過氧化氫體(Peroxisome)為細胞代謝之重要胞器，其功能包括協助細胞進行呼吸作用、長碳鏈脂肪酸代謝、活性氧類(ROS)代謝等。細胞中過氧化氫體生合成蛋白(Peroxisomal biogenesis factor, Peroxins)負責過氧化氫體的增生及發育，主要的增生方式分成由 Pex3, Pex16, Pex19 為主的過氧化氫體新生合成(de novo synthesis)以及由 Pex11ß 為主的過氧化氫體分裂增生(Peroxisome fission) 這兩種方法。在實驗室先前的研究中發現，隨著肌肉生成過程，過氧化氫體的數量也會逐漸增加，暗示了肌肉生成過程與過氧化氫體之間的相互關係；而在癌症惡質症環境中，過氧化氫體相關基因也呈現下降的趨勢。因此，本篇便著重探討過氧化氫體於肌肉生成及癌症惡質症之間的關聯性。
實驗中我們發現，在肌肉生成過程中，不論是 in vivo 或 in vitro，過氧化氫體相關基因的表現皆呈現逐漸上升的趨勢，且過氧化氫體的新生數量也逐漸增加。同時觀察到，MRFs(Myogenic regulatory factor)會直接對 Pex3, Pex16, Pex19 啟動子進行調控。而上述實驗結果指出，Pex3 在肌肉生成過程中似乎扮演著很重要的角色。在藉由 knockdown Pex3 的細胞株中觀察到，大部分大量表現 Pex3 細胞株中觀察到除了過氧化氫體數量及形態上的改變，過氧化氫體及粒線體的功能並沒有太大的改變，然而在大量表現 Pex3 後細胞的分化效率卻有顯著地下降，指出在肌肉中，Pex3 對於過氧化氫體及粒線體扮演著舉足輕重的角色，並且在肌肉分化過程需要被精準地調控。此外，在經過有氧呼吸運動訓練後發現，快縮肌與慢縮肌之間過氧化氫體新生合成因子表現方式不同，並且觀察到在肌肉中過氧
化氫酶並不位於過氧化氫體內。
在早發性癌症惡病質小鼠內，觀察到在快縮肌內 Pex16 及 Acox1 異常上升，同時藉由 RNAseq 分析發現在晚發性小鼠快縮肌可以觀察到過氧化氫體相關基因大量被影響。此外，早發性與晚發性小鼠之間，大部分的 MRFs 表現趨勢類似，唯獨 MyoG 在早發性小鼠中出現異常上升，並且在快縮肌組別中出現較嚴重的症狀。將大量表現 MyoG 細胞株培養於癌症惡質症的環境中觀察到，兩者的加成性會影響肌肉分化並導致肌肉萎縮，造成癌症惡質症的惡化。

Peroxisome is an important organelle for cell metabolism, including reducing ROS(Reactive oxygen species), very long-chain fatty acid, lipid metabolism, and so on.Peroxisomal biogenesis factors (Peroxin) are involved in several procedures of peroxisome biogenesis, and in the two major ways regulating the number of peroxisomes, de novo synthesis is regulated by Pex3, Pex16, and Pex19 while growth and division is mainly regulated by Pex11β. As the number of peroxisomes has been found to be increased during myogenesis in our previous study, therefore, in this thesis, I want to clarify how the peroxisome is regulated in myogenesis and cancer cachexia.
Here I found the expression of many peroxisome related genes is increased during myogenesis both in vitro and in vivo. Meanwhile, MRFs (Myogenic regulatary factors) show their ability to regulate Pex3, Pex16, Pex19 promoters derectly. These data all points out that Pex3 plays an important role in myogenesis. In Pex3 knockdowned cells, neither peroxisome nor mitochondria was functioning properly, and the number and morphology of both organelles were changed significantly. Furthermore, less total and tubular shape peroxisomes were observed in these cells. On the other hand, in Pex3 over-expressed cells, the number of peroxisome was increased, and more tubular shape peroxisomes were found. However, the function of peroxisome and mitochondria were
both the same as that in controll cells. However, over-expression of Pex3 lead to poorer myogenesis, indicating that Pex3 level is critically regulated during myogenesis.
Interestingly, after aerobic exercise training, peroxisome related genes were differentially regulated in different muscle types, the expression level of Pgc1α is similar to Pex19 level but not Pex3. We were also surprised to find the location of Catalase out of peroxisome in myofibers in vivo.
In Cancer cachexia, the expression of Pex16 and Acox1 was abnormally increased in fast-twitch SKM. With RNAseq analysis, the peroxisome related genes were found
significantly regulated in fast-twitch SKM of late onset mice. Meanwhile, MRFs were mostly the same in both early onset and late onset groups, but only MyoG was abnormally absent in fast-twitch SKM of early onset group. In MyoG over-expressed cells, myogenesis was functionally normal, but serious muscle atrophy and myogenesis dysfunction were observed when MyoG and cachexia signals were combined.

1 Fujiki, Y., Okumoto, K., Mukai, S., Honsho, M. & Tamura, S. Peroxisome
biogenesis in mammalian cells. Front Physiol 5, 307, doi:10.3389/fphys.2014.00307
(2014).
2 Romagnoli, C. & Brandi, M. L. Muscle Physiopathology in Parathyroid Hormone
Disorders. Front Med (Lausanne) 8, 764346, doi:10.3389/fmed.2021.764346 (2021).
3 Antonenkov, V. D., Grunau, S., Ohlmeier, S. & Hiltunen, J. K. Peroxisomes are
oxidative organelles. Antioxid Redox Signal 13, 525-537,
doi:10.1089/ars.2009.2996 (2010).
4 Lodhi, I. J. & Semenkovich, C. F. Peroxisomes: a nexus for lipid metabolism and
cellular signaling. Cell Metab 19, 380-392, doi:10.1016/j.cmet.2014.01.002 (2014).
5 Van Veldhoven, P. P. Biochemistry and genetics of inherited disorders of
peroxisomal fatty acid metabolism. J Lipid Res 51, 2863-2895,
doi:10.1194/jlr.R005959 (2010).
6 de Duve, C. Peroxisomes and related particles in historical perspective. Ann N Y
Acad Sci 386, 1-4, doi:10.1111/j.1749-6632.1982.tb21402.x (1982).
7 Lazarow, P. B. & Fujiki, Y. Biogenesis of peroxisomes. Annu Rev Cell Biol 1, 489-
530, doi:10.1146/annurev.cb.01.110185.002421 (1985).
8 van der Zand, A., Gent, J., Braakman, I. & Tabak, H. F. Biochemically distinct
vesicles from the endoplasmic reticulum fuse to form peroxisomes. Cell 149, 397-
409, doi:10.1016/j.cell.2012.01.054 (2012).
9 Motley, A. M. & Hettema, E. H. Yeast peroxisomes multiply by growth and division.
J Cell Biol 178, 399-410, doi:10.1083/jcb.200702167 (2007).
10 Schrader, M., Bonekamp, N. A. & Islinger, M. Fission and proliferation of
peroxisomes. Biochim Biophys Acta 1822, 1343-1357,
doi:10.1016/j.bbadis.2011.12.014 (2012).
11 Agrawal, G. & Subramani, S. De novo peroxisome biogenesis: Evolving concepts
and conundrums. Biochim Biophys Acta 1863, 892-901,
doi:10.1016/j.bbamcr.2015.09.014 (2016).
12 Susin, S. A. et al. Molecular characterization of mitochondrial apoptosis-inducing
factor. Nature 397, 441-446, doi:10.1038/17135 (1999).
13 Duchen, M. R. Mitochondria and calcium: from cell signalling to cell death. J
Physiol 529 Pt 1, 57-68, doi:10.1111/j.1469-7793.2000.00057.x (2000).
14 Andersson, S. G. et al. The genome sequence of Rickettsia prowazekii and the origin
of mitochondria. Nature 396, 133-140, doi:10.1038/24094 (1998).
15 Anderson, S. et al. Sequence and organization of the human mitochondrial genome.
Nature 290, 457-465, doi:10.1038/290457a0 (1981).
66
16 Appling, D. R., Anthony-Cahill, S. J. & Mathews, C. K. Biochemistry : concepts
and connections. (Pearson, 2016).
17 Osellame, L. D., Blacker, T. S. & Duchen, M. R. Cellular and molecular mechanisms
of mitochondrial function. Best Pract Res Clin Endocrinol Metab 26, 711-723,
doi:10.1016/j.beem.2012.05.003 (2012).
18 Birch-Machin, M. A. The role of mitochondria in ageing and carcinogenesis. Clin
Exp Dermatol 31, 548-552, doi:10.1111/j.1365-2230.2006.02161.x (2006).
19 Tulah, A. S. & Birch-Machin, M. A. Stressed out mitochondria: the role of
mitochondria in ageing and cancer focussing on strategies and opportunities in
human skin. Mitochondrion 13, 444-453, doi:10.1016/j.mito.2012.11.007 (2013).
20 Oyewole, A. O. & Birch-Machin, M. A. Mitochondria-targeted antioxidants.
FASEB J 29, 4766-4771, doi:10.1096/fj.15-275404 (2015).
21 Schrader, M., Costello, J., Godinho, L. F. & Islinger, M. Peroxisome-mitochondria
interplay and disease. J Inherit Metab Dis 38, 681-702, doi:10.1007/s10545-015-
9819-7 (2015).
22 Costello, J. L., Passmore, J. B., Islinger, M. & Schrader, M. Multi-localized Proteins:
The Peroxisome-Mitochondria Connection. Subcell Biochem 89, 383-415,
doi:10.1007/978-981-13-2233-4_17 (2018).
23 Fransen, M., Lismont, C. & Walton, P. The Peroxisome-Mitochondria Connection:
How and Why? Int J Mol Sci 18, doi:10.3390/ijms18061126 (2017).
24 Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J. & Subramani, S. A conserved
tripeptide sorts proteins to peroxisomes. J Cell Biol 108, 1657-1664,
doi:10.1083/jcb.108.5.1657 (1989).
25 Jones, J. M., Morrell, J. C. & Gould, S. J. Multiple distinct targeting signals in
integral peroxisomal membrane proteins. J Cell Biol 153, 1141-1150,
doi:10.1083/jcb.153.6.1141 (2001).
26 Ma, C., Agrawal, G. & Subramani, S. Peroxisome assembly: matrix and membrane
protein biogenesis. J Cell Biol 193, 7-16, doi:10.1083/jcb.201010022 (2011).
27 Schueller, N. et al. The peroxisomal receptor Pex19p forms a helical mPTS
recognition domain. EMBO J 29, 2491-2500, doi:10.1038/emboj.2010.115 (2010).
28 Sato, Y. et al. Structural basis for docking of peroxisomal membrane protein carrier
Pex19p onto its receptor Pex3p. EMBO J 29, 4083-4093,
doi:10.1038/emboj.2010.293 (2010).
29 Fransen, M. et al. Analysis of human Pex19p's domain structure by pentapeptide
scanning mutagenesis. J Mol Biol 346, 1275-1286, doi:10.1016/j.jmb.2005.01.013
(2005).
67
30 Fang, Y., Morrell, J. C., Jones, J. M. & Gould, S. J. PEX3 functions as a PEX19
docking factor in the import of class I peroxisomal membrane proteins. J Cell Biol
164, 863-875, doi:10.1083/jcb.200311131 (2004).
31 Agrawal, G. & Subramani, S. Emerging role of the endoplasmic reticulum in
peroxisome biogenesis. Front Physiol 4, 286, doi:10.3389/fphys.2013.00286 (2013).
32 Honsho, M., Yamashita, S. & Fujiki, Y. Peroxisome homeostasis: Mechanisms of
division and selective degradation of peroxisomes in mammals. Biochim Biophys
Acta 1863, 984-991, doi:10.1016/j.bbamcr.2015.09.032 (2016).
33 Sugiura, A., Mattie, S., Prudent, J. & McBride, H. M. Newly born peroxisomes are
a hybrid of mitochondrial and ER-derived pre-peroxisomes. Nature 542, 251-254,
doi:10.1038/nature21375 (2017).
34 Farre, J. C., Mahalingam, S. S., Proietto, M. & Subramani, S. Peroxisome biogenesis,
membrane contact sites, and quality control. EMBO Rep 20,
doi:10.15252/embr.201846864 (2019).
35 Schrader, M., Costello, J. L., Godinho, L. F., Azadi, A. S. & Islinger, M.
Proliferation and fission of peroxisomes - An update. Biochim Biophys Acta 1863,
971-983, doi:10.1016/j.bbamcr.2015.09.024 (2016).
36 Olson, E. N. & Klein, W. H. bHLH factors in muscle development: dead lines and
commitments, what to leave in and what to leave out. Genes Dev 8, 1-8,
doi:10.1101/gad.8.1.1 (1994).
37 Otto, A., Schmidt, C. & Patel, K. Pax3 and Pax7 expression and regulation in the
avian embryo. Anat Embryol (Berl) 211, 293-310, doi:10.1007/s00429-006-0083-3
(2006).
38 Kodaka, Y., Rabu, G. & Asakura, A. Skeletal Muscle Cell Induction from
Pluripotent Stem Cells. Stem Cells Int 2017, 1376151, doi:10.1155/2017/1376151
(2017).
39 WISELY, I. WHO report on cancer: setting priorities, investing wisely and
providing care for all., (World Health Organization, 2020).
40 Argiles, J. M., Busquets, S., Stemmler, B. & Lopez-Soriano, F. J. Cancer cachexia:
understanding the molecular basis. Nat Rev Cancer 14, 754-762,
doi:10.1038/nrc3829 (2014).
41 Ruas, J. L. et al. A PGC-1alpha isoform induced by resistance training regulates
skeletal muscle hypertrophy. Cell 151, 1319-1331, doi:10.1016/j.cell.2012.10.050
(2012).
42 van der Ende, M. et al. Mitochondrial dynamics in cancer-induced cachexia.
Biochim Biophys Acta Rev Cancer 1870, 137-150,
doi:10.1016/j.bbcan.2018.07.008 (2018).
68
43 Jauregui, M. & Kim, P. K. Probing peroxisome dynamics and biogenesis by
fluorescence imaging. Curr Protoc Cell Biol 62, Unit 21 29 21-20,
doi:10.1002/0471143030.cb2109s62 (2014).
44 Bonetto, A., Rupert, J. E., Barreto, R. & Zimmers, T. A. The Colon-26 Carcinoma
Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia. J Vis Exp,
doi:10.3791/54893 (2016).
45 McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W. & Looger, L.
L. A bright and photostable photoconvertible fluorescent protein. Nat Methods 6,
131-133, doi:10.1038/nmeth.1296 (2009).
46 De Zitter, E. et al. Mechanistic Investigations of Green mEos4b Reveal a Dynamic
Long-Lived Dark State. J Am Chem Soc 142, 10978-10988,
doi:10.1021/jacs.0c01880 (2020).
47 De Zitter, E. et al. Mechanistic investigation of mEos4b reveals a strategy to reduce
track interruptions in sptPALM. Nat Methods 16, 707-710, doi:10.1038/s41592-
019-0462-3 (2019).
48 Zemirli, N., Morel, E. & Molino, D. Mitochondrial Dynamics in Basal and Stressful
Conditions. Int J Mol Sci 19, doi:10.3390/ijms19020564 (2018).
49 Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using
CRISPR-Cas9. Nat Rev Genet 16, 299-311, doi:10.1038/nrg3899 (2015).
50 Walton, P. A., Brees, C., Lismont, C., Apanasets, O. & Fransen, M. The peroxisomal
import receptor PEX5 functions as a stress sensor, retaining catalase in the cytosol
in times of oxidative stress. Biochim Biophys Acta Mol Cell Res 1864, 1833-1843,
doi:10.1016/j.bbamcr.2017.07.013 (2017).
51 Beltra, M., Pin, F., Ballaro, R., Costelli, P. & Penna, F. Mitochondrial Dysfunction
in Cancer Cachexia: Impact on Muscle Health and Regeneration. Cells 10,
doi:10.3390/cells10113150 (2021).
52 Moresi, V. et al. Myogenin and class II HDACs control neurogenic muscle atrophy
by inducing E3 ubiquitin ligases. Cell 143, 35-45, doi:10.1016/j.cell.2010.09.004
(2010).
53 Sun, R. et al. Valproic acid attenuates skeletal muscle wasting by inhibiting
C/EBPbeta-regulated atrogin1 expression in cancer cachexia. Am J Physiol Cell
Physiol 311, C101-115, doi:10.1152/ajpcell.00344.2015 (2016).