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The foundation of science lies in investigating the events that happen around us. Researchers utilise a variety of processes to learn about the natural phenomena. The fundamental investigation process is based on the scientific method. The scientific method is a guide scientists use to logically examine and interpret the information collected by the following steps sequentially, (Castillo, 2013), to utilise their knowledge gained from their study in an attempt to explain the occurrences around the world. The primary objective of the scientific method is to ensure that the research taken place is conducted in a objective, replicable and justified manner. The exact process may vary according to the field of investigation and the type of questions that needs to be answered, but six necessary steps can sum up the entire process of a scientific method. These steps are listed below:
• Ask a question:
Curiosity is the beginning of a scientific approach. The scientific method begins when we observe the things around us and ask questions such as what the event is. E.g. How is the event happening? Why is it happening? What are the causes of a particular illness such as cancer? How can we prevent or cure disease? It is essential that we find something which is unanswered and then moves to the process of finding the answers to the questions. These questions are the primary motive of the study that researchers carry out.
• Define the problem:
In this step, researchers learn about the phenomena and identify the question which needs to be tackled. Researchers carry out background research around the topic, and they may locate specific research areas that have not been researched in detail. For example, if we observed a particular illness and current study only defines the causes of the disease, but there is a lack of information or research about the mechanism of the disease, then researchers may want to work out the arrangement of the illness (Bradford, 2017). This step is vital in any research because it helps in defining the area of study for researchers to investigate in which will aid them when identifying a hypothesis as well as enhancing their knowledge in the field.
• Propose a hypothesis:
A hypothesis is a statement made by a researcher based on an assumption and needs to be tested further to be proved. In this step, the researcher constructs a hypothesis by making an educated guess, after doing their background research, about what they expect from the research/results of the experiment, which is evaluated in the further steps.
• Experiment:
The next step is to set up an method where quantitative and qualitative data based on evidence will be collected to test the hypothesis. Here we act to find results to our question. An analysis contains an independent and a dependent variable. ‘An independent variable is something which can be manipulated by the experimenter, and a dependent variable is a quantity or the thing being observed’ (Caparlar & Donmez, 2016). For example, the severity of a disease is the dependent variable, and the malfunctions that occurred in the immune system is an independent variable in the case that we are considering. The data we would collect in our experiment will be about the level of defects in the immune system about the incidence and severity of illness.
• The Formulate a theory:
In this step, researchers study and analyse the data collected to conclude. In the above example, we need to examine if there is any correlation between the defects in the immune system and the occurrence of the illness. We may find that there is indeed a relationship between the flaws in the immune system with the disease proving the hypothesis to be right. Alternatively, the data may indicate that the condition has no relation to the malfunctions of the immune system which means that our theory is wrong. We have to reject or accept a hypothesis depending upon the results of the conducted experiment. By this analysis, researches formulate a method which is the general principles drawn from the facts of the investigation.
• Replicate the results:
To get the validity and credibility of the results obtained, it is necessary that we communicate the results of the experiment (Leung, 2015). Replicate experiments assures that the results obtained are fair and free from experimental bias. Having other scientists test the hypothesis through their own set of operations is also beneficial in ensuring the correctness of our results. This step is essential as any research is considered to be reliable only if it is reproducible.

Figure 1: The research cycle
‘During the process of scientific research and experiment, we need to develop tools that represent the subject of our study for a variety of purposes’ (Caparlar & Donmez, 2016). These representations are called the scientific models. An experimental model is usually a small scale, a simplified illustration of an organism, object, process, natural phenomena or a complex system that shares its characters with its real-world counterpart and is used in researches in place of them. The models are central, in both the communication of the explanations and the research itself. Models are used for scientific study as it’s not efficient to perform scientific experiments on complex and significant objects due to many reasons (Jonsson ; Sheiner, 2002). For example, models also help to minimise the cost and are usually less time-consuming. Also, many believe it’ ethically incorrect to experiment on a living organism. For example, to understand the development process of a baby, we cannot conduct operations in the real baby. So, we make use of models that share similar characters with the human baby. Albeit the experimental model is the modern science’s central component; at best they are approximations of systems represented by them that cease to be precise as replicas. Therefore, constantly scientists have been working in improving and refining models.
One of the animal models that is currently being widely used in scientific researches, especially in the studies regarding inflammatory and metabolic diseases, is the Zebrafish which is a tropical freshwater fish native to South Asia. The characteristics that make it an excellent experimental model is described below:
• The genetic analogy to humans:
The genome sequence of a Zebrafish was sequenced entirely in 2013, and 70% of human genes are found in it (Chen ; Zon, 2009).
• Embryonic characters:
The embryo and larvae of a zebrafish are relatively large and transparent at the early stage which allows viewing the development of fertilised eggs in real time. The embryogenesis is also rapid, and the entire body plan is established in less than two days post fertilisation (Kawakami et al., 2004). The size of a zebrafish embryo is such that they can be manipulated easily. The propagation is external, unlike mice which allow observation of the ordinary or abnormal development of the living embryos, without physically affecting the mother to reach the embryo.
• High fecundity rate:
Each mature female zebrafish can produce 200-250 eggs per mating, and the coupling is not seasonal (Lang et al., 2009). Having an animal that provides a large number of offspring is helpful for the replication of scientific experiments.
• Ease of housing and care:
Zebrafish are used for many research purposes such as the study of ‘wound healing, gastrointestinal diseases, microbe-host interactions, genetic diseases and drug discovery’ (Truong et al., 2011). ‘There are several advantages of using a zebrafish as a model such as reduced cost, low space requirement, low feeding cost, ease of transportation between labs, ease of genetic manipulation and injection, a higher level of tolerance for chemical mutagens and absorbing regenerative capacity’ (Goldsmith ; Jobin, 2012). Also, the zebrafish larvae live depending on its innate immune system because the adaptive immune system is functional only after 4-6 weeks’ post-fertilisation. However, there are some features which may be disadvantageous to scientific research. One of these disadvantages particularly for human research is that it is not a mammal which leads to biological differences. Additionally, it is not useful to study human diseases without the presence which cause it, especially for the study of conditions that take place in a tissue type or a body part that is absent in zebrafish (Burke, 2016).
Inflammation is a body’s complex reaction to the tissue injury and infection for the repairing of damage and to normalise the tissue to a healthy status. This is our first line of defence of our innate immune system simulated ‘by pathogens, mechanical and chemical agents and the autoimmune response'(Fujiwara ; Kobayashi, 2005). The inflammatory response is characterised by redness, swelling, warmth and pain. Zebrafish are used to study inflammation because of all its transparent embryo and the similar innate immune system.
The nuclear factor- ?B (NF- Kb) pathway has an vital role to play in B cell and T cell development and proliferation, diseases such as ‘asthma, atherosclerosis, AIDS, tumorigenesis, diabetes, muscular dystrophy, Alzheimer’s disease and several other health conditions (Kumar et al., 2004)’, it’s also one of the crucial regulators of the proinflammatory gene expressions. The most significant role of this pathway is observed in the pathogenesis of inflammation. Different therapies targeting the blockage of NF-KB activity is also being studied in animal models. This pathway is known to be regulated by the NF-inducible inhibitor protein I?B?. ‘Functional NF-?b is a dimeric molecule composed of five proteins’ (Hayden ; Ghosh, 2012). In most cell types, this dimer remains inactive by binding to the inhibitor I?B protein, i.e. ? and ?. These proteins inhibit the transcription factor’s DNA binding activity and inhibit its nuclear accumulation. The activation of the pathway depends upon signal-dependent phosphorylation and degradation of I?B proteins which then transports the NF-KB dimer. ‘The negative feedback loop involving the I?B? protein regulates different inflammatory responses mediated through the NF- Kb pathway’ in attempt to bring the environment back to normal conditions (Karin et al., 2004). The NF-KB pathway itself facilitates the down-regulatory factor of the NF-KB protein due to the inhibitory I?B protein being produced due to this pathway (Hoesel & Schmid, 2013). Thus, the negative feedback loop is mediated through the path as shown in the figure below:

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Figure 2: Schematic diagram of the negative feedback loop in the NF-KB pathway
(Source: Hoesel and Schmid, 2013)
One of the critical immune cells is the macrophages is that it plays a diverse role in the inflammatory response as well as in the wound healing. Following an injury or infection, the nearby immune cells are activated by the pro-inflammatory mediators as well as the Damage Associated Molecular Pattern Molecules (DAMPs) which results in the recruitment of neutrophils, mast cells and macrophages. ‘Macrophages originate from the circulating peripheral monocytes present in the blood’ (Gordon & Martinez-Pomares, 2017). During an immune response, the monocytes are rapidly transported to the site of injury where they undergo rapid cell division resulting in the formation of specialised tissue macrophages. This results in the restoration of the healthy tissue function.
The macrophages play a significant part in our immune system by:
Promotion of Inflammation: Under normal circumstances, the macrophages are generally inactive and produce low levels of pro-inflammatory mediators. However, following the activation by the pro-inflammatory mediators the pro-inflammatory response of macrophages is activated leading to the release of a large number of mediators and cytokines which acts as chemo-attractant and recruit’s immune components.
Promotion of Wound healing: The macrophage team are involved in promoting the wound healing process where the macrophages ‘transits from a pro-inflammatory phenotype to a reparative phenotype referred to as wound macrophages’ (Verschoor et al., 2012).
Removal of neutrophils/apoptotic load of the wound: The critically important function of the wound macrophages is facilitating the non-phlogistic removal of the neutrophils which reduces the oxidative stress on the site of injury/infection.
Promotion of angiogenesis, the proliferation of fibroblast and ECM synthesis: Certain studies reveal that macrophages simulate the wound healing process via the generation of growth factors, protein synthesis, production of proteases and proteases inhibitors influencing the ECM content and remodelling.
The study of the macrophages is done by use of different microscopic tools that includes intravital imaging tools such as fluorescence microscopy and confocal microscopy. The essential step of studying the macrophages by microscopy is labelling them with a fluorescent dye which enables to view the activities within a cell. These dyes are called fluorophores which are used to mark proteins, tissues and cells that are to be examined by fluorescence microscopy (Martynov et al., 2016). They absorb the energy of a specific wavelength and re-emits that energy in another particular wavelength region in the form of light called fluorescence. This emitted light allows the proteins tagged with the fluorophore to be visible under microscopes. The Jablonski diagram explains the mechanism of fluorescence of the fluorophore. This is an energy diagram where we can see that a fluorophore is excited to a higher energy level after absorption of radiation. However, it returns to the ground state within 10-8 seconds, and fluorescence emission occurs when the fluorophore atoms return to its ground energy state. The phosphorescence is also emission of the absorbed energy, but it happens slowly and is usually present when the energy gap is higher (Chan et al., 2017).

The use of Green fluorescent protein (GFP) is extensive in the immunological studies, and different variants of this protein with variable spectra are being developed. The energy spectra of fluorophores is a diagram that depicts the wavelengths where these chemicals absorb radiation and show maximum fluorescence (Shorter et al., 2017). GFP fluoresces maximum when excited at 400 nm with a smaller peak at 475 nm and fluorescence emission peaks at 509 nm. The excitation and emission spectra of the most common fluorophores are shown below:

Figure 3: Excitation and emission spectra of GFP where dotted lines represent excitation spectra, and the solid line represents emission spectra

Figure 4: Spectra of GFP and Red Fluorescent Protein (RFP)

Figure 5: Spectra of common fluorophores
In the context of macrophages study, GFP is used to study its behaviours such as cell proliferation, cell migration, cell division, interactions with the pathogen and in immunoassays. Zebrafish can be a good model for studying macrophages as they also contain similar cells that migrate to sites of injury in larvae. Studies using zebrafish allow the real-time study of macrophage recruitment and phagocytosis in a non-invasive manner. Using transgenic zebrafish lines that label the leukocyte populations with GFP or similar other fluorophores, the roles of macrophages during infection control has been studied. As the larvae are transparent, zebrafish acts as an ideal system for use in GFP and confocal microscopy. More importantly ‘the tissue regeneration capability of these models and the role of immune systems in the process is one widely studied mechanism to understand the behaviour of macrophages’ (Yoshida et al., 2017).
For instance, if we want to investigate the time taken for macrophages to be recruited to caudal fin amputation sites we can design an experiment which makes use of transgenic zebrafish line where the macrophage population is labelled with the GFP. The first step of the research will be to maintain a zebrafish population that has been genetically modifying to visualise macrophages. Then, the caudal fin of the zebrafish larvae will have to be amputated using sterile scalpel and anaesthesia. Then, the timer will be started, and the larvae with amputated fins will be placed under a fluorescent microscope. An observation about the period within which the macrophages were recruited to severed caudal fin sites will then be made. This way we can learn about the function of macrophages in damaged tissue sites promptly. The scientific research process is hence facilitated by the use of animal models and little tools.
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PART 2
NF-?b signalling is essential for the migratory behaviour of macrophages towards caudal fin amputation. By being a ubiquitous transcription factor, it controls the countenance of some genes included in the physiological processes. There are five members of the transcription factor protein family identified namely p65(RelA), RelB, c-Rel, NF-?B1 and NF-?B2. This factor binds to different DNA regions. When they’re activated, they produce various proteins involved in the immune responses in conditions which are unstimulated. This signalling pathway activates some passages that guide the stress-responsive inflammatory responses, developmental programming and cancerous cell growth activities. In the inactive state, the NF-KB factor has a dominant presence in the cytoplasm bound to its inhibitor proteins called the I?B proteins. The I?B family includes p105, p100, I?B– ?, ?, ? and Bcl-3 proteins, present in a tissue-dependent manner. Phosphorylation is then carried out by IkB kinases (IKK1/IKK? and IKK2/IKK?) alongside the non-catalytic accessory protein such as NEMO (NF-Kb Essential Modulator) which leads to the proteasomal degradation of the inhibitors. After degradation of the inhibitory proteins, the NF-kB transcription factor is further phosphorylated and translocated to the nucleus where its function is to act as an activator or repressor of different target gene segments. The usual stimulating molecules are tumour necrosis factor ? (TNF?), cell wall components, and interleukin-1? (IL-1?). Abrignother pathway of NF-kB activation known as the non-canonical path starts from the binding of the stimulatory cytokines to receptors including the B-cell activation factor (BAFFR), CD40, receptor activator for nuclear factor kappa B (RANK) or lymph toxin ?-receptor (LT?R). ‘The NF-Kb pathway regulates the transcription of more than 300 different genes including cytokines, growth factors, micro RNAs, ant apoptotic proteins cell adhesion molecules and it also controls the expression of its inhibitor that is the expression of the IkB proteins’ (Hoesel & Schmid, 2013).

Figure 6: Schematic representation of the pathways leading to NF-kB activation
This signalling pathway is also thought to be involved in some physiological processes in all vertebrates such as fishes including the regeneration of caudal fin. The caudal fin is made of bony rays, mesenchymal tissue, blood vessels, nerves and it has full regeneration ability. This regeneration consists of three stages where wound healing occurs first followed by the creation of the regeneration blastema. Regeneration blastema is a mass of highly multiplying lineage-restricted progenitor of mesenchymal cells and terminates with the patterning of new tissue. NF-kB signalling has recently been shown to be involved in the cardiomyocytes during heart regeneration in zebrafish. The cardiomyocyte-specific inhibition of this pathway has also resulted in cardiomyocyte proliferation that led to defective heart regeneration. Research has shown that the monocytes and the neutrophils migrate towards the stimuli in zebrafish (Sehring et al., 2016). In the case of tail fin amputation, it has been seen that macrophages appear later than the neutrophils to the site of injury. They carry out roles of phagocytosis and also take part in rapid cell regeneration and remodelling of the damaged section.
To examine the NF-kB signalling pathway’s role in the migration of macrophages to the site of caudal fin amputation, zebrafish would be a great model. Majority of scientists believe that the macrophages that take part in the tissue regeneration and wound healing processes migrate towards the damaged site due to the NF-kB signalling pathway being activated (Karin et al., 2004). The experimental strategy to prove this hypothesis will make use of different experimental sets that will relate the level of NF-kB activation to the rate of migration of macrophages towards the caudal fin amputation.
The first thing that we will require for this experiment is a transgenic line of zebrafish. In this experiment, we will view the macrophages throughout the regeneration process after amputation of the caudal fin. Live images will be taken, and the use of fluorophores will be done in conjunction with microscopy to have a direct look at the migration of macrophages. ‘To examine macrophages’ functional role in fin regeneration, it is required to develop a transgenic zebrafish that expresses any fluorophore such as GFP, YFP or EGFP under the control of the mpeg1 promoter’ (Chen & Zon, 2009). This will enable us to visualise the migration of macrophages towards the site of amputation. It is also necessary that we can imagine the activation of the NF-kB signalling pathway in the fishes that we use for experimentation. The genes that controlled by the NF-kB pathway will have to be conjugated to any fluorophore reporter gene such that the cells with the activated pathway will be readily imaged. If any correlation between the activation of this pathway and the macrophage migration is observed, it will be a successful outcome of the experiment. The use of a transcriptional reporter line of the NF-kB signalling pathway will monitor cells with the active NF-kB pathway (Ruland, 2011). The gene expression levels of the inhibitors of the NF-Kb signalling pathway can also be assessed to observe the direct effects of the signalling pathway on the macrophage migration route. Genes that are characteristic of this pathway can be upregulated and compared with controls who have reasonable regulations, to see if this upregulation has any effect on the migration pattern of the macrophages and the rate of regeneration. Fluorescent microscopy or confocal microscopy will be a useful tool in this experiment as it allows live imaging and accurate visualisation of the expression of fluorophores as explained before.
The next strategy would be to make use of the ligands that bind to the receptors and stimulate the NF-Kb pathway. This experiment will make use of the molecular targets such as TNF receptor, IL-1 receptors, IKK, NF- Kb translocation, chromatin remodelling and transcription with a motive of understanding the role of each step of the NF-Kb pathway in the migration of macrophages. We can upregulate or downregulate the expression of these proteins by gene manipulation and observe its effects on the macrophages. Western blot can be used to monitor the levels of these receptors and ligands. E.g., If we increase the level of expression of the kinases that degrade the NF-kB inhibitors and observe that the macrophages migration has been affected by this change, then the role of NF-Kb signalling in migration can be deduced.
The transgenic zebrafish lines can also be treated with the drug inhibitors of the NF-kB pathway to study the function of this pathway in macrophage transportation. Inhibitors such as Lactacystin, epoxomicin, parthenolide, carfilzomib may be used for this purpose. When the channel is inhibited, there will be a disruption in the macrophage migration pattern. This may result in the direct deformations in the caudal fin amputation. Such deformities will indicate that the activation of the NF-kB pathway has an essential role in the regeneration of the caudal fin in zebrafish.
The next method that can be used is Flow cytometry on the fins so that observation of the macrophages about the activated NF-kB signalling reporter fluorescence is possible.
The methods that are used in the experimental strategy is described below in detail:
Cell Fractionation:
Cell fractionation is the process where a cell is broken up to separate its cell components and organelles so that these organelles can be studies in their forms. In this method, the cells are first broken up by using a variety of methods depending upon the type of experiment and the cell. ‘Detergents such as SDS, extensive agitation, use of ultrasonic waves or use of blenders may be done to break the cells’ (Radka et al., 2018). Then, the cells are subjected to differential centrifugation where the cells are rotated under high speed for some times such that the larger cellular particles settle at the bottom called the pellet. The lighter particles remain at the top and are called the supernatant. This supernatant is separated, and a series of centrifugation is done to collect the smaller parts of a cell successively. These fractions are collected by methods that keep it pure and then analysed using microscopy, biochemical methods, Bradford assay or western blot. In the experimental strategy devised above, this method can be used to determine the level of proteins produced and to analyse the level of NF-kB in the nucleus. We can also compare the activity of the cytokines and chemokines in different cell organelles using this method.

Western Blot:
Western blot is a widely used technique for the identification of any specific protein of interest from a mixture of protein that is extracted from a cell. This process is also known as immunoblotting. In this method, we make use of antibodies specifically designed to bind with the protein of interest. Binding of the antibody with the protein of interest allows us to separate the protein of interest from a mixture of proteins as the protein of interest will be attached to an antibody that is tagged for its recognition. The tagging of antibody is done by the use of an enzyme or by attaching a fluorophore to the antibody. In this method, you would need to extract the protein from the desired cells (Mahmood & Yang, 2012). After protein extraction, gel electrophoresis that is SDS-PAGE of the mixture of proteins is done which separates the proteins by its molecular weight, electric charge or isoelectric point or a combination of all of these factors. Then blotting of the proteins is done by placing a nitrocellulose membrane on the gel. Here, the proteins get transferred into the nitrocellulose paper using capillary action. Electroblotting can also be used for faster blotting of the proteins to the paper. The fourth step is the most important step of western blotting where the nitrocellulose paper is blocked by saturating it with Bovine Serum Albumin(BSA). After this, the primary antibody which is specifically designed for the protein of interest is added. If the desired protein is present in the membrane, then a protein-antibody complex is formed. This antibody is called the primary antibody. The secondary antibody added after the primary antibody is tagged with an enzyme and this is made specific to the primary antibody so that it can bind with the protein-antibody complex. Finally, the reaction mixture is incubated with a specific substrate that is converted by the labelled enzyme to give a visible coloured product so that we can be assured about the presence of the protein of interest. The quantitative amount of the protein of interest can also be estimated by observing the amount of substrate utilised. Western blot can be used in or experimental strategies to observe the expression of transcription factors, cytokines, receptors, NF-kB responsive proteins and other proteins of interest. The western blot can also confirm the upregulation and downregulation of the specific proteins that we are going to investigate in this process. Increased levels of expression shown in the mutant types that we had manipulated to observe the effect of NF-kB signalling will indicate that the genetic manipulation has been successful.

FRET: Fluorescence Resonance Energy Transfer (FRET) is a physical phenomenon that is being widely used in biomedical research. It is the transfer of energy from a donor molecule to an acceptor molecule without the presence of any radiation in a distance-dependent manner. The donor molecule is usually a dye or a fluorophore that absorbs the energy initially. Later, this energy will be transferred to the other fluorophore without any molecular collision, and it leads to the reduction of the donor’s fluorescence intensity (Broussard et al., 2013). The emission intensity of the acceptor increases after the acceptance of energy. FRET occurs when the donor and acceptor are close in a distance of 10-100 Å. Also, the absorption or excitation spectra of the acceptor must match with the fluorescence emission spectra of the donor.
This phenomenon is most useful for the study of biological molecules as the required distance matches with the diameter of the thickness of membranes, proteins and the distances between active sites of multi-complex proteins. The FRET is also called a spectroscopic ruler genetically encoded fluorescent dyes such as GFP, or YFP is used to perform FRET invitro. These proteins are allowed to form pairs in the living cells, and can also be attached to other proteins of interest. The resultant chimeric proteins contain a fluorophore and the protein of interest. With donor and acceptor chimeric proteins, the interaction of proteins can be studied. If the donor and acceptor interact then only FRET would occur which means that the proteins of interest are also communicating with each other.
Additionally, this phenomenon can also be used to assess the properties and binding of nucleic acid properties. DNA hybridisation studies can also be done using FRET. It is also utilised as a tool for identifying the distance and supramolecular organisation of cell surface and cell adhesion molecules. In vivo ion binding and interaction of transmembrane receptor proteins can also be monitored using FRET.

Flow Cytometry: Flow cytometry is another method that can be used to assess the cellular characteristics such as cell size, cell count or the cell cycle on a cell-by-cell basis. It is a technique that is useful in analysing the physical and chemical characters of particles in a fluid as it passes through laser. The cellular components are fluorescently labelled, and the fluorophore is then excited by the laser light at different wavelengths. The flow cytometry is used for immunophenotyping where the different types of cell subsets in a heterogeneous sample is measured. This is done by tagging specific proteins of one cell population with a fluorescent tag in the cell surface. Which can also be used to ‘distinguish between necrosis or apoptosis for each cell based on the morphological, biochemical and molecular modifications occurring in the dying cells’ (Houston et al., 2010). It is also applicable in the cell proliferation assays as it can measure the cell multiplication by labelling the undividing cells with a cell membrane fluorescent dye called the carboxyfluorescein succinimidyl ester (CFSE). After the division of the cell, half of the colour is passed to daughter cell which results in a reduction of the fluorescent signal which can be studied through a flow cytometer. The flow cytometer also can monitor the intake of calcium into the cell and the response of the cell to the stimulus. In our experimental strategy, flow cytometry can be used to analyse the macrophage populations as it moves towards the caudal fin amputation site. It can also be used to observe the response of the cells to the stimulus provided in the form of cytokines and interleukins.
In this way, the role of NF-kB signalling in the movement of macrophages towards the caudal fin amputation can be studied by the use of the techniques and methods described above.
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