THE CIRCADIAN RHYTHM OF THE CELLS IS DEPENDENT OF THE PH OF THE CELL

THE CIRCADIAN RHYTHM OF THE CELLS IS DEPENDENT OF THE PH OF THE CELL

Bachelor Thesis Project in Biomedicine 30 ECTS

Spring term 2020

Maria Ines Berrojo Romeyro Mascarenhas Supervisor: Afrouz Behboudi

Examiner: Ferenc Szekeres

Abstract

Circadian rhythms are endogenic autonomous oscillators of physiological activities resulting from 24- hour day/night cycles. Circadian rhythms regulate a wide variety of metabolic and physiological functions. This allows the organism to control its molecular, biochemical, physiological, and behavioural processes. Some studies have recently been performed on the relationship between cancer and circadian rhythm. In this paper we analyse the relationship between pH, the Pentose Phosphate Pathway, and the circadian through two databases of RNA sequence, GSE101988 and GSE74439. It was found that the Period and Cryptochrome family genes, which are linked to DNA damage response pathways, are more expressed than the control groups. At the same time, CLOCK and BMAL genes were inhibited. This, therefore, forms our supposition that the pH, through several mechanisms, does affect the circadian and thus the tumour progression. This is a very important study focus, because acidosis and alkalosis could be a biomarker for early tumour apparition in local tissues.

In this databased research, BMAL1 (BMAL2) was observed to be more active than the key circadian regulator at lower pH. Together, the CRY family of genes was downregulated in both datasets.

However, in the analysis involving Pentose Phosphate Pathway inhibition, p53 was up-regulated and G6PD was without any statistically significant improvement.

Abbreviations

• 6-Aminonicotinamid (6AN)

• Arginine vasopressin (AVP)

• Basic helix-loop-helix (bHLH)

• Bis(2-ethylhexyl) phthalate (DEHP)

• Cryptochrome (Cry)

• Dorso-medial part (DM)

• Gamma-aminobutyric acid (GABA)

• Glucose-6-phosphate dehydrogenase (G6PD)

• Nicotinamide-adenine dinucleotide phosphate (NADPH)

• Optical chiasm (OP)

• Period-Arnt-Single-minded (PAS)

• Pentose Phosphate Pathway (PPP)

• Period (PER)¹

• post-translational modifications (PTMs)

• Sleep and circadian rhythm disruption (SCRD)

• Suprachiasmatic nucleus (SCN)

• Transcription translation feedback loop (TTFL)

• Vasoactive intestinal polypeptide (VIP)

• Ventro-lateral part (VL)

1Period (PER): Abbreviation for the Period genes, not period as a noun

Popular Scientific Summary

Circadian rhythms are endogenic autonomous oscillators of physiological activities resulting from 24- hour day / night cycles. Circadian rhythms regulate a wide variety of metabolic and physiological functions. This allows the organism to control its molecular, biochemical, physiological, and behavioural processes.

The core circadian clock is controlled by the suprachiasmatic nucleus (SCN), which located the anterior hypothalamus. The SCN is anatomically located to obtain sensory stimuli from the light-dark cycle as well as non-photic information from other neuronal tracts, an example of this, is temperature.

Nevertheless, experiments have shown that peripheral clocks are also capable of oscillating independently and preserving their internal rhythms. The prevailing theory is that the SCN, as the master pacemaker, is capable of controlling peripheral clock rhythms. Examples of other endogenous oscillators that maintain their own rhythms are the heart, the liver, the kidney, and isolated cells, such as cancer cells. Within cells, circadian mechanisms regulate and/or influence fundamental processes.

Almost every aspect of physiology is regulated by circadian pacemakers, including neural function.

Circadian timekeeping is the key to the functioning of neural function, most notably in the sleep-wake cycle.

In this study, it was found that the paralog of BMAL1, (BMAL2) was more active than the main regulator of the circadian at lower pH. Together, the CRY family of genes were downregulated in both the datasets. However, in the study involved with the inhibition of the PPP, the p53 was upregulated whereas G6PD was without any statistical significant change.

The effect of pH in this mechanism is highly unexplored; the effects it may have could be interesting for future research to perceive the effects of environmental pollutants or drugs to treat several diseases. The pH in the cell could be key, in many other cellular processes that can be affected due to the dysregulation of the circadian rhythm.

Content

Introduction ... 1

Materials and methods ... 5

Results ... 6

Discussion ... 12

Acknowledgements ... 15

References ... 16

1

Introduction

Nearly all organisms coordinate their physiology, behaviour, and metabolism in accordance with the 24-hour solar cycle. Time-dependent changes in these variables have evolved to allow animals and plants alike to optimize their fitness according to environmental circumstances. Circadian rhythms are endogenic autonomous oscillators of physiological activities resulting from 24-hour day / night cycles that allow organisms to adapt to a changing environment. Circadian rhythms regulate a wide variety of metabolic and physiological functions (Sahar & Sassone-Corsi, 2009). This allows the organism to control its molecular, biochemical, physiological, and behavioural processes. (Canaple et al., 2003). The circadian rhythm has been researched over the years, and some studies have recently been performed on the relationship between cancer and circadian rhythm.

The mechanism usually consists of three parts in mammals, which include the input pathway, the core circadian clock, and the output pathway. The input path senses external timing signals, such as light / dark, and sends information to the core circadian clock. The core circadian clock forms an endogenic circadian rhythm with external stimuli to enable adaptation to the environment. In response to changes in the core circadian clock, the output pathway regulates physiological activity in various organs and tissues through neuro-humoral regulation. (Xie et al., 2019) The core circadian clock is controlled by the suprachiasmatic nucleus (SCN), which is in the anterior hypothalamus. The SCN is anatomically located to obtain sensory stimuli from the light-dark cycle as well as non-photic information, such as temperature, from other neuronal tracts. (Green, 2012) Connections to the periphery through parasympathetic and sympathetic nerves also serve its purpose of transmitting signals from the SCN. In addition to receiving photic stimuli, SCN must incorporate internal feeding signals, locomotive activity, and photobiotic hormones like melatonin. (Greene, 2012) The SCN is divided into two identical hemispheres (left and right), each composed of two groups of neurons (core and shell, shown on the right hemisphere), distinguished by the type of neurotransmitters released. In the ventro-lateral part (VL), the neurons mainly express vasoactive intestinal peptides (VIP) (displayed on the left hemisphere) while in the dorso-medial part (DM), arginine vasopressin (AVP) is expressed.

The two components also vary in their coupling properties. In addition, only a subset of VL neurons is light-sensitive and energized by light cues from the optical chiasm (OC). (Hafner, 2012) The VL-SCN, which controls essential physiological activities such as exercise, body temperature, heart rate, and hormone synthesis, serves to couple the circadian system. VIP is released periodically from the VL core region, binding to VPAC2 on the neuronal surface, resulting in cell depolarization and induction of PER1 and PER2 (Xie, et al., 2019) VIP deficiency negatively affects the synchronization of the cells, leading to the deterioration of the rhythms of the whole body. AVP-deficient rats show weakened rhythms, but do not experience a change in circadian pacemaking. (Xie, et al., 2019) SCN is known to be the product of VIP and AVP deficiency. Certain neurotransmitters such as glutamate and GABA in the SCN also help to regulate the circadian clock system. (Xie, et al., 2019) Nevertheless, experiments have shown that peripheral clocks are also capable of oscillating independently and preserving their internal rhythms. The prevailing theory is that SCN (Figure 1), as the master pacemaker, is capable of controlling peripheral clock rhythms, in order for all the peripherial clocks to function in harmony. Examples of other endogenous oscillators that maintain their own rhythms are the heart, the liver, the kidney, and isolated cells, such as cancer cells. (Canaple et al., 2003)

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Figure 1: A representation of how the suprachiasmatic nucleus controls the peripheral clocks as the master pacekeeper. The SCN sends signals to the Autonomic Nervous System (ANS) and Neuroendocrine system (NES). These send the signal that will release neurotransmitters, cytokines and different kind of hormones, such as glucocorticoids. These hormones, through different pathways, regulate the gene expression of circadian genes in the oeripheral clocks of the body.

Within cells, circadian mechanisms regulate and/or influence fundamental processes. Almost every aspect of physiology is regulated by circadian pacemakers, including neural function. Circadian timekeeping is the key to the functioning of neural function, most notably in the sleep-wake cycle.

(Hastings, Maywoood, & O'Neill, 2008) At the beginning of the 1990s, studies in Drosophila melanogaster and Neurospora crassa contributed to a coherent model of pacemaker as self-regulatory trancription translation feedback loop (TTFL). Following the cloning of different homologues of invertebrate clock genes and the discovery of the mouse clock gene, the TTFL model of circadian cellular pacemaking was extended to mammals. Clock proteins are capable of imposing metabolic rhythms on cells and organisms on a daily basis. (Hastings, Maywoood, & O'Neill, 2008)

The circadian rhythm is made from transcriptional and post-translational feedback loop. (Ko &

Takahashi, 2006) Most of the clock components identified function as transcriptional activators or repressors. Two transcriptional activators / repressors, CLOCK and BMAL1, are at the core of the oscillator. Multiple proteins control the stability and/or nuclear accumulation of clock components.

There are several clock gene paralogues, such as BMAL2 and NPAS2, which may regulate unique target genes due to differential spatial expression. (Baggs, 2009) The CLOCK gene has been shown to be a key component of circadian clock development. The circadian clock is thought to play a key role in regulating gene expression in the human brain and several other tissues. The transcription factors are CLOCK and BMAL1 which initiate the transcription of Cryptochrome genes (Cry) and Period genes (Per).

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These also act as negative regulators by directly interacting with CLOCK and BMAL1. (Greene, 2012) Transcription and translation of core clock components (CLOCK, NPAS2, BMAL1, BMAL2, PER1, PER2, PER3, CRY1 and CRY2) play a critical role in the generation of rhythms, while delays imposed by post- translational modifications (PTMs) are important for the determination of rhythm time (tau refers to rhythm time and is the length, in time, of one complete cycle). (Hastings, Maywoood, & O'Neill, 2008) In the principal feedback loop, the positive elements include genes of the basic helix-loop-helix (bHLH)- PAS (Period-Arnt-Single-minded) family transcription factor, CLOCK and BMAL1. CLOCK and BMAL1 heterodimerize and activate transcription of target genes containing E-box cis-regulatory enhancer sequences, including Period (Per1, Per2 and Per3 mammals) and Cryptochrome (Cry1 and Cry2 mammals) Negative feedback is achieved by PER-CRY heterodimers which convert back to the nucleus to repress their own transcription by acting on the CLOCK-BMAL1 complex. (Jagannath, 2017)

A common reason of human circadian disorder is misalignment between the environmental rhythm and the endogenous circadian oscillators. In animal studies, when the intrinsic clocks of animals are desynchronized from external timing cues, disorders in many organs or tissues can occur. Genetically, since the molecular clock is highly conserved, diseases caused by mutations of clock genes (which are often used in animal models to study clock gene-related dysfunction) are rare in humans. Circadian clock gene single nucleotide polymorphisms related to metabolic syndrome, hypertension, and diabetes mellitus have also been reported in genome-wide association studies. (Xie, et al., 2019) Circadian clock disorders also affect the development of organs, such as the brain and bone, leading to dysplasia. Circadian rhythm is essential for the development of the brain. (Jagannath, 2017) Circadian clocks are involved in the regulation of neurogenesis. Sleep and circadian disturbances during pubescence can have an impact on brain development and can lead to mood susceptibility and substance use disorders. (Jagannath, 2017)

There is considerable evidence that patients with neuropsychiatric diseases, such as bipolar disorder, schizophrenia and depression, have sleep and circadian rhythm disruption (SCRD). This disruption involves a wide variety of sleep disturbances, including disrupted sleep, decreased total sleep time, and changes in standard sleep architecture. (Wulff, Gatti, Wettstein, & Foster, 2010) Remarkably, fibroblasts isolated from schizophrenic patients display a lack of rhythm in CRY1 and PER1 expression, and their peripheral blood leukocytes have decreased and/or reduced diurnal expression of CLOCK, PER1/2/3, CRY1 and functional CLOCK homologue NPAS2 relative to healthy controls. (Jagannath, 2017)

Evidence that individual genes of the circadian clock play a role in the control of tumorigenesis has been provided as well. (Kiessling, 2017) Circadian gene abnormalities such as mutations, deregulated expression and even translocation of PER genes have been reported in various cancers including breast, colorectal, endometrial, lung and different types of lymphoma and leukemia. (Cadenas, et al., 2014) Two of these circadian rhythm regulators, PER1 and PER2, have now been linked to DNA damage response pathways. Overexpression of either PER1 or PER2 in cancer cells inhibits neoplastic growth and increases their apoptotic rate. (Chen-Goodspeed & Chi Lee, 2007) (Gery & Koeffler, 2007) PER2 mutant mice have increased susceptibility to spontaneous and radiation-induced tumor growth. These mutant mice show aberrant temporal expression of cell cycle genes such as p53, c-myc, cyclin A, and MDM2, leading to deregulated cell division and reduced apoptosis levels. (Gery & Koeffler, 2007) In

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fact in the study of (Miki, Matsumoto, Zhao, & Lee, 2013) it was found that PER2 is directly regulated by p53, p53 binds to the PER2 promoter response element. Furthermore, the p53 response element is evolutionarily conserved and overlaps with the E-Box element critical to the BMAL1 / CLOCK binding and its transcriptional activation of the PER2 expression.

The major glucose catabolism pathway in the organism, is the Pentose Phosphate Pathway (PPP). The PPP redirects glucose flow to its oxidative area and contributes to a reduced form of NADPH and nucleotides. (Jin & Zhou, 2019) PPP plays a crucial role in the regulation of cancer progression and involves numerous enzymes. The oxidative branch transforms glucose 6-phosphate (G6P) to ribulose 5-phosphate (Ru5P), CO2 and NADPH. NADPH is essential to maintain a reduction-oxidation (redox) equilibrium under stress conditions and to allow cells to proliferate rapidly. (Jin & Zhou, 2019) G6PD catalyzes the irreversible oxidation of G6P to 6-phosphogluconolactone at a rate-limiting point. The p53 tumor suppressor binds to G6PD and prevents the development of active dimer and suppresses the growth, absorption of glucose and biosynthesis of NADPH resulting in PPP inhibition (Jin & Zhou, 2019). Elevated expression of G6PD is indicative of low survival in cancer patients. G6PD activity is increased in several types of cancers, including bladder, breast, cervical, colorectal, endometrial, esophageal gastric, hepatic, lung, ovarian, prostate, renal cancers, as well as glioblastomas, glioma and leukemia. (Yang, et al., 2019) These findings suggest that G6PD could be a possible prognostic biomarker and a promising target for cancer therapy. In a recenty study, it was found that the PPP regulated the expression of the circadian rhythm through NADPH. In the study they found that the redox disturbances increased the DNA-binding activity of BMAL1 / CLOCK, which in turn led to profound quantitative changes in circadian gene expression. (Rey, et al., 2016)

The aim of this study is to study the gene expression of the circadian rhythms in redox disturbances, either by extracellular culture manipulation of the pH or the inhibition of the NADPH to manipulate the intracellular pH. Since the studies are done in vitro, the circadian rhythm studied will be in the periferal clocks of osteosarcoma cells. This is a revelant topic in the tumor progression because the control of the PPP can be pharmaceutical and could be another treatment for cancer progression.

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Materials and methods

Two databases were used for this study, GSE101988 and GSE74439, these are the studies of (Walton, et al., 2018) and (Rey, et al., 2016). The sequencing data was submitted to the Galaxy cloud site, and I used the public repository at usegalaxy.org to analyze the data.

Both databases used the cell line U2OS, an osteosarcoma cell line that in previous studies, (Zhang, et al., 2016), it was shown that has its own circadian rhythm.

The cells from the, GSE101988 were processed with Illumina HiSeq200, the source was transcriptomic, the samples were processed with cDNA and had a single layout. The protocol they followed was the following: TRIzol extraction (Invitrogen) according to the manufacturer's instructions. Media was aspirated from cells growing in 35 mm dishes. 1 mL of TRIzol Reagent was added to plates. A cell scraper was then used to collect cells and lysate which was frozen at -80 until RNA isolation following the manufacturer’s instructions with substitution of 1-Bromo-3-chloropropane for chloroform. RNA integrity was verified by bioanalyzer (Agilent Technologies) (RNA integrity number (RIN) 8.7-10.0, median = 9.7). Illumina TruSeq Stranded mRNA Library Preparation kit (Walton, et al., 2018).

The cells from the GSE74439 were analyzed with Illumina HiSeq2500. The source was transcriptomic, the samples were done with cDNA and the layout was single. The protocol they used was the following RNA-Seq: Standard RNA extraction with Zymo Direct-zol™ RNA kits Illumina TrueSeq platform compatible dual-indexed multiplex library protocol was followed. (Rey, et al., 2016)

First the data was transformed from the SRA database to FASTQ, BAM and FASTA. It was then aligned using the HISAST2 option under RNA seq. The databased that was used was Human Homo sapiens (b38) hg38. Single paired and stranded forward, the rest of the parameters were the default. HISAT uses the Bowtie2 interface to perform most of the FM index operations.

Afterwards, the data was processed in String Tie, with the following parameters, stranded forward, without using the GTF/GFF3 and default job parameters. String Tie is a fast and highly efficient RNA- Seq alignment assembler for potential transcripts. It uses a new network flow algorithm as well as an optional de novo assembly step. It assembles and quantifies full-length transcripts representing multiple splice variants for each gene locus.

With the FPKM (fragments per kilobase per million mapped reads) data it was performed a t-test comparing the two groups in the two different databases. FPKM data is directly proportional to the gene expression. The FPKM was processed either by t-test or Mann Whitney U test as statistical analysis depending if the data was normalized or not. The data was normalized by a Shapiro Wilk test.

This statistical analysis and graphical representation in boxplots were done with SPSS platform.

The fold change and log2FC were done by calculating the average of the treatment divided by the average of the control. Afterwards, the log2FC was calculated with the value of the division and with a logarithm with base 2.

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Results

In this section, the image on the left, illustrates the study of (Walton, et al., 2018). The graph represents the comparison of the fold change, between the control group which was cultured in DMSO and the ones cultured in 6AN, which inhibits the Pentose Phosphate Pathway.

The image on the right portrays the study of (Rey, et al., 2016). The graph displays the comparison of the two different pH levels that the cells were cultured in, the neutral pH (7.4) which is the control group, and the acidic culture media of pH (6.3).

All of the graphs represent the distribution of FPKM (fragments per kilobase of transcript per million reads). Therefore, it represents the amount of activity changes the gene had over time depending on how narrow or broad the box is.

Figure 1: Distribution of the CLOCK gene expression. Fist image: FC: 1.33 log2FC: 2.4. Second image:

FC: 1.43 log2FC: 1.94. The study of the inhibition of the PPP in the CLOCK gene had the following statistics. Shapiro-Wilk test p=0.338, null hypothesis retained, data normally distributed. Levene’s test p=0.649, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=1.04e^-7 95%CI (1.46-2.52). Null hypothesis rejected, alternative hypothesis accepted. The second study about the effect of different pH media culture in the CLOCK gene had the following results. Shapiro-Wilk test p=0.0679, null hypothesis retained, data normally distributed. Levene’s test p=0.865, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=1.3e^-4 95%CI (6.16-16.40). Null hypothesis rejected, alternative hypothesis accepted. There is a statistical difference between groups.

The CLOCK gene is found to be upregulated in both studies since there is a statistical difference between them. Overtime since the box plot is narrow, shows that not many changes occurs , except on the graph of the right in figure 1, there we can see that the box of the 7.4 is broader, this would correspond to undisturbed circadian activity. However, in the pH 6.3, the box is narrower and has two outliers corresponding to the time points, 4 hours and 16 hours after the beginning of the study.

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Figure 2: Distribution of the BMAL1 gene expression. First image: FC: 1.02 log2FC: 3.86. Second image:

FC: 1.62 log2FC: 1.43. The study of the inhibition of the PPP in the BMAL1 gene had the following statistics. Shapiro-Wilk test p=0.487, null hypothesis rejected, data normally distributed. Levene’s test p=0.076, null hypothesis rejected, alternative hypothesis accepted, no equal variances between groups. T-test, 2 tail, p=0.879. Null hypothesis accepted, there is no statistical differences between groups. The second study about the effect of different pH media culture in the BMAL1 gene had the following results. Shapiro-Wilk test p=0.054, null hypothesis rejected, data normally distributed.

Levene’s test p=0.003, null hypothesis rejected, alternative hypothesis accepted, no equal variances between groups. T-test, 2 tail, p=0.008, 95%CI (2.875-16.74). Null hypothesis rejected, alternative hypothesis accepted. There is a statistical difference between groups.

BMAL1 gene is found to be slightly downregulated in the second study, however, there is no statistical difference in the study of the PPP. We can observe that there is no statistical difference since the error bar of DMSO are overlapping with the error bars of 6AN. Since the box of BMAL1 are broad, this means that the gene had significant changes depending on the hour, we can specially notice I the graph of the right where the pH of 7.4 is clearly bigger than the pH of 6.3.

Figure 3: Distribution of the BMAL2 gene expression. First image: FC: 1.31 log2FC: 2.57. Second image:

FC: 1.51 log2FC: 1.68. The study of the inhibition of the PPP in the BMAL2 gene had the following statistics. Shapiro-Wilk test p=0.777, null hypothesis retained, data normally distributed. Levene’s test p=0.137, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=0.017 95%CI (0.40- 3.69). Null hypothesis rejected, alternative hypothesis accepted. The second study about the effect of different pH media culture in the BMAL2 gene had the following results. Shapiro-Wilk test p=0.108, null hypothesis retained, data normally distributed. Levene’s test p=0.231, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=0.001 95%CI (6.99-16.75). Null hypothesis rejected, alternative hypothesis accepted. There is a statistical difference between groups.

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BMAL2 gene is found upregulated in both studies. In BMAL2 all the boxes in the graph are very narrow, since BMAL2 is a paralog of BMAL1, it is normal to have lesser activity than BMAL1, and therefore have less changes over time.

Figure 4: Distribution of the PER1 gene expression. First image: FC: 0.66 log2FC: -0.97. Second image:

FC:1.34 log2FC:-0.44. The study of the inhibition of the PPP in the PER1 gene had the following statistics. Shapiro-Wilk test p=0.021, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=2.00, z=-4.04, p=5.3e^-5, q=3.0e^-6. Null hypothesis rejected. Alternative hypothesis accepted, there is a statistical difference between groups. The second study about the effect of different pH media culture in the PER1 gene had the following results.

Shapiro-Wilk test p=5.0e^-6, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=41.00, z=-2.23, p=0.026, q=0.026. Null hypothesis rejected. Alternative hypothesis accepted, there is a statistical difference between groups.

PER1 gene is found downregulated in both studies. The changes overtime increased in the 6AN cultures, and in the DMSO cultures maintain a narrower box. Therefore, the inhibition of PPP disrupts the gene expression of PER1.

Figure 5: Distribution of the PER2 gene expression. First image: FC: 0.66 log2FC:-1.68 Second image:

FC: 0.37 log2FC:-0.69. The study of the inhibition of the PPP in the PER2 gene had the following statistics. Shapiro-Wilk test p=0.007, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=23.00, z=-2.83, p=0.005, q=0.004. Null hypothesis rejected. Alternative hypothesis accepted, there is statistical difference between groups. The second study about the effect of different pH media culture in the PER2 gene had the following results.

Shapiro-Wilk test p=0.004, null hypothesis rejected, alternative hypothesis accepted.

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Data not normally distributed. Mann Whitney U test, U=84.00, z=0.00, p=1.00, q=1.00. Failed to reject Null hypothesis. There is no statistical difference between groups.

PER2 gene is found downregulated in the first study. The box of the 6AN is broader than the DMSO, which means that there were more changes on the gene expression overtime in this culture. DMSO has a low outlier at the time point 24H. Nevertheless, in the study of the extracellular pH effects there were no statistical differences. This is represented in the graph as the error bars and boxes are similar to each other.

Figure 6: Distribution of the PER3 gene expression. First image: FC: 0.90 log2FC:-6.36. Second image:

FC: 0.85 log2FC:-4.22. The study of the inhibition of the PPP in the PER3 gene had the following statistics. Shapiro-Wilk test p=0.666, null hypothesis retained, data normally distributed. Levene’s test p=0.061, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=0.359. Null hypothesis accepted, there is no statistical differences between groups. The second study about the effect of different pH media culture in the PER3 gene had the following results. Shapiro-Wilk test p=0.005, null hypothesis retained, data normally distributed. Levene’s test p=0.535, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=0.428. Failed to reject null hypothesis, there is no statistical differences between groups.

PER3 gene was not found to have any statistical difference in any of the studies, this is represented with the overlapping error bars and box plots. The DMSO bars are enclosed by the 6AN error bars (left) and the box of the 6.3 pH is enclosed by the box of 7.4pH culture (right).

Figure 7: Distribution of the CRY1 gene expression. First image: FC: 0.71 log2FC: -1.98. Second image:

FC: 0.44 log2FC:-0.84. The study of the inhibition of the PPP in the CRY1 gene had the following statistics. Shapiro-Wilk test p=0.005, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=25.00, z=-2.71, p=0.007, q=0.006. Null hypothesis rejected. Alternative hypothesis accepted, there is statistical difference between groups.

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The second study about the effect of different pH media culture in the CRY1 gene had the following results. Shapiro-Wilk test p=0.002, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=20.00, z=-3.308, p=0.001, q=4.9e^-4. Null hypothesis rejected. Alternative hypothesis accepted, there is statistical difference between groups.

CRY1 gene is found to be downregulated in both studies. In both treated groups (6AN [left graph] and 6.3pH [right]) the gene had more oscillations overtime than the control groups. This is translated as the changes of pH have disrupted the circadian regulation of the CRY1 gene.

Figure 8: Distribution of the CRY2 gene expression. First image: FC: 0.61 log2FC:-1.43. Second image:

FC: 0.38 log2FC:-0.71. The study of the inhibition of the PPP in the CRY2 gene had the following statistics. Shapiro-Wilk test p=0.044, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=6.00, z=2.2e^-4, p=1.4e^-4, q=-3.811. Null hypothesis rejected. Alternative hypothesis accepted, there is statistical difference between groups. The second study about the effect of different pH media culture in the CRY2 gene had the following results.

Shapiro-Wilk test p=3.0e^-4, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=23.00, z=-3.154, p=0.002, q=0.001. Null hypothesis rejected. Alternative hypothesis accepted, there is statistical difference between groups.

The CRY2 gene is found to be downregulated in both studies. In both treated groups (6AN [left graph]

and 6.3pH [right]) the gene had more oscillations overtime than the control groups. This is translated as the changes of pH have disrupted the circadian regulation of the CRY2 gene. This is the same results as in the family gene CRY1.

Figure 9: Distribution of the p53 gene expression. First image: FC: 1.06 log2FC: 10.68 Second image:

FC: 1.34 log2FC: 3.35. The study of the inhibition of the PPP in the p53 gene had the following statistics.

Shapiro-Wilk test p=0.777, null hypothesis retained, data normally distributed. Levene’s test p=0.137, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=0.0.018 95%CI (0.385-

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3.702). Null hypothesis rejected, alternative hypothesis accepted, there is statistical differences between groups. The second study about the effect of different pH media culture in the p53 gene had the following results. Shapiro-Wilk test p=0.009, null hypothesis retained, data normally distributed.

Levene’s test p=1, null hypothesis retained, equal variances between groups. T-test, 2 tail, p=1. Failed to reject null hypothesis, there is no statistical differences between groups.

P53 gene is found to be upregulated in the PPP inhibition study. In this study the boxes are very narrow compared to the second study. Nonetheless the box from the 6AN is bigger than DMSO, which means there were more changes compared to the control group. In the second study, we can see there were many changes overtime but also that the error bars are overlapping each other, therefore p53 had no statistical differences.

Figure 10: Distribution of the G6PD gene expression. First image: FC:1.02 log2FC:32.95. Second image:

FC:1.23 log2FC:3.29. The study of the inhibition of the PPP in the G6PD gene had the following statistics. Shapiro-Wilk test p=0.002, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=72.00, z=0.00, p=1.00, q=1.00. Null hypothesis accepted, there is no statistical difference between groups. The second study about the effect of different pH media culture in the G6PD gene had the following results. Shapiro-Wilk test p=0.009, null hypothesis rejected, alternative hypothesis accepted. Data not normally distributed. Mann Whitney U test, U=64.00, z=-1.051, p=0.293, q=0.311. Failed to reject null hypothesis, there is no statistical difference between groups.

G6PD gene was not found to have any statistical difference between the groups, which can be see with overlapping error bars and box plots in the graph, therefore the findings of G6PD were not significant.

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Discussion

In the Pentose Phosphate Pathway study, CLOCK and BMAL2 had a significant difference between the PPP inhibited group and the control group. Both of them were upregulated Figure 1 and 3, (p=1.04e^-7 and p=0.017 respectively) However, in BMAL1 it was not found any statistical difference in the groups (p=0.88). In PER1 and PER2 (p=5.30e^-4 and p=0.005) together with CRY 1 and 2 it was also found a statistical difference, Figure 4, 5, 7 and 8. But on the contrary to the CLOCK and BMAL2 genes they were downregulated. Since PER and CRY are in charge of inhibiting CLOCK and BMAL (Lagmesser, et al., 2008), it could be a reason why these genes are upregulated and PER and CRY were downregulated.

PER3 was not found with any statistical difference (p=0.36) PER3 is it is more linked to sleep disorders (Landot, 2011), than onto cancer cells such as the cells osteosarcoma cells of the study. Thus, it’s understandable why its gene expression would not change, given its not related to the PPP.

An interesting observation, however, is that the gene p53 is upregulated (p= 0.018). This gene is a tumor supressor, as a matter of fact it when it binds to G6PD it acts as an inhibitor of the PPP, preventing the tumors from growing. (Gottlieb , 2011) In fact, 6AN has also been linked as a potent inhibitor of G6PD. (Ntsala & Lemmerer, 2012) These two phenomenon would explain how sucessfuly was G6PD inhibited, however, the gene was not found with no statistical difference to the control group. Which could mean that p53 and 6AN where competitors to bind to G6PD. In the original study (Rey, et al., 2016), it was found that the lower the quantity of NADPH, implied that the activity of CLOCK and BMAL increased.

In this acidosis study analysis it was found that CLOCK and BMAL2 were upregulated, in contrast to BMAL1 that was downregulated. All of the three genes were statistically different from their control group (p=1.3e^-4 , p=0.001, p=0.008 respectively). The reason why BMAL1 might have been downregulated, is due to the fact that BMAL2 was upregulated. BMAL2 is a paralog of BMAL1 and it can replace it. (Shi, et al., 2010)

PER1 was the only in the PER family that was statistically significant (p=0.026). This gene was also upregulate. (PER2 p=1.0, PER3 p=0.43) Given PER1 has the ability to regulate the cell cycle, if it’s upregulated it would imply that it’s promoting cell division.(Zhao, et al., 2016) This is not surprising as the U2OS cell line are tumour cells. Furthermore, there is a connection that mTOR and PER1 work together in the circadian regulation in mice. (Cao, et al., 2011) In the original study, they in fact concentrate in the expression of mTOR when is inactivated due to the low pH and it’s effect on the circadian. (Walton, et al., 2018) This could be the reason why PER1 is upregulated.

CRY 1 and 2 were downregulated (p=0.001, p=1.4e^-4). Since the CLOCK-BMAL complex is upregulated, these genes are not inhibiting the complex of CLOCK and BMAL, which would explain why they are downregulated.

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Figure 11: Figure that illustrates how the studies modified the pH and how did it affect the circadian rhythm. The spiked line represents interaction between the genes. PER and CRY interact as a complex as it occurs with CLOCK-BMAL. The increment of the positive feedback (CLOCK-BMAL complex), increases the activity of the circadian rhythm, furthering the tumour progression.

In Figure 11, we can also see the illustrations of the intracellular pH changing as a consequence of lower amount of NADPH and the effect of the lower extracellular pH. In both studies, as consequence of the intracellular pH changes, the CLOCK and BMAL genes are upregulated. This translates as an increase of the circadian activity. The increase of the circadian rhythm activity has been linked to tumour progression. (Zhou, Yu, Sun, Zhang, & Wang, 2018) (Silke Kiessling, 2017) (Lee, et al., 2019) PER2 downregulation in previous studies has been linked in non-small cell lung cancer to tumor progression and metastasis. (Xiang, et al., 2018). Moreover, the CRY family of proteins were negatively impacted, since the low pH seems to downregulate these genes. In the same cell line (U2OS) in (Zhou, Yu, Sun, Zhang, & Wang, 2018) study, it was found that the inhibition of CRY1, had as a result the upregulation of CLOCK and BMAL, but also enhanced the proliferation and migration of the osteosarcoma cells.

It is also of importance the fact that like in other studies, BMAL and p53 were both upregulated, which consequently downregulates PER2. (Kawamura, et al., 2018) BMAL and p53 will form a complex to help the cell cope with the oxidative stress, these results are consistent with the same stress that is produced with UV radiation.

The clock-driven circadian oscillations at pH in mammalian tissues require the interesting possibility of a conserved, bi-directional acid-clock crosstalk reinforcement. (Walton, et al., 2018) Since this has not been suffienctly explored (Walton, et al., 2018). The vast effects this could have on the peripheral clock in cancer cells, might be crucial to the development of new therapies to treat cancer.

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There were no ethical aspects to be considered in this study. This is to due that not only the study was done through public databases but also because these publications were done with an osteosarcoma comercial cell in vitro. There was no involvement with neither animals or humans. However, the authors of the studies, must have followed the laws that are implemented in relation to the purchase and selling of human tissue cells. Commercial cell lines, have passed rigurous burocratic and quality standards, in order to be able to reproduce, storage, and distribute this cell lines. In this studies, it was used the U2OS cell line. As it was osteosarcoma, the cells belong to cancerous bone tissue. For this particular cells, the primary cells belonged to a caucasian girl of 15 years old. The cells were donated or extracted in 1964. One of the ethical questions that can arrise due to this topic, since it was so long ago, is until what point can we consider this cell donation to be consented, but this question should have been directed to the researchers that collected the samples of this girl.

As mentioned earlier in the paper, the circadian rhythm is linked to many conditions apart from cancer, such as diabetes, sleep disturbances, malformation in some organs, hypertension or schizofrenia.

(Jagannath, 2017) (Canaple, Kakizawa, & Laudet, 2003) (Wulff, Gatti, Wettstein, & Foster, 2010). The significance of this study lies in raising awareness of the graveness of the effects that circadian disruptions can cause. Also, this could be an aproach to individualize treatment. For example in cancer patients depending on why the tumor appeared (due to the different point mutations ) it would be easier to tackle the tumor by inhibiting or stimulating the genes and have a more effective response to the treatment. This would raise the chances to have a more successful recovery after developing this disease. Furthermore, another effect that can be studied with the circadian rhythm, is the effect that a medication can have on different hours of the day and when is the most appropiate time to take the relevant medication. One example of this could be homone therapy in change sex treatments, treatments in order to conceive a child or prevent it, or the insulin intake. This effects can be studied even further, since the considerations usually taken are due to their method of absorption and possible medication interaction. If a step further would be taken, it would be benefitial for those extensive treatments that are much more sensitive to this changes.

It would be also interesting to look at how environmental pollutants may affect this mechanism, and if it’s through a chain of consequences or if it directly affects the expression of the circadian genes. As an example there has been linked that Bis(2-ethylhexyl) phthalate (DEHP) and the PPP. This pollutant has been classified as an endocrine disruptor and toxic for the reproductive ability according to the European Agency of Chemicals. In recent studies it was found that it affected the PPP in mice (Amara, et al., 2019), but also the circadian rhythm in drosophila. (Cao, 2018) Hormones are highly dependent of the circadian rhythm as they follow a cycle, this could be linked to the series of changes due to the effect of the PPP disruption together with the circadian, causing the disruption in the endocrine system. (Hastings, Maywoood, & O'Neill, 2007)

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Acknowledgements

I want to thank my parents, to pushing me towards my dreams and give me the opportunity to study with the best conditions I could ever ask for. I also want to thank my brother for proofreading all my work and always give motivation when necessary. In addition, I am also thankful to Helgi Schiöth for welcoming me in his research group and Wen Liu, for teaching me many things while staying in the laboratory in Uppsala even though the report won’t be able to include it. As well, I am thankful to all my professors during my bachelor’s for instructing me along the period of my studies. Finally, a big thank you to my classmates, friends, and the rest of my family that have supported me through this journey. To all of you thank you.

This work is dedicated to my grandparents, for being one of the strongest people I know and an example that age does not limit us in the pursuit of our passions and dreams.

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