**4. Pharmacodynamic changes in children**

Results obtained from clinical trials and experimentation with the animals show that receptor development leads to changes in drug response. Serotonin is a neurotransmitter paying important role in the behavioral and psychiatric disorders. Serotonin at the brain steadily decreases with the increasing age. The pharmacodynamic response of Dopamine,

Protein binding may change with age and concomitant illness. In certain circumstances, an understanding of protein binding may be needed to interpret the data from a blood level measurement and to determine appropriate dose adjustments. In vitro plasma protein binding studies can determine the extent of binding of the parent and the major active metabolite(s) and identify specific binding proteins, such as albumin and alpha-1 acid glycoprotein. Optimal estimates of the degree to which protein binding is linear may be obtained by testing

The main reason that age affects drug action is that drug elimination is less efficent in newborn babies and in old people, so that drugs commonly produce greater and more prolonged effects at the extremes of life. Other age-related factors, such as variation in

Drug excretion by the kidney is controlled by glomerular filtration, tubular secretion, and tubular re-absorption (Buxton & Benet, 2011). Because these processes mature at different rates in the pediatric population, age can affect systemic exposure for drugs where renal excretion is a dominant pathway of elimination. Consideration should also be given to the maturation of other excretory pathways, including biliary and pulmonary routes of excretion. Glomerular filtration rate (GFR) in the newborn, normalized to body surface area, is only about 20% of the adult value, and tubular function is also reduced. Accordingly, plasma elimination half-lives of renal eliminated drugs are longer in neonates than in adults. In babies born at term, renal function increases to values similar to those in young adults in less than a week, and indeed continues to increase to a maximum of approximately twice the adult value at 6 months of age. The increase in renal function occurs more slowly in premature infants. Renal immaturity in premature infants can have a very large effect on drug elimination. For example, in premature newborn babies the antibiotic Gentamicin has a plasma half-life of 18 hours or greater, compared with 1-4 hours for adults, and approximately 10 hours for babies born at term. It is, therefore, necessary to reduce and/or

Drugs and their metabolites are excreted through sweat, urine, stools, or enzymes. By the time kidney functions develop, disposing drugs via urinary system is limited. The glomerular filtration speed and the circulation in kidneys are 30-40% of adults; this ratio is even smaller for infants delivered before 34 weeks. Following the first two weeks, the glomerular filtration speed is doubled, eventually reaches to adult-levels in 2.5 – 5 months (Çetinkaya &Tengir, 2006). Glomerular filtration speed meets adult levels in 6 – 12 months

Results obtained from clinical trials and experimentation with the animals show that receptor development leads to changes in drug response. Serotonin is a neurotransmitter paying important role in the behavioral and psychiatric disorders. Serotonin at the brain steadily decreases with the increasing age. The pharmacodynamic response of Dopamine,

(Pala & Baktr, 2011). The half-lives of drugs also change (Pala & Baktr, 2011).

maximumand minimum observed concentrations (Buxton & Benet, 2011).

pharmacodynamic sensitivity, are also important with some drugs.

space out doses to avoid toxicity in premature babies.

**4. Pharmacodynamic changes in children**

**3.4 Protein binding**

**3.5 Excretion**

also an important neurotransmitter, varies largely in the newborn and adult test animals. Many neurological, psychiatric and behavioral disorders are related to the dopamine at SSS. Among the pharmacodynamic responses of the drugs which are being used against this type of disorders may show significant differences during infancy and childhood.

Major factors affecting the newborn's response to a treatment: Gestation age, chronological age, weight, development phase, liquid-electrolyte balance, disorder level at the organ systems and functions, presence of co-existing diseases, accompanying other medication (Oval, 2008).


*3.2.1 Development of early time BOD biosensors and accomplishment by Dr. Isao Karube in this field*

As described above, the BOD5 method is time-consuming and requires tedious operations. For example, it is not possible to detect the abnormality of wastewater before and after the treatment because it takes 5 days to obtain the measurement result. In other words, even if abnormal wastewater flows into the treatment facility or is not sufficiently treated at the treatment facility, it can be detected only after the wastewater has flowed out to the environmental water. In addition, the tedious operations of the BOD5 method also make it difficult to make accurate and accurate measurements.

In 1977, Dr. Karube studied a practical microbial biosensor for BOD [58]. The key technique was the immobilization of microbes to a thin collagen membrane. The microbial membrane was put onto the surface of a DO electrode. By the addition of a sample solution into a batch system, microbial respiration was activated by the decomposition of organic matters, and the degree of DO consumption by the microbes was determined by the DO electrode. The microbial BOD biosensor indicating DO consumption (BODDO) could successfully determine the BOD value at drastically shortened incubation and measurement times (ca. 30 minutes). By the study, the possibilities for solving the problems of wastewater control were enhanced.

In 1979, Hikuma and Karube et al. developed a flow system of the BODDO biosensor [59]. In the study, omnivorous yeast *Tricbosporon cutaneum* (*Tc*), as a practical microbe, was used in this BODDO-*Tc* biosensor. The microbes were immobilized onto a microporous membrane and attached to the surface of the DO electrode. Based on the study, a flow-type BODDO-*Tc* biosensor was available for practical applications.

Both the desktop-type for rapid measurements and the installation-type for continuous monitoring were sold from the Central Kagaku Co. in 1983. These BODDO-*Tc* biosensor instruments enabled wastewater control by both real-time measurement and continuous monitoring at sewage plants and factories. The BODDO-*Tc* biosensor was established as one of the JIS methods (JIS K 3602) in 1990 [60]. Since then, the BODDO-*Tc* biosensor instrument has also been used in educational settings as a science teaching material [61].

Since the first microbial biosensor was reported, many kinds of microbial biosensors have been studied for not only environmental applications but also food applications, including fermentation. These studies on both environments and foods by microbial biosensing methods were reviewed [3, 10, 15, 62].

Dr. I. Karube was widely studied all-fields of biosensor development as one of the leading scientists in the world. His notable study on biosensor development was summarized in the review [10] and the detailed history of his study on microbial biosensor development was described in one chapter of Encyclopedia [57].

The notable studies on the microbial BOD biosensor development performed by Karube et al. are briefly described as follows. As the study on the BOD biosensors, a microbial fuel cell (MFC) type biosensor has also been developed. However, the MFC biosensor at that time used expensive materials, easily deteriorated electrodes, anaerobes, etc., and had low cathode reaction efficiency due to low electron transfer from the anaerobes to the cathode. In addition, a flow-type cathode chamber of the MFC biosensor has a low exchange efficiency of sample solution, making it difficult to repeat and rapid BODMFC measurements.

After two types of BODDO-*Tc* biosensor instruments were practically used, the other types of BOD biosensors have been widely studied and developed. One of the practical studies was the development of a bioluminescence BOD biosensor using a luminous bacterium *Photobacterium phosphoreum* (*Pph*) [63]. In 1993, Hyun et al. studied a BODBL-*Pph* biosensor; however, the emission intensity of BL released by *P. phosphoreum* decreased with each measurement. Thus, a reagent-type BODBL-*Pph* biosensor instrument was practically used by Tamiya et al.

Another practical study was the development of portable type instruments for on-site monitoring. To realize the on-site monitoring, it was required to stop using air-supply equipment, to reduce the size of measurement devices, to miniaturize and single-use biosensors, to employ omnivore and vital microbes, etc. Dr. Hiroaki Suzuki has been studying the miniaturization of the biosensors to be used as disposable sensor chips [64]. In 1996, Suzuki and Yang et al. studied BODDO-*Tc* biosensors for on-site monitoring and developed a low-cost paper-based DO electrode [65] and a disposable BODDO-*Tc* biosensor chip [66]. However, in general, the BODDO-*Tc* biosensors were difficult to accurately measure the high BOD value of a sample solution, because the solubility of oxygen into water is limited (8.11 mg O2/L at 25°C and 1 atm).

To solve the problem, in 2000, Yoshida et al. studied two types of BOD biosensor principles for on-site monitoring. One was a single mediator (SM) type of an electrochemical BOD biosensor [67]. In the study, omnivorous bacteria *Pseudomonas fluorescens* (*Pf*) was isolated from a sewage plant. By using potassium ferricyanide as a highly soluble mediator in water (460 g/L), a BODSM-*Pf* biosensor that does not require air-supply equipment was developed. The principle was applied to a portable type instrument and a disposable BODSM-*Pf* biosensor chip for on-site monitoring [68]. As a result, a wide linear range of calibration (15 and 260 mg O2/L) was obtained. Another type was an optical BOD biosensor using the redox electron acceptor 2,6-dichlorophenolindophenol (DCIP) sodium salt as a redox color indicator

### *Developmental Studies on Practical Enzymatic Phosphate Ion Biosensors and Microbial BOD… DOI: http://dx.doi.org/10.5772/intechopen.104377*

(RCI). Before the development of a portable type optical instrument, an absorptiometric high-throughput BODRCI-*Pf* measurement method was studied using a microplate reader, which is able to measure 96 samples simultaneously [69]. Based on the principle, the portable type optical BODRCI-*Pf* biosensor instrument was constructed using three pairs of light-emitting diodes (LEDs; 600 nm) and silicon photodiodes (Si-PDs), and a transparent disposable chip containing three biosensing spots [70]. As a result, a linear relationship was observed below 176 mg O2/L, and the detection limit was 14 mg O2/L (*n* = 6, RSD = 10.3%). An excellent correlation coefficient (*r* 2 = 0.989) was obtained after 600 seconds incubation time. By the study of Yoshida et al., two portable types of the practical BOD*Pf* biosensor instruments were developed using two types of redox electron acceptors.

On the other hand, in 1999, Chee et al. studied highly sensitive BODDO biosensors for low BOD measurements [71]. The background of this study was that the spread of sewage treatment facilities improved the water quality of rivers at that time, and it became necessary to measure low BOD including persistent organic matters in the sample solution. To properly measure the river water quality at that time, isolation of the microbe that biodegrades persistent organic matters was needed, and they isolated *Pseudomonas putida* (*Ppu*) from a sewage plant. To enhance the biodegradability of the persistent organic matters by *P. putida*, they examined pretreatment methods of the sample solution and finally established the photocatalytic UV-TiO2 method [72]. As the result, a practical linear range of calibration (0.5 and 8 mg O2/L) was obtained using artificial wastewater containing persistent organic matters.

The studies on the practical BOD biosensor were successfully performed by Dr. Karube et al. and the practical instruments including the potable instruments for on-site monitoring were developed.

#### *3.2.2 Development of BOD biosensors that the author principally conducted*

After these excellent studies on the practical BOD biosensors by Dr. Karube et al., the author explored other possibilities of BOD biosensors with better functionality, for example, improvements of (1) detection limit, (2) signal repeatability of the microbial biosensor, and (3) suitability of microbe used.

Then, the author tried to satisfy these requirements by employing the absorptiometric BODRCI measurement method and a temperature-controlled three-cuvette-stir system [73, 74]. As the usable (easily available, omnivorous, and vital) microbe, Baker's dry yeast *Saccharomyces cerevisiae* (*Sc*) was used after liquid culturing. *S. cerevisiae* is budding yeast; therefore, it was suitable for forming uniform suspension in the cuvette. On the other hand, *T. cutaneum*, which is conventionally used for the BOD biosensors and makes flocks by sticking together, was not suitable for this study because it was filamentous fungi. In this method, DCIP was used as RCI of a high absorption coefficient (*epsilon* = 1.45 × 105 cm−1 M−1) to enhance the sensitivity of the absorptiometry. Then, the absorbance of DCIP decreased due to degradation of organic matters by *S. cerevisiae* in the measurement suspensions, and the absorbance change between before and after incubation was measured as the time difference method (**Figure 3**).

In general, the suspension is not suitable for absorptiometry due to the occurrence of light scattering. However, by a combination of the cuvette-stir system and the time difference method, the influences of the light scattering were efficiently canceled and only absorbance change of DCIP was accurately determined. Further, by repeating the exact same measurement operation three times, the fluctuation of the measured value became small, and the reproducibility was improved, so that highly precise measurement became possible. As a result, the significant difference from the blank value became large, and highly sensitive measurement became possible.

BODRCI-*Sc* measurement method: As a result, a calibration curve between 1.1 and 22 mg O2/L (*r* = 0.988, six points, *n* = 3, RSD = 1.77%) was obtained by this highly sensitive BODRCI-*Sc* measurement method when the incubation mixture was incubated for exact 10 minutes at 30°C. Employing salt-tolerant yeast *S. cerevisiae*

#### **Figure 3.**

*A key principle for the simple absorptiometric measurement method using redox color indicator [73].*

*Developmental Studies on Practical Enzymatic Phosphate Ion Biosensors and Microbial BOD… DOI: http://dx.doi.org/10.5772/intechopen.104377*

ARIF KD-003 (*Sc*II), a BODRCI-*Sc*II measurement method was also studied for seawater [75]. In these studies, the highly sensitive BODRCI-*Sc* measurement method was developed by enhancing the repeatability of the measurements and by employing the usable yeast. From the future perspective of this study, the BODRCI-*Sc* measurement method might be improved by applying it to an absorptiometric-FIA system.

BODDM-*Sc* biosensor: As the other application of *S. cerevisiae* to a BOD biosensor, the author also studied a mediator type BOD biosensor. By applying a double mediator (DM) system coupled with ferricyanide and a lipophilic mediator, menadione, electrochemical signals were obtained from the eukaryote *S. cerevisiae* cells [76]. For practical use, the author designed a package-free disposable microbial biosensor chip containing living microbial cells [77] and the biosensor chip was applied to a DM-type BODDM-*Sc* biosensor [78]. Under the optimal conditions, a calibration curve was obtained with a practical range of 6.6–220 mg O2/L (five points, *n* = 3, RSD = 6.6%). Thus, the BODDM-*Sc* biosensor was developed as the third generation of the BOD biosensor [3, 10].

BODCL-*Sc* measurement method: By applying the principle of the BODDM biosensor, the author next studied a chemiluminescence BOD (BODCL-*Sc*) measurement method. Because Yamashoji et al. in 2004 already established luminol CL assay for the viable microbial detection method using ferricyanide and menadione [79]. Their principle was based on hydrogen peroxide (H2O2) determination produced by the reaction of the viable microbes and menadione. After optimization of the measurement conditions, a practical correlation between BODCL-*Sc* value and amount of organic matters assimilated by *S. cerevisiae* was observed with a range of 11–220 mg O2/L (six points, *n* = 3, RSD = 3.71%) at the incubation time of only 5 minutes [80, 81]. Then, the detection limit was 5.5 mg O2/L.

BODDM:Trinder-*Sc* measurement method: As one of the reagents for H2O2 measurement, there is a modified Trinder's reagent. It is an absorptiometric H2O2 measurement method using peroxidase (POD), 4-aminoantipyrine (4-AA), and *N*-ethyl-*N*-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt (MAOS). Applying this reagent, we already developed a surface plasmon resonance (SPR) biosensor for H2O2 measurement using a modified Trinder's reagent as a color indicator [82]. This reagent had high selectivity, however, POD was unsuitable for applying to the microbial biosensor due to take cost. On the other hand, H2O2 reacted as the reactant in BODCL-*Sc* method. This meant that H2O2 or active oxygen species might also react as the reactant in BODDM-*Sc* method. To verify the possibility, MAOS and 4-AA, which were used in the modified Trinder's reagent, were added to the principle of the BODDM-*Sc* method. As the result obtained without the use of POD as the biocatalyst, correlations between BODDM:Trinder-*Sc* value and amount of organic matters assimilated by *S. cerevisiae* were observed in several measurement conditions, although further practical examinations are required [83].

In the studies that the author principally conducted, excellent functions in the BOD biosensor were achieved in (1) the practical detection limit, (2) the signal reproducibility, and (3) the suitability of the microbe used. In addition, some principles of both microbial BOD biosensors and measurement methods have been studied for practical use, but none of the studies the author have principally conducted has reached practical use. The most practical BOD measurement method in the studies might be the BODRCI-*Sc* measurement method. The reasons are as follows:

For future study, automatic instrumentation of the BOD biosensor would be required having the features that we obtained. Then, further suitability of the microbe might be needed to be considered, for example, use of thermally killed microbes [84] or cell crushed microbes, or direct use of available dry yeast [85].


*3.1. Boundary Conditions*

The school year begins in mid-September. This study was developed one month after the beginning of classes, with the autumn just begun. Autumn 2019 presented itself as a time of slightly higher temperatures than usual. The average temperature of this season tends to be around 20 ◦C, but

in this case, it has been recorded at an average of 1 ◦C more. No rainfall was recorded in the days leading up to the survey.

On the day of the survey, the outdoor temperature variation exceeded 15 ◦C. In the morning at the beginning of classes, 18 ◦C was measured and at the exit, 34 ◦C was reached. This difference has a great influence on the variation in the thermal sensation of the students throughout the morning (Figure 3).

**Figure 3.** Outdoor temperature evolution in Seville during the study period.

Figure 4 shows a slight deviation from the CS1 and CS2 class towards heat sensations with respect to the CS3 class, who were more comfortable thermally because their class schedule was 10 to 11 hours and the average temperature measured was 26.2 ◦C. Meanwhile, for the CS1 and CS2 groups, the time frame in which they did the questionnaires and the discussion was 11:30–12:30 and 12:30–13:30, respectively, and the average temperature measured was 27.4 ◦C.

**Figure 4.** Thermal sensation reported by students.

Despite this, when assessing overall comfort (Figure 4), CS1 gets a rating somewhat worse than CS3. This is indicative that there are factors that are affecting comfort beyond temperature.

Figure 5 shows the comfort sensation reported by students. The valuations were affected by several overall comfort parameters, as well as the schedules and time-frames for the assessments, resulting in different distribution of rating among CS1, CS2 and CS3.

**Figure 5.** Comfort sensation reported by students.

Another value that can influence the results is the clothing of the occupants, as it has been evaluated and established that the conditions in this aspect are very similar, since the variation is minimal (Figure 6).

**Figure 6.** Clothing index (clo) in the classrooms.


4.8 CH4 gas sensing response

The first step was the preparation of CH4 gas sensor. In the fabrication of CH4 gas sensor, a thick layer (200 nm) of Au was coated by ion sputtering technique on Si (100) wafers. A small amount of ZnO nanostructures was put on a pair of interdigitated electrodes on Si substrates having a gap of 55 μm. A small drop of methanol was dropped on the nanomaterials so that a thick paste was formed. The annealing of sensors was carried out in an open furnace tube for 2 h at 400°C before performing the gas sensing experiments, for the purpose of attachment of oxygen on the surface of sensors. The sensing experiment was performed at 200°C with 5-min cycles of dry air and 400 ppm CH4 gas concentration. The sensing response (S = Ra/ Rg) of the device was measured by resistance change upon exposure to air (Ra) and CH4 gas (Rg). Figure 10(a) and (b) shows the sensitivity response of CH4 (methane gas) at 200°C for undoped ZnO nanowires and for Mg-doped ZnO nanobelts, respectively. Research papers showed that the sensitivity of the resistive sensors is highly affected by the Mg doping. The sensors were tested in a temperature range of 50–200°C for 400 ppm of CH4 gas. Sensors show some response magnitude from 100°C temperature. Undoped ZnO nanowire sensors get its optimal point at 200°C

a huge sensing response to acetone (14), and those of the other solvents are no

The Higher Education Commission (HEC) of Pakistan is acknowledged for financial support through project No. 9294/NRPU/R&D/HEC/2017. Thanks to Prof. Dr. Syed Zafar Ilyas and Dr. Waqar. A. Syed. The authors would also be thankful to

COMSATS University Islamabad for necessary funds for the project.

Synthesis of Metal Oxide Semiconductor Nanostructures for Gas Sensors

DOI: http://dx.doi.org/10.5772/intechopen.86815

There is no conflict of interest in this chapter.

\*, Majeed Gul2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Thin Films Technology Laboratory, Department of Physics, COMSATS

2 Centre of Excellence in Science and Applied Technologies, Islamabad, Pakistan

© 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,

, Murrawat Abbas3 and Muhammad Amin2

greater than 8.6.

Acknowledgements

Conflict of interest

Author details

119

Nazar Abbas Shah1

University, Islamabad, Pakistan

3 NUST University, Islamabad, Pakistan

provided the original work is properly cited.

Figure 10.

(a) CH4 gas sensing response of undoped ZnO nanowires (S1). (b) CH4 gas sensing response of Mg-doped ZnO nanobelts.

with response magnitude of 1.84. Doped ZnO nanobelts also get its optimal operating point at 200°C. Its response magnitude was obtained at 2.06. The best sensing signal response of CH4 was found at 200°C. The sensing response at 50, 100, and 150°C (not shown) was comparatively negligible. The sensing response of undoped ZnO nanowires and Mg-doped ZnO nanobelts was found to be 1.84 and 2.06 at 200° C for the same concentration, respectively. The enhanced sensitivity response was observed for the ZnO nanostructures as shown in Figure 10(b). Large amount of oxygen molecules and atoms are adsorbed on Mg-doped ZnO nanobelts due to large surface area (i.e., large defects are created) due to which interaction chance of CH4 gas increases as compared to undoped ZnO nanostructures [60, 61]. On exposing the surface of the ZnO nanostructures to air, oxygen is adsorbed at the ZnO nanostructures surface by capturing an electron from conduction band of surface sites of undoped and Mg-doped ZnO nanostructures [62, 63]. Reactive O2 (oxygen molecules) are chemisorbed or trapped by these ZnO nanostructures from the air, forming active oxygen species O2 and O; as a result transformation of electrons takes place, due to which a wide space charge is formed that results in a decrease in carrier concentration due to which the resistance of the material is increased.

### 5. Conclusions

Growth of 1-D ZnO nanostructures was presented in the present chapter. Vaporliquid-solid mechanism has been employed for the synthesis of ZnO nanostructures. It was found that the morphologies tuned with change in temperature which leads to the formation of nanowires at 850°C, nanorods at 900°C, nanobelts at 950°C, and nanobelts with needle-like ends at 1030°C. The dimensions of the morphologies have been measured by SEM. The length of the structures from 2.93 to 319.48 μm, thickness of the structures from 0.05 to 1.88 μm, and diameter of the structures from 0.95 to 12.66 μm have been obtained successfully. XRD peaks show that the crystallinity and intensity increase with increase in temperature. Doping of magnesium acetate (0.05 g) in ZnO through vapor transport method was successfully achieved. The sensing response of doped ZnO nanostructures for UV light at room temperature and CH4 gas at 200°C has increased. ZnO nanowires show great selectivity response toward different volatile organic compounds (ethanol, methanol, and acetone). At the same concentration and temperature, the ZnO nanowires show Synthesis of Metal Oxide Semiconductor Nanostructures for Gas Sensors DOI: http://dx.doi.org/10.5772/intechopen.86815

a huge sensing response to acetone (14), and those of the other solvents are no greater than 8.6.


**3.1 Medicinal and aromatic plants for human life**

 The magical compound of medicinal and aromatic plants keeps saving human until present, such as medicine, food, healing, and recreation. One of the huge benefits from medicinal and aromatic plants was to overcome many difficult illnesses, such as contagious disease, cancer, and AIDS/HIV. The National Cancer Institute (NCI) screens plants for the possibility of new drugs and active plant chemicals for cancer and AIDS/HIV in several ongoing collaborative programs [9]. **Table 3** indicated anticancer drugs which have received FDA approval for commercial production.

**Table 3.**

*Plant-derived anticancer drugs have received FDA approval for commercial production.*


**2. Evolution of the genome: why thymine was key**

The genome of all organisms on earth today is coded by 4 nucleobases, which are the two purines, adenine (A) and guanine (G), and the 2 pyrimidines, cytosine (C) and thymine (T). The nucleobases are commonly conjugated to (deoxy)ribose, which are termed nucleosides, and they can then be further conjugated to phosphate groups, giving them the name nucleotides. As deoxynucleotide triphosphates (dNTPs), the nucleobases A, T, G and C are commonly incorporated into DNA, held in a sequence via covalent attachments to the DNA backbone (made up of covalently attached deoxyribose and phosphate in a chain), making a DNA strand. A DNA strand is paired with another DNA strand (forming the classic double-helix structure of DNA) which are complementary to one another via hydrogen bonds between nucleobases within adjacent strands of the DNA helix. In 'normal' DNA, A:T and G:C always pair with one another. Uracil (U) is another nucleobase that is mostly found in RNA, which is synthesised via nucleotide triphosphates (NTPs). RNA can form similar structures to DNA, except that A:U pair together instead of A:T and that the RNA backbone incorporates ribose instead of deoxyribose. RNA is also able to encode genetic information (mRNA) but is more diverse and carries out functions similar to proteins (tRNA and ribozymes). One might ask what the need is for these two similar systems for carrying genetic information and why are they different? To answer these questions, it helps to explore the evolution of the genome.

Life on earth is thought to have originated ~3.8 billion years ago from what is termed the 'primordial soup' (or prebiotic soup). In this prebiotic world the 'RNA world' hypothesis states that the first complex organic molecules to form were RNA based. To support this hypothesis, analysis of carbonaceous meteorites that have fallen to earth have found to contain a range of carbon-molecules, including U, A and G (but not C), which is thought to represent the composition of a very young earth (reviewed here [7]). Additionally, a formamide-based scenario has purposed that formamide (available in the prebiotic earth [8]) could be the starting point for generating all the RNA-components, under conditions that are thought to be present in the prebiotic earth, with U being generated in good yield [9–12]. This leads to the belief that the hereditary genetic information might have been RNA. It is thought that, eventually, there was transition to a DNA-based hereditary system since the deoxyribose-containing DNA backbone is much more stable than ribose-containing RNA backbone [13–15] and RNA replication seems to be far more error prone than DNA replication [16]. In turn, this allowed the evolution of larger, more complex genomes and, therefore, complex multicellular organisms to form.

Potentially, at some point RNA and other molecules were concentrated in a membrane-like structure (making certain catalytic reactions feasible), forming the first RNA cells with metabolism. RNA can catalyse reactions (ribozymes), encode genetic information, transport amino acids (tRNA) and catalyse peptide-bond formation (ribosomes). The idea that RNA was first to carry out these critical functions of the cell is based on ribosomal RNA being extensively involved in peptide-bond formation, suggesting that proteins potentially became essential later in evolution [17]. To allow for the transition from an RNA to DNA cell, the evolution of a mechanism to convert NTPs (containing ribose) to dNTPs (containing deoxyribose) must have occurred. Ribonucleotide reductases (RNRs) frequently catalyses the conversion of nucleotide diphosphates (NDP)/NTP in eukaryotic/prokaryotic cells into dNDP/ dNTP, respectively, and are thought to have a common ancestor [18, 19]. Interestingly, it is thought that the first DNA cell would have incorporated U (instead of T) into its genome. This is backed up by the fact that RNR can directly convert ATP, UTP, GTP

## *The Importance of the Fifth Nucleotide in DNA: Uracil DOI: http://dx.doi.org/10.5772/intechopen.110267*

and CTP into its corresponding dNTPs; however, dTTP needs extra steps involving deoxyuridine monophosphate (dUMP) conversion to dTMP, via thymidylate synthase (TS or TYMS), and two phosphorylation steps by kinases to produce dTTP [20]. Due to this convoluted route to produce dTTP, yet dUTP is synthesised in a simpler manner, it would make sense in an evolutionary context that the initial DNA cell first incorporated U into its DNA, which was then later replaced by T.

One might ask why the need of T-based DNA when U-based DNA performs the same task and is energetically easier to make? C deamination produces U, which happens at a relatively fast rate. A deaminated C will produced a G:U mismatch and led to mutated DNA during replication. In U-based DNA, a U produced via C deamination and U that is normally incorporated in DNA are chemically identical and, therefore, a G:U mismatch would be difficult to identify as damaged DNA, in the context of primitive cells without sophisticated DNA repair mechanisms. To overcome this issue, cells evolved to incorporate T instead of U into their DNA, making U produced from C deamination completely foreign instead. This meant that T-based DNA produced more stable genomes, which is especially important for evolving and maintaining large complex genomes found in, for example, multicellular organisms.

For T-based DNA to be viable, the cell evolved three key enzymes: dUTP nucleotidohydrolase (dUTPase), thymidylate synthase (TS) and uracil-DNA glycosylases (UDGs). After dUTP synthesis via RNR, dUTP is potently dephosphorylated to dUMP by dUTPase. In a two-fold mechanism, this reduces dUTP levels, reducing U misincorporation into the DNA, and produces the TS substrate (dUMP, as discussed in the previous paragraph), leading to an increase in dTTP. The relative levels of the dUTP:dTTP pools determines the rate of U misincorporation into DNA, due to DNA polymerase having difficulty distinguishing between dTTP and dUTP, which are identical molecules except for a single methyl group. In fact, DNA polymerases readily incorporate dUTP as well as dTTP based on their representative concentration and nucleotide availability [21]. If U is misincorporated or C deamination occurs, a UDG removes U, creating an abasic site where no nucleobase is present in a strand of DNA, and a primitive form of DNA damage repair could have corrected it. With the evolution of these 3 proteins, cells were able to detect C deamination (which is always mutagenic) rapidly, repair it and reduce U misincorporation as well.


**4. Goats in the texts, art and imagery of the ANE**

Primary sources available to the modern scholar of the ANE cultures include objects, architecture and written texts that have survived environmental extremes. This investigation seeks to determine whether, if goats appear in these material

artefacts, there is some indication that they were considered more than merely useful as a livestock species with utilitarian value but also as an object in myth, legend or fairy tale.