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Mutations are constantly occurring in our genome from generation to generation. They lead to genetic variation between individuals of a population. Not all mutations are equal though, and most mutations go unknown and unseen by most of the population. This is because there are several different types of mutations that can occur. One of these mutations is a missense mutation, which results in a change in one DNA base pair. This causes one amino acid to become another and can either be harmful or unnoticeable. Nonsense mutations are when an amino acid is changed into a stop codon, prematurely ending the protein. This can be catastrophic for the protein involved. Finally, insertions or deletions result in frameshift mutations, which change every amino acid after the affected area. This can completely change protein function and be devastating. 

Genetic drift is a unique evolutionary force. Genetic drift acts entirely random and has no bias towards any phenotype. Populations that undergo genetic drift experience fluctuating phenotype and genotype ratios. What really sets genetic drift apart is the it always moves towards fixing a certain allele. It can be either the dominant or recessive since it is random selection. After a certain number of generations only undergoing genetic drift there will only be one allele type left. This is because as individuals are selected to reproduce, allele frequencies change. Even if both alleles start out equal, one will be favored over the other due to random chance. This will lead to a divergence that eventually leads to allele fixation. Genetic drift causes populations to differentiate. 

Phloem sap is difficult for scientists to study. This is because plants are highly evolved to minimize phloem loss in the case of external damage. When a sieve tube is penetrated, it signals throughout the plant, triggering the release of callose which clogs the pores of the sieve plate. This causes all phloem movement through the sieve tube to stop. Luckily, scientists have figured out two ways to study the phloem uninterrupted. The first is to use a normal extraction tool that has been coated in an anti-slime. This allows scientists to observe the plant’s phloem without the interruption of sliming. The second way is by zapping aphids off of the plant after they have inserted their stylet. Aphids are able to access the phloem without triggering the release of callose. By removing the aphid without removing the stylet it acts as a phloem pump. Both of these methods are effective at extracting phloem from a plant without sliming occurring. 

The cell wall provides many functions to the well being of a plant. First, the cell wall is strong and durable. This helps to protect the plant from the environment as well as pathogens. This durability also helps to support the plant during growth. If it wasn’t for the strength of the cell wall, the plant would wilt and not be able to grow upright. The strength of the cell wall also helps to anchor the plant to its environment. This allows the plant to survive without being easily uprooted. Aside from mechanical function, the cell wall also aids in transporting water and nutrients throughout the plant, as well as signaling and transferring information throughout the plant.

There are three major components of the cell wall. These components are cellulose, hemicellulose, and pectin. Each of these components is made up of different monomers. Cellulose consists of β (1-4) linked D-glucose. Hemicellulose consists of glucose, xylose, mannose, galactose, rhamnose, and arabinose. Finally, pectin consists of α-(1-4)-linked D-galacturonic acid. Of all of these components in the cell wall, cellulose is the most abundant and strongest. 

Cells treated with different stimuli demonstrate different patterns of cytosolic calcium fluctuations due to the nature of the stimuli interaction with the cell. ATP stimulated cells demonstrate oscillatory calcium concentrations due to the negative feedback loops of the signaling pathway that allow calcium influx and export to be halted and resumed on a continuum. Bradykinin stimulated cells are stimulated by G-protein receptors, which when stimulated commence a signaling cascade which opens calcium ion channels on the plasma membrane of the cell. The nature of G-protein coupled receptors can be variable with respect to response, so this delayed and segmented response of fluo-4 is expected. Vasopressin stimulated cells exhibited no change in cytosolic concentration over time, despite common scientific literature stating an oscillatory response is expected. This lack of response in the cells to vasopressin could be due to insufficient time of cells observed, in which the cytosolic calcium did in fact fluctuate, but later in time and was not visualized in the time lapse. Another reason for this disappearance in fluo-4 intensity fluctuation could be due to inadequate addition of the vasopressin stimulus, so that cells were unable to react to the vasopressin.

Three additional samples of live LLC-Pk1 cells were treated with fluo-4. Control group cells were submerged in HBS buffer, while ATP and bradykinin experimental groups were submerged in calcium free HBS buffer, eliminating significant extracellular calcium. Cells were then treated with respective stimuli, and sub sequentially treated with ionomycin. Time lapse images were taken at 10X magnification for 270 s (1 frame every 2s) to capture the cellular response to the stimuli and ionomycin via fluorescent activity (Figure 4). Fluorescence intensity versus time data reveal patterns of fluorescence intensity (and therefore, cytosolic calcium level) fluctuation based on specific stimuli introduced to the extracellular space (Figure 2). Control sample (ATP/ionomycin treated cells in HBS) exhibited an oscillating fluctuation in fluorescence intensity over time (Figure 3A) and did not demonstrate a relative fluo-4 intensity increase after ionomycin treatment (Table 1). Bradykinin experimental cells   demonstrated no response over time after being treated with stimuli, whereas a majority of ATP experimental cells exhibited a response (0/101, 152/154, respectively, Table 2). However, after ionomycin introduction, both bradykinin and ATP experimental cells experienced an increase in fluorescence intensity (800, 500, respectively, Table 2) for the remainder of the time lapse imaging (32s, Table 2). Differences in control and ATP experimental groups are noted in the duration of initial calcium spike (12 s, 8 s, respectively, Table 2), avg. time to first peak of cells responding to initial stimulus (42 s, 32 s, respectively, Table 2) and increase in intensity at peak of response to the stimuli (240, 850, respectively, Table 2).

Four samples of live LLC-Pk1 cells were treated with fluo-4 (a calcium sensitive fluorophore). Each sample was then exposed to a different stimulus while submerged in HBS buffer, Time lapse images were taken at 10X magnification for 120 s (1 frame every 2s) to capture the cellular response to the stimuli via fluorescent activity (Figure 3). Fluorescence intensity versus time data reveal patterns of fluorescence intensity (and therefore, cytosolic calcium level) fluctuation based on specific stimuli introduced to the extracellular space (Figure 1). Cells treated with HBS (control group) expectedly demonstrate no significant fluctuation in fluorescence intensity overtime, and therefore were not able to quantitatively analyzed any further (Figure 1), Cells treated with bradykinin appear to have a delayed rise in fluorescence intensity over time (44s, Table 1) whereas ATP treated cells appear to have an immediate and oscillating fluctuation in fluorescence intensity over time (3s, 18s, respectively, Table 1). Only a small number of cells responded to bradykinin introduction with an increase in fluorescence intensity, while a majority of the cells treated with ATP demonstrated a response (11/154, 97/99, respectively, Table 1). Additionally, the relative increase of fluorescence intensity in cells treated with bradykinin appears to be much larger than that of cells treated with ATP (589, 331, respectively, Table 1). Vasopressin treated cells demonstrated no fluorescent fluctuation over time, and therefore were not able to be quantitatively analyzed any further (Figure 1). Reasons for this absence of an expected response are detailed in the discussion.

It is necessary for cells to be able to communicate at both intracellular and extracellular levels in order to grow and survive in a given environment. This laboratory exercise investigates the mechanisms of cellular response to internal and external stimuli, with respect to calcium ion transport, using a variety of agents (ATP, bradykinin, vasopressin). Exploration to determine whether a particular stimulus mediates calcium transport via plasma membrane channels or via release from intracellular stores was conducted using ionomycin (a calcium ionophore) to gain further insight on both levels of cellular communication.

“We are formed by our environment, and our environment is formed by us”. Mammals are influenced by climate, changing geography, and plants, and the influence their surroundings. One example of this is the Columbian mammoth, which adapted to its surroundings over time and lived 5 million—4,500 years ago. The primitive species of mammoths had fewer ridges on their teeth while newer species evolved with more ridges as more abrasive foods because available. Woolly mammoths, related to Columbian mammoths, had long fur to insulate it from the cold since it lived in an ice age. Another example of the environment affecting a species and the species affecting the environment are horses in North America. The Spanish re-established horses in North America in the 1500s after they had gone extinct 8,000 years before. The original North American horses underwent evolution over time as their environment changed, and they grew larger as forests turned into grassy plains. Today, there are concerns about mustangs damaging the biotic crusts of the grasslands, causing erosion. Sometimes, this two-way evolution is not a positive development.