizobia, plants produce individual flavonoid signals that can stimulate or inhibit the nod-genes of rhizobia [28]. The identification of factors by microorganisms which are re‐ leased by plants initiates physiological processes required for the infection of the host plant. In turn, the microbial signals induce the plant to express the genes required for the forma‐ tion of responses [5, 18, 25, 29]. Amongst the signals produced by the bacteria, the most studied are the lipoсhitooligosaссharide Nod-factors [14, 24]. During the formation of sym‐ biotic relationships lectins on the plant recognize the nod-factor signal released by the bacte‐ ria. It is believed that lectins binding to the bacteria whith the surface of root hairs are able to identify the rhizobial signaling molecules, through surface polysaccharides on the rhizo‐ bia [14, 30]. It has been shown that the lectin of dolichos roots, which is localized on the sur‐ face of root hairs, is able to connect with some of the Nod-factors [14]. Furthermore, from the soybean root tissue there has been found and characterized a chitin-binding protein of the plasma membranes of cells. The presence of this protein and the specificity of the induc‐ tion of the biological response to the binding of ligand *Bradyrhizobium japonicum* indicates its

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

importance in the initiation of response to the binding of chitin in soybean [31].

improved upon these mechanisms [26].

Relationships

64

the plant, but are not necessary for the infection [35].

Therefore Nod-factors play the role of a trigger mechanism for initiating bacterial invasion and nodulation of the plant [24]. Lipopolysaccharides inform the plant about the transition to a symbiotic interaction and the formation of the so called functional areas [32]. Legume plants are unique in their ability to form a response to Nod-factors, which consist of a prepa‐ ration for the penetration of bacteria into the roots. Unfortunately, we do not know how the representatives of this family developed the ability to recognize such signals and how they

An important stage of preinfection is the adsorption of bacterial cells on the surface of root hairs [33, 34]. The process of attachment of the bacteria from the *Rhizobiaceae* family consists of several stages with the participation of bacterial surface proteins in the first stage (rickad‐ hesin, porin) and polysaccharide fibrils in the second stage. The first stage, involving pro‐ teins, is more decisive for the success of infection than the second stage, mediated by specific cellulose fibrils. The latter promote the retention of bacterial cells on the surface of

The adsorption of rhizobia has also been associated with the ability of legumes to synthesize specific glycoproteins called lectins, which bind to polysaccharides on the surface of rhizo‐ bia cells [36, 37]. It is believed [33, 34] that the ability of rhizobia to be adsorbed on the sur‐ face of the roots of the host plant (like the chemotaxis) may be either specific or nonspecific. The initial binding of rhizobial cells to the surface of root hairs occurs by the means of exo‐ polysaccharides (EPS). The degree of affinity between EPS microsymbionts and host plant lectin determines the degree of homology between the symbiotic partners and provides an

advantage over other homologous strains in the process of plant infection.

The "Lectin" hypothesis which explains the specificity for formation of nodules as resulting from the complementary interaction of the surface structures of bacterial and plant cells. This forms an "antigen-antibody" complex, which was first recognized in the early 1980's. Although initially a controversial subject, Diaz and co-writer [39] provided strong genetic evidence that the root lectins of legumes determine the specifics of symbiosis development. They injected special strains of *Agrobacterium* into the roots of clover through transfer of the psl gene (codes for synthesis of pea lectin). This resulted in clover acquiring the ability to form nodules with pea rhizobia. A similar study ten years later had the same result [40]. Transgenic lucerne plants carrying genes encoding soybean or pea lectin generated a struc‐ ture similar to that of the root nodule in response to incoulation with pea and soybean nod‐ ule bacteria producing the specific Nod-factor. However not all of the structures created were able to form complete infection threads. The results confirm the importance of the presence of lectin in establishing contact between the symbionts and demonstrates the unique role of exopolysaccharides (EPS) in the formation of nodules. *Galega officinalis* and *orientalis* are other examples demonstrating the importance of lectins in legume-rhizobial symbiosis. The validity of this result is confirmed by sequencing the amplifiers of DNA ex‐ tracted from the seeds of the mentioned crops from different geographical locations [41].

Therefore numerous experimental data confirms the important role of lectins in the early stages of the symbiosis and suggests the involvement of these proteins in a variety of phys‐ iological processes of plants.

Once they have prepared each other with molecular signals, the partners begin to form the structural basis of symbiosis - a nodule. This leads to a morphophysiological differentiation of bacterial and plant cells [18, 25]. Nod-factors control the phenotypic changes that occur in the roots of the host plant (the initial stage of symbiosis.) In addition there is also the defor‐ mation and proliferation of root hairs [25], the expression of early nodule genes [5], the in‐ duction of mitotic divisions in the cortex, followed by the onset of the first stages of histogenesis of nodules [42, 43].

Based on the assumption regarding the participation of lectins in various physiological proc‐ esses in the formation of symbiosis, much attention was paid by researchers to the study of the direct effects of plant lectins on the manifestation of the symbiotic properties of nodule bacteria. In particular, it has been shown that the incubation of nodule bacteria *Bradyrhizobi‐ um japonicum* whith soybean root exudates and lectin from its seeds increased the activity of nodule formation in the mutated soybean HS 111, characterized by delayed nodulation, and in doubling the number of nodules formed by wild strain USDA 110 [44]. Later it was

shown that treatment of rhizobia by specific plant lectin increases their virulence and com‐ petitiveness [34], and also increases the quantity of infectious threads in the roots of legumi‐ nous plants [45]. The particular modulating effect of the plant lectin on the formation and functioning of symbiosis has been established, which is manifested in the stimulating effect on partners, homologous data of the proteins, and the neutral or suppressive reaction of lec‐ tin which is not in accordance with the symbionts [46]. Moreover it is shown that the incuba‐ tion of nodule bacteria with homologous lectin has a positive effect on the symbiotic properties of the active strain *Bradyrhizobium japonicum* 634b, and the intensity of the basic physiological processes in plants - nitrogen fixation and photosynthesis. The introduction of the same protein into an inactive strain of rhizobia suspension *Bradyrhizobium japonicum* 604k has a suppressive effect on the symbiotic system which it took part in forming as well as the stated processes [47]. It has also been found that the nature of the influence of homol‐ ogous lectin on the growth of nodule bacteria in a axenic culture and the biosynthesis of ex‐ tracellular carbohydrates [48], the ability of rhizobia to form nodules and their nitrogenase activity, as well as its impact on the productivity of host plants depends on the concentra‐ tion of this protein in the bacterial suspension [49].

Co-incubation of nodule bacteria with lectin enhances soybean growth processes in the early stages of ontogeny as well as in the functioning of the symbiotic system, increases the nitro‐ gen-fixing activity and, consequently, the productivity of plants [47, 49, 50]. This is why in recent years work was done on the selection of the optimal concentration of the homologous lectin, and the length of time of its co-incubation with the culture of *Bradyrhizobium japoni‐ cum* in the production of bacterial fertilizers both in liquid and solid form. Lectin is used as a growth stimulating biologically active substance, which is introduced into the culture of bacteria under certain conditions [51].

In the bacterial model *Bacillus subtilis* where antibiotics were used as metabolic inhibitors, which block the processes of replication, transcription or transmission, there was established an ability of carbohydrate-binding proteins, lectins of plant origin, to have various effects on intracellular processes. Among the presumed processes affected by lectins were reparative functions [52]. It was also found that lectins from the seeds of leguminous plants that have a high molecular weight (> 100 000 Da) can stimulate the respiration of some *Rhizobium*. Ac‐ cording to the authors [53], this effect is induced by the lectins, and significantly increases the interaction of lectin-*Rhizobium* due to the physiological properties of the bacteria.

The presented data gives cause to consider the homologous lectin, not only as a receptor or a signaling molecule in the initial stages of symbiosis, but also as a molecular signal that changes the metabolism of rhizobia, which significantly affects their symbiotic properties and the physiological status of the host plant.

In the process of development of nodules in legume plants, lectins are localized at least in three different places. Their possible functions are related to areas of infection on the surface of the roots. In the nodule primordia lectins can stimulate mitotic activity reducing the threshold of sensitivity to the rhizobial Nod-factors. At the same time, in the central part of the mature nodule, lectins can function as spare nitrogen compounds [54].

It is thought that legume lectins are involved in the formation of nodules. The activating ef‐ fect of lectin on the synthesis of extracellular and capsular polysaccharides of rhizobia is shown, which in turn induces the formation of infection threads [55]. Data for the study of lectin gene expression during the development and functioning of root nodules of lucerne provided evidence supporting the involvement of these proteins in the early stages of the ontogenesis of nodules [56]. A study has been made of a number of genes and plant proteins encoded by them, which play a role in the formation and functioning of root nodules. Among them is a lectin related to the pea *ps*1 gene with the presumed function as a mitotic stimulator and soybean lectin *le*1, which takes part in the attachment of cells [19].

shown that treatment of rhizobia by specific plant lectin increases their virulence and com‐ petitiveness [34], and also increases the quantity of infectious threads in the roots of legumi‐ nous plants [45]. The particular modulating effect of the plant lectin on the formation and functioning of symbiosis has been established, which is manifested in the stimulating effect on partners, homologous data of the proteins, and the neutral or suppressive reaction of lec‐ tin which is not in accordance with the symbionts [46]. Moreover it is shown that the incuba‐ tion of nodule bacteria with homologous lectin has a positive effect on the symbiotic properties of the active strain *Bradyrhizobium japonicum* 634b, and the intensity of the basic physiological processes in plants - nitrogen fixation and photosynthesis. The introduction of the same protein into an inactive strain of rhizobia suspension *Bradyrhizobium japonicum* 604k has a suppressive effect on the symbiotic system which it took part in forming as well as the stated processes [47]. It has also been found that the nature of the influence of homol‐ ogous lectin on the growth of nodule bacteria in a axenic culture and the biosynthesis of ex‐ tracellular carbohydrates [48], the ability of rhizobia to form nodules and their nitrogenase activity, as well as its impact on the productivity of host plants depends on the concentra‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Co-incubation of nodule bacteria with lectin enhances soybean growth processes in the early stages of ontogeny as well as in the functioning of the symbiotic system, increases the nitro‐ gen-fixing activity and, consequently, the productivity of plants [47, 49, 50]. This is why in recent years work was done on the selection of the optimal concentration of the homologous lectin, and the length of time of its co-incubation with the culture of *Bradyrhizobium japoni‐ cum* in the production of bacterial fertilizers both in liquid and solid form. Lectin is used as a growth stimulating biologically active substance, which is introduced into the culture of

In the bacterial model *Bacillus subtilis* where antibiotics were used as metabolic inhibitors, which block the processes of replication, transcription or transmission, there was established an ability of carbohydrate-binding proteins, lectins of plant origin, to have various effects on intracellular processes. Among the presumed processes affected by lectins were reparative functions [52]. It was also found that lectins from the seeds of leguminous plants that have a high molecular weight (> 100 000 Da) can stimulate the respiration of some *Rhizobium*. Ac‐ cording to the authors [53], this effect is induced by the lectins, and significantly increases

the interaction of lectin-*Rhizobium* due to the physiological properties of the bacteria.

the mature nodule, lectins can function as spare nitrogen compounds [54].

The presented data gives cause to consider the homologous lectin, not only as a receptor or a signaling molecule in the initial stages of symbiosis, but also as a molecular signal that changes the metabolism of rhizobia, which significantly affects their symbiotic properties

In the process of development of nodules in legume plants, lectins are localized at least in three different places. Their possible functions are related to areas of infection on the surface of the roots. In the nodule primordia lectins can stimulate mitotic activity reducing the threshold of sensitivity to the rhizobial Nod-factors. At the same time, in the central part of

tion of this protein in the bacterial suspension [49].

bacteria under certain conditions [51].

Relationships

66

and the physiological status of the host plant.

It is established that the proteins extracted from lupine and soybean nodules are capable of hemagglutinating activity. A comparative analysis of the nitrogen-fixation of soybean nod‐ ules and the lectin activity of the protein extracts from these nodules revealed a link be‐ tween these rates in ontogenesis [57]. This data suggests that the proteins which are capable of lectin activity may be directly involved in the processes of nodule functioning.

A hypothesis was made by Antoniuk and his co-writer that the lectin of wheat germs acts as a signal for *Azospirillum brasilense*, which changes the metabolism of the bacteria in a direc‐ tion favorable to the growth and development of the host plant [58, 59]. According to the authors, the level of lectin in the plant depends on a number of conditions and is one of the factors responsible for the variability in the results of wheat inoculation.

Nodule bacteria penetrate the plant cell (as opposed to *Azospirillum*) and transform into bac‐ teroids, which do not divide, but only increase in size. Nonetheless a number of analogies can be made between the influence of the wheat germ lectin on *Azospirillum* and the influ‐ ence of a specific legume lectin on *Rhizobium*. In the first as well as in the second case, the homologous lectin has a positive effect on the symbiotic properties of bacteria, which pro‐ motes a more efficient interaction between the partners in the different symbiotic systems that they create. There remains the little-studied question regarding the participation of lec‐ tins in the functioning of the symbiotic apparatus of legumes, but it is possible that the pres‐ ence of proteins with lectin activity in the nodules is associated with the biosynthesis of proteins (including nitrogenase) in bacteroids and in ensuring conditions for the effective functioning of their symbiotic system.

Legume lectins may influence the receptor and signaling molecules during the stages of the symbiosis. Hemagglutinating activity of the proteins contained in the nodules, varies de‐ pending on the efficiency of their functioning. Specific lectins are able to modify the symbi‐ otic properties of the nodule bacteria which positively affect the physiological status of the host plant and, ultimately, the effectiveness of the symbiotic system. Uncovering the role that lectins have in the functioning of the symbiotic apparatus of legumes requires further research. At the same time these proteins can be regarded as one of the factors for the effec‐ tive symbiosis, which must be considered when developing and implementing new ap‐ proaches to the management of production process in legume plants.


4.3.2. In Vitro Stability and Plasma Protein Binding for [18F]6

A total of 40 μL of [18F]**6** was incubated in 0.01 M PBS at room temperature, in a microtube for 90 min, with mixing (400 rpm). At selected time-points (*t* = 5, 30, 60 and 90 min, *n* = 1), the samples were injected into an HPLC with PDA- and radiodetector, for analysis. Radiotracer [18F]**6** (40 μL) was incubated in 50% human plasma (anonymous donor FFP-24 plasma provided by the Finnish Red Cross Blood Service, Helsinki, Finland) in PBS at 37 ◦C, and in mouse plasma (separated from CD-1 mouse blood). At selected time-points (5, 60, 120, and 240 min, *n* = 2 for each), 100 μL of the samples were taken, diluted with 200 μL of cold acetonitrile, and centrifuged at 10,000 g for 5 min. The radioactivity in the precipitated pellet (protein-bound fraction) and supernatant (free fraction) were measured with a gamma counter and 100 μL of the samples were injected into HPLC, for radio-HPLC analysis.

#### *4.4. Biological Studies*

All animal experiments were conducted under a project license approved by the National Board of Animal Experimentation in Finland (license number ESAVI/12132/04.10.07/2017). The animals were group-housed in standard polycarbonate cages, on aspen bedding, in a HEPA-filtered housing unit (UniProtect, Ehret, Emmendingen, Germany), with food (Envigo Teklad Global Diet 2016) and tap water, available ad libitum. Conditions were maintained at 21 ± 1 ◦C and 55 ± 15% relative humidity, with a 12:12 lighting cycle. The biodistribution studies were conducted in healthy, female CD-1 mice (weight 25–33 g, 11 to 12 weeks, Charles River). The radiotracers [18F]**6** and [18F]**10** were injected via the tail vein to CD-1 mice, in the following formulations—10% EtOH and 0.5% Solutol HS 15 in 0.01 M PBS pH 7.4 for [18F]6 and 0.01 M PBS pH 7.4 for [18F]10. At selected time-points (5, 60, 120, and 240 min), the mice were euthanized with CO2 asphyxiation, followed by cervical dislocation, and selected tissues were collected, weighed, and the radioactivity was measured on an automated gamma counter.


Abstract

In developing countries, rural electrification in areas with limited or no access to grid connection is one of the most challenging issues for governments. These areas are partially integrated with the electrical grid. This poor electricity distribution is mainly due to geographical inaccessibility, rugged terrains, lack of electrical infrastructure, and high required economic investment for installing large gridconnected power lines over long distances to provide electricity for regions with a low population. On the other hand, rapid depletion of fossil fuel resources on a global scale and progressive increase of energy demand and fuel price are other motives to reduce the reliance on fossil fuels. Hybrid renewable energy system (HRES) can be a suitable option for such remote areas. The objective of this chapter is to develop a methodology for sizing hybrid power generation systems (solardiesel), battery-backed in non-interconnected zones, which minimizes the total cost and maximizes the reliability of supply using particle swarm optimization (PSO). The proposed methodology assists the sizing and designing process of an HRES for an off-grid area minimizing the cost of energy (COE) and maximizing the reliability of the system. Economic incentives offered by the Colombian government are considered in the model.

Keywords: hybrid renewable energy system (HRES), stand-alone systems, off-grid areas, particle swarm optimization (PSO), photovoltaic energy, power dispatch strategy


**1. Introduction**

Renewable energy sources have been gaining more importance in the last few decades as part of the strategies adopted by countries all over the world to limit the use of fossil fuel and fight pollution and global warming. In particular, wind energy has grown rapidly, resulting in the increasing size of modern wind turbines with the objective of reducing the cost of the produced energy [1]. Nevertheless, the adoption of more slender blades has also led to higher deflections during normal operation, and consequently, more interest toward the fluid–structure interaction (FSI) phenomenon in modern wind turbines. Recent works have computed that, while operating in design conditions, the flapwise deflection of a modern horizontal-axis wind turbine (HAWT) blade is in the order of 6–7% of its span [2–4]. Furthermore, their deflections have a sensible impact on the produced power, affecting its oscillation or introducing a reduction up to 6% [2–6].

However, due to the complexities involved, when the FSI of modern wind turbine blades is to be analyzed, simplified models are often adopted for this task. On the aerodynamic side of the problem, among others, blade element momentum theory (BEM) is widely used in the FSI modeling of wind turbines [7–9]. Despite its low computational cost, BEM theory is affected by many limitations, including the need to include tip-loss corrections to account for a blade of finite length [10]. Another class of widely used models are the actuator models, where the blades are represented by lines or surfaces exchanging momentum with the incoming wind flow [11–13]. This strategy has also been

used in FSI simulations of a multi-megawatt turbine, coupling it with a structural model based on non-linear beam theory [14].

In order to increase the level of detail in the extracted results, a higher computational cost is required. Computational fluid dynamics (CFD) simulations, performed by solving the Navier–Stokes equations on a computational grid rendering the geometry of the wind turbine, have recently been used in literature within FSI frameworks. In particular, Reynolds-averaged Navier–Stokes (RANS) models are often used to account for turbulence in the atmospheric wind flow [15–19]. This class of turbulence models are computationally cheap when compared to more complex turbulence models, which are also reported in recent literature within FSI simulations of wind turbines [2–4,20,21] but have an extremely high computational cost.

On the structural side of the FSI problem, the complex nature of modern composite blades [22] often leads to the adoption of simplified models. Among others, FSI works relying on multi-body dynamics [21] or using one-dimensional beam elements [14,15,19] can be found, whereas the implementation of detailed finite element models (FEMs), loyally reproducing the composite structure of modern blades, is still limited to only a few works [3,4,6,20] by two distinct research groups.

Despite its importance, the atmospheric boundary layer (ABL), namely the velocity gradient leading to higher wind speeds at higher heights, is very often neglected in FSI simulations of wind turbines. To the best of the authors' knowledge, only a very limited number of works account for it [6,21,23]. These works remark upon the importance of the ABL, highlighting how it induces oscillating loads, power, and deflections on modern blades.

Furthermore, given the aleatory nature of the wind, wind turbines always operate in unsteady and rapidly changing conditions. In particular, local oscillations in the wind speed, referred to as "gusts," lead to the varying performance and structural responses of the blades, which is particularly important in the fatigue life estimation of modern structures. Gusts can be of various shapes, sizes, lengths, and intensities [24], and can be induced by the terrain morphology, as well as by thermal or turbulent effects [25]. Despite their relevance in relation to large HAWTs, the majority of works currently available in the literature focus on vertical-axis wind turbines (VAWTs) or on small HAWTs. Wu et al. [26] carried out 2D RANS simulations to obtain the lift–drag polars of the sections of a VAWT in both steady wind and unsteady (i.e., affected by a gust) wind conditions and used them to obtain the performance of the turbine by means of a BEM-like strategy. Onol and Yesilyurt [27] analyzed a small VAWT by means of a 2D unsteady RANS (U-RANS) model, changing the approaching wind velocity according to the gust time variation prescribed by the IEC-64100 standards for wind turbines. Where 3D simulations are concerned, Bhargav et al. [28] performed CFD simulations of a VAWT while changing the inlet wind speed to follow a sinusoidal function but without considering the FSI.

Regarding to FSI simulations, the work of Timme et al. [29] analyzed the aeroelastic response of a straight wing impacted by a vertical gust. The shape of this gust corresponds to the "1 − cos" shape, also prescribed in the IEC-64100 standards. Also operating in an aeroelastic framework, Svacek and Horacek [30] analyzed the response of a flexibly supported airfoil to a vertical wind gust, mimicking the dynamics of a wing by means of 2D U-RANS simulations.

The available literature on the gust aeroelastic response to HAWT is extremely limited. First in time, the work of Younsi et al. [31] featured a BEM code in combination with a structural model reproducing the blade as being homogenously made of an elastic and isotropic material. A single blade was modelled, neglecting the ABL and simulating an extreme gust impacting on the whole blade. More recently, Castellani et al. [32] carried out both experimental and numerical works to investigate the response of a small HAWT (2-m diameter) to a periodical change in the incoming wind speed, aiming at the investigation of an optimal control strategy for such a machine. On the other hand, Ebrahimi and Sekandari [33] investigated the aeroelasticity of a multi-megawatt wind turbine to a sudden change in wind, coupling an unsteady vortex-lattice method (VLM) with a structural model relying on a modal approach. The entire turbine was subjected to a change of velocity magnitude and

direction, also accounting for the response of the control systems that react by changing the pitch of the blades and the yaw of the whole machine.

All the aforementioned works on HAWTs neglect the ABL and analyze a wind gust bigger than the analyzed turbine by means of simplified models. The investigated gust impacts on the whole rotor can be counteracted by the turbine control systems [33]. No work about the aeroelastic response of the blades of a large HAWT immersed in the ABL and locally impacted by a wind gust (i.e., soliciting only one blade) was found in the current literature. In this work, for the first time, two high-fidelity models, one for the CFD side and one for the FEM side of the FSI problem, were strongly coupled to dynamically analyze the aeroelasticity of the blade of a 100-m diameter HAWT immersed in the ABL and impacted by wind gusts of different intensities and morphologies. The proposed methodology is believed to be well-suited for advanced engineering applications, and in particular, to analyze the response of modern wind turbines to extreme load cases as part of the design process. This is an added value compared to the available literature, in which the aeroelastic analysis of a wind turbine blade attacked by a wind gust is scarcely reported.

The paper is structured as follows. The CFD model is presented in Section 2.1, the structural FEM model is described in Section 2.2, the coupling strategy is addressed in Section 2.3, followed by the gust model in Section 2.4. Section 3 contains the extracted results and their discussion, and the conclusions are drawn in Section 4.


*2.4. Pollution Assessment*

Two indices were used to assess pollution in the studied soils [13]. The enrichment factor (EF) compares the concentration of an element in the studied soil with the background concentration, both concentrations standardized against a reference element (Al, Fe, or Ti). We chose Fe as the reference element, so that EF was calculated as:

$$\text{EF}\_{\text{X}} = (\text{C}\_{\text{X}} / \text{C}\_{\text{Fe}})\_{\text{soil}} / (\text{C}\_{\text{X}} / \text{C}\_{\text{Fe}})\_{\text{banks/ground}}$$

where X represents the element of interest; EFX, the EF of element X; CX, the total concentration of element X; CFe, the total concentration of iron (a lithogenic reference element); "soil" refers to the assessed soil, and "background" to the situation of natural unpolluted soils in the study area, taken as a reference. EF values below 2 indicate minimum enrichment; values between 2 and 5 denote moderate enrichment; significant enrichment is attributed to values between 5 and 20; values above 20 show very high enrichment; while values above 40 reveal extremely high enrichment [13].

The geoaccumulation index (Igeo) for a given element is calculated as:

$$\mathbf{I}\_{\text{geo}} = \log\_2 \left( \mathbf{C}\_{\text{X}} / 1.5 \,\mathrm{B}\_{\text{x}} \right)$$

where CX is the total concentration of element X; BX is the background value for element X; and the factor 1.5 is used to minimize the effect of possible variations in the background due to lithological variations. According to Igeo values, the soils are classified into seven categories: <0 = practically unpolluted, 0–1 = unpolluted to moderately polluted, 1–2 = moderately polluted, 2–3 = moderately to strongly polluted, 3–4 = strongly polluted, 4–5 = strongly to extremely polluted, and >5 = extremely polluted [13].

As background concentrations to calculate EF and Igeo, we used the quality reference values (QRVs) established by Rodríguez Alfaro et al. [26] for Cuban soils:

$$\begin{aligned} \text{EF}\_{\text{X}} &= (\text{C}\_{\text{X}} / \text{C}\_{\text{Fe}}) / (\text{QRV}\_{\text{X}} / \text{QRV}\_{\text{Fe}}) \\\\ \text{I}\_{\text{gco}} &= \log\_2 \left( \text{C}\_{\text{X}} / 1.5 \text{ QRV}\_{\text{x}} \right) \end{aligned}$$

QRVFe equals 54,055 mg kg−1 [26].

The EF and Igeo for Ti were calculated using the average concentrations of Ti (4.4 g kg−1) and Fe (50 g kg−1) in the earth's crust, instead of the QRVs for Cuban soils, because there is no reference value for Ti in Cuban soils:

$$\rm EF\_{Ti} = (C\_{\rm Ti} \%\_{\rm Fe})\_{\rm soil} / (C\_{\rm Th} \%\_{\rm Fe})\_{\rm earth\ crust}$$

where CTi represents the Ti concentration and CFe represents the Fe concentration.


*2.13. Pharmacological Treatments (In Vivo)*

The following pretreatments were applied into mice in order to investigate LiRecTCTP effects on edema formation and vascular permeability in vivo: cromolyn (mast cell degranulation inhibitor), 30 mg/kg, administered i.p. in three consecutive days before exposure to LiRecTCTP; promethazine (histamine type 1 receptor (H1R) antagonist), 5 mg/kg, administered i.v. 30 min before exposure to LiRecTCTP; cimetidine (histamine type 2 receptor (H2R) antagonist), 15 mg/kg, administered i.p. 2 h before exposure to LiRecTCTP; thioperamide (histamine type 3 and 4 receptor (H3/H4R) antagonist), 20 mg/kg, administered i.p. 30 min before exposure to LiRecTCTP [23]. Sterile PBS was used as a negative control for pharmacological treatments and LiRecTCTP injections.


**7. Vasopressin system**

Vasopressin (ADH) is released by the hypothalamus as a result of baroreceptor, osmotic, and neurohormonal stimuli. It normally maintains body fluid balance, vascular tone, and regulates contractility. Heart failure causes a paradoxical increase in AVP. The increased blood volume and atrial pressure in heart failure suggest inhibition of vasopressin secretion, but it does not occur. This phenomenon is related to SNS and RAAS activation overriding the volume and low-pressure cardiovascular receptors and osmotic vasopressin regulation causing increase in AVP secretion. It contributes to the increased systemic vascular resistance (V1 receptors) and to renal retention of fluid (V2 receptors). Stimulation of V1 receptors can also case vasoconstriction of the peripheral vessels, platelet aggregation, and adrenocorticotrophic hormone stimulation. Low-dose arginine infusion initiated in the operating room after complex neonatal cardiac surgery was associated with decreased fluid resuscitation and catecholamine. The vasopressin levels are usually high in the early phase of septic shock, but it's deficiency was noted in vasodilatory shock.

The important mechanism of vasopressin action in stress states is its potentiating effect on ACTH secretion leading to cortisol release. Although vasopressin is a powerful vasoconstrictor it dilates the pulmonary, cerebral, and myocardial circulations helping to preserve vital organ blood flow.

In our group of patients with single ventricle, there was a significant correlation between vasopressin concentration and disturbances of water – electrolyte balance in single ventricle patients. Higher vasopressin plasma levels were connected with greater propensity for fluid retention and prolonged pleural effusions (6).


**1. Introduction**

In the new knowledge economy, innovation plays a fundamental role in the socio–economic development of regions [1]. This growth is associated with the dynamism of companies, especially in the roles played by small and medium businesses [2]. In this context, entrepreneurship is a factor that favors the search for opportunities and innovation with a positive impact on the countries' wealth [3]. Additionally, a sufficient mixture of specific conditions of the entrepreneur, a favorable business climate, and the needed infrastructure is required, as are legal and political conditions that encourage people to create new businesses, especially in the case of Latin America, where this performance has been weak [4].

To achieve this growth, the interaction of diverse actors of the innovation ecosystem is necessary; elements such as access to information, training of human capital through universities and research centers, access to funds, and business opportunities related to the market, customers, and suppliers affect the creation of new ventures [5]. In this interaction, institutions such as universities include educational programs for entrepreneurship to develop creative and innovative talent, which provides opportunities to generate knowledge and networks [6].

As mentioned earlier, human capital formation is a fundamental element of economic growth and innovation [7]. Therefore, previous studies have determined how training projects in universities help to increase the culture of innovation through the promotion of entrepreneurship. One of the studies conducted by Diamantini and Tommasone [8] in Brazil analyzes, through a case study, the contribution of the Master's degree in Innovation Management for Local Development offered by the Fluminense University of Rio de Janeiro and taught by experts from the University of Milano-Bicocca of Italy.

This Master's program provides students with tools to build new businesses and to transform their environments, especially seeking to analyze the main elements of innovation and technology transfer, which promote entrepreneurship and the relationship with other actors, such as incubators, small and medium enterprises, and technology parks. The research concluded that interaction among the different actors is needed to achieve economic growth, although it is not possible to define correct policies without knowing the ideas of those who are experiencing the situation.

Another study was made by the KICKSTART group, integrated by nine institutions of higher education from Latin America and Europe. The research objective was to seek new practices that generate innovation in the countries of the Latin American region [9,10]. In this study, strategies were used to improve the quality of the teaching–learning process to seek the development of professionals with an innovative approach. For instance, in research conducted in Tecnológico de Monterrey, Mexico, leadership was determined as a fundamental element that must exist in the professionals responsible for developing innovative strategies. Furthermore, this leadership should seek interaction with the tools that allow the transfer of innovative products and processes to generate projects that solve problems in their environment. However, not all teachers knew how to implement these approaches [11].

Therefore, the university needs to be involved in the innovative and entrepreneurial talent training processes, and one of its functions is to prepare citizens to join the productive processes and the generation of wealth [12]. Entrepreneurship education should support the student to learn by doing, incorporating mentors, networking, and increasing the interest of creating a new business [13,14]. However, authors consider that there are still programs that are closer to theory than to practice [15–18]. Thus, understanding the best practices in developing different skills in individuals to meet the new requirements becomes an indispensable factor for improving competitiveness [19].

In this context, the IC2 Institute of The University of Texas at Austin developed the Master of Science in Technology Commercialization (MSTC) in 1997 and began a similar program (MCCT) in 2009 in the city of Monterrey, Mexico, managed by the Center for Global Innovation and Entrepreneurship (CGIE). This center has graduated 250 students who have developed 54 technology-based entrepreneurship projects in Mexico. The cohorts and team membership were designed to evenly mix as managers and directors in national and multinational companies, professors and researchers in universities, and entrepreneurs. Also, a number of former students have started new ventures after taking the program.

Analyzed literature indicates that there is a gap between what universities teach and what students can apply in their lives [20,21]. Some programs are more theoretical than practical, which does not allow students to prepare for the needs of today's society [20]. It is also necessary to know the effects of this training on the development of the regions, but few studies have analyzed them. It is especially relevant for Latin America, where the dynamism of innovation has been limited [4,22]. Hence, this study aims to know the impact on professional development and venture creation from the viewpoint of those who received training in innovation. Also, it seeks to answer the following question: How can higher education institutions have an impact on the development of entrepreneurship and innovation? By understanding the vision of people involved, it is possible to know the contribution that this type of training has in the creation of new companies and in the processes of innovation that can generate a transformation in the social, economic, and political problems of the region.

This article is structured as follows. First, it presents the literature review related to training for innovation and technology transfer. Second, it describes the context in which it was developed. Third, research methods explain how the research was conducted. The following section presents the results

and the analysis of the data obtained with the instrument. Finally, it develops the discussion and conclusions of the research.

#### **2. Literature Review**

In recent years, different studies related to the development of innovation and technology transfer have appeared, and they consider the following elements relevant for improving the economy and promoting innovation and entrepreneurship.

#### *2.1. Training for Innovation*

According to the Oslo Manual, innovation is a new or improved product or process, different from the original and available to users [23]. These changes promote economic and scientific progress through research and the development of new strategies [15]. Additionally, innovation is associated with the formation of human capital that has the necessary skills, because professionals who are part of the innovation processes of the public and private sectors are required to be competitive in the products and services they offer [20]. Therefore, to train future innovators, it is crucial to establish strategies that encourage creativity and promote the required skills to participate in innovation processes.

In this regard, the training processes should bring the student closer to what they will experience in real life, through a pedagogy of innovation where elements known as meta-innovations converge [21]. These elements are, according to Keinänen and Kairisto–Mertanen [20], active teaching–learning methodologies, multidisciplinary learning environments, integration of working life, research and development (R and D), flexible curriculums, entrepreneurship, and internationalization. The path of the training of students to participate in innovation is in Figure 1.

**Figure 1.** Path of the training of students to participate in innovation, according to Keinänen and Kairisto–Mertanen [20].

The meta-innovation processes are active learning methodologies that allow students to build knowledge and meaning to the situations they experience in the educational process, and the orientation of working life that enables learning around real-life situations working on projects with other colleagues. This participation of other people must be multidisciplinary, so that students can share competencies and interact with each other. The flexibility of the curriculum allows following different trajectories according to the needs and interests of the students. It should also give the possibility to promote entrepreneurship, which implies risk management and the search for opportunities. Finally, internationalization will make it easier to develop competencies to participate in an increasingly globalized world [24].

Entrepreneurship is a relevant factor for the economic development of a region [25]. Also, the entrepreneur's role is looking for solutions to problems and bringing these answers to the market. Schumpeter [26] states that the entrepreneur creates new ideas that lead to changing the market with new ways of doing things. This type of innovation is known as a technology push, where the entrepreneur proposes the change and can introduce a new product or a new production method, open

a new market, or make a new industry organization. On the other hand, Kirzner [27] promotes market pull, where the needs of the market lead the entrepreneur to propose a solution [28].

For Rypestøl [25], these classifications are useful to distinguish entrepreneurs and companies, but they do not allow for determining the impact that new ventures have on the development of a region. Besides this, it is necessary to consider the effects of entrepreneur training, as well as the conditions that favor interaction with other actors such as universities, accelerators, incubators, and research centers to generate the expected development and economic growth.

#### *2.2. Technology Transfer*

Latin America has had an increase in entrepreneurship in recent years. However, despite its potential, this region has not achieved the dynamism of innovation that other areas have, in addition to the rise of informal enterprises [22]. As reported by Amorós et al. [4], the little development of innovative companies on this region is caused by three main reasons: 1) a lack of connection of research and development with the creation of new companies, which does not allow the transfer of technology; 2) little application of technology in business models; and 3) lack of public policies that support technology-based companies.

It is crucial to promote entrepreneurship training and innovation to reduce these causes. Furthermore, it is essential that the transfer of knowledge takes place in universities and that research and the search for solutions to society's problems make it easier to commercialize these innovations [29]. Recent studies found a gap between education for entrepreneurship and technology transfer because, in the university, commercialization of technology occurs in controlled environments and its development outside of this context is scarce [30].

An environment to form successful entrepreneurs, in which they can innovate and develop their businesses, is required. The existence of an innovation ecosystem is essential, where the conditions facilitate and give support to the entrepreneur, with an agenda that promotes the processes of entrepreneurship. The degree of success will depend on the development of a virtuous circle to improve economic development, as well as the conditions for innovation, entrepreneurship, public policies for competitiveness, and the growth of a knowledge-based economy [31].

Lackéus and Williams [32] notice that the programs that look for the creation of companies have potential to form interdisciplinary groups of students, offering a diversity of problems to solve, and a portfolio presented by the teachers. These characteristics challenge academic institutions because they require coordination among different disciplines, integration of other actors, and responsibility for the intellectual property of the ideas generated in the programs.

This study analyzes theoretical aspects, based on the opinions of the interviewees and the reality of the context in which they work. Good practices can be known to reduce the identified gap by presenting activities that are being used by higher education centers, such as CGIE, as well as showing the impact on the development of students' innovation and entrepreneurship skills, making these experiences visible in order to continue exploring their effects. The next section presents this context and contains ways in which the research gap is addressed.


3.6.1. Flavonoids against SARS-CoV-2 MPro

Using molecular docking, Cherrak et al. identified quercetin-3-O-rhamnoside, myricetin 3-rutinoside, and rutin as the potential inhibitors of SARS-CoV-2 Mpro in decreasing order with binding energies of −9.7, −9.3, and −9.2 kcal.mol−1 respectively [142]. Another report also confirmed that rutin is a potential flavonoid against the SARS-CoV-2 MPro with a binding energy of −11.187 kcal/mol [143]. A recent study supported the inhibitory effect of rutin on SARS-CoV-2 Mpro via molecular docking with a binding energy −15.63 kcal/mol. Furthermore, ADMET analysis, combinatorial molecular simulations, and hybrid QM/MM approaches concluded that rutin binds very strongly at the active sites of SARS-CoV-2 MPro by forming three hydrogen bonds at His 163, Glu 166, Gln189 residues [144]. Rakshit et al. screened various flavonoids against MPro and identified the top five potential flavonoids in order of rhoifolin, 5,7-dimethoxyflavanone-40-O-b-d-glucopyranoside, baicalin, luteolin, and kaempferol based on their binding energies of −9.28, −8.81, −8.29, −8.14, −8.11 kcal/mol [145]. The inhibitory effect of luteolin against the Mpro is recently confirmed [146]. Fayyaz et al. screened several flavonoids and identified three potentially active flavonoids, whose activities against SARS-CoV-2 Mpro were in the order of rhodiolin > baicalin > silymarin based on their binding energy (−9.05, −8.85, −8.71 kcal/mol respectively) and dissociation constant (0.23, 0.33, 0.41 µm, respectively) using molecular docking and simulation studies [147]. Thioflavonol also inhibits Mpro [148] along with other flavonoids such as apigenin, daidzein, quercetin, kaempferol, luteolin, epigallocatechin, and kaempferol using molecular docking and simulation analysis [104].

The MPro is considered the most promising drug target for SARS-CoV-2 due to its proteolytic activity, cleaving viral polyprotein into independent functional proteins required for SARS-CoV-2 replication [149–151]. The other reason for its therapeutic importance is its dissimilarity to any human cell protease [152–154] and its similarity with the MPro of SARS-CoV [155]. The most common active site residues of MPro were Glu166, His163, and Met165, which were involved in the interaction with most of the flavonoids.

#### 3.6.2. Flavonoids against SARS-CoV-2 Spike Glycoprotein

Rutin inhibits the SARS-CoV-2 spike glycoprotein but with less binding energy (−7.9 kcal/mol) [143]. On the other hand, naringin inhibits the spike glycoprotein more effectively with binding energy −9.8 kcal/mol compared to standard drug dexamethasone with the binding energy of −7.9 kcal/mol [156]. Fayyaz et al. described potentially active flavonoids, whose activities against SARS-CoV-2 spike protein were in the order of rhodiolin > hesperidin (with active site 1) > hesperidin (with active site 2) > silyhermin based on their binding energy (−8.68, −8.53, −8.18, −8.05 kcal/mol, respectively) and dissociation constant (0.43, 0.56, 1.01, 1.25 µm respectively) [147]. The authors showed that hesperidin could bind to two different active sites on the spike glycoprotein with different binding energies. Teli et al. highlighted that rutin could serve as a dual receptor inhibitor against the Mpro and spike glycoprotein of SARS-CoV-2 with improved ADMET parameters.