Banca de DEFESA: YGOR AMARAL CAMOSSATO

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : YGOR AMARAL CAMOSSATO
DATE: 04/07/2023
TIME: 10:00
LOCAL: on-line
TITLE:

Proteomic analysis of sleep restriction on murine melanoma development

PAGES: 100
BIG AREA: Ciências Biológicas
AREA: Bioquímica
SUBÁREA: Química de Macromoléculas
SPECIALTY: Proteínas
SUMMARY:

Cancers comprise a set of more than 100 diseases, characterized by disorderly and uncontrolled cell proliferation from accumulated mutations in specific genes. Among then, melanoma is one of the most aggressive and affects the skin and the cells responsible for pigmentation, the melanocytes. An important characteristic is its high capacity to generate metastases. The diagnosis is usually clinical, but because they often resemble small skin signs, patient look for medical assistance when melanoma is already in more advanced stages.
Many cancer diagnoses occur together with a very high emotional burden for patients, which can, in many cases, lead to sleep cycle dysregulation. Several studies demonstrated that sleep deprivation or restriction affect the immunity, leading to a reduction in phagocytosis, antibody production and antigen presentation. Thus, deprivation, restriction and debts in the sleep cycle can trigger events related to tumor progression and the development of metastases, since the immune system is the first to be affected.
In addition, sleep disorders are very common due to many factors related to people's life quality and the high emotional burden that can affect cancer patients. Thus, it is important to understand and correlate the tumors progression in these patients, for the development of new methods for detecting and monitoring the disease.
Proteomic techniques applied to oncology allow high throughput protein expression studies and identification and characterization of changes in molecular levels in tumor microenvironment, as well as detect and quantify protein changes in sleep restricted patients, representing a significant advance in the understanding of melanoma molecular mechanisms.
Based on the interaction of sleep and immune system and the importance of sleep in inflammatory conditions, the objective of the present study was to evaluate the effects of sleep restriction on neoplastic development using B16F10 murine melanoma model.
Sixteen mice (C57Bl/6 males) were distributed into two groups: control group (non-sleep restricted) and sleep restricted (SR). All animals received 3x105 cells in the right flank subcutaneously and only the restricted group was submitted to the sleep restriction protocol. Cages with water and platforms were used so that the animals could move freely. However, falling into the water prevented them from entering deeper stages of sleep. The animals were sleep restricted for 18 hours a day for 21 consecutive days. At the end of the experiment, animals were euthanized by decapitation and their tumors removed and kept in 70% alcohol.
The tumors were dissected into smaller fragments and these were submitted to fixation and paraffin embedding. Histological sections were performed for staining and proteins extraction to be analyzed by mass spectrometry.
The tissues were stained with hematoxylin and eosin and the tumors were compared with a healthy region of the opposite flank. The lack of tissue structure was observed in tumors, when compared to healthy epithelial structures. In addition, the presence of two distinct regions was observed in the tumors, the most peripheral with a bluer cytoplasm, more regular and clear nuclei and decondensed chromatin (Euchromatin). These regions represent active tumor cells division, indicating tumor integrity.
The tumor inner region showed reddish cytoplasm staining, often irregular and dark colored nuclei and condensed chromatin (heterochromatin). These regions represent cell death process, with less or no cell division activity, indicating tumor degeneration. These regions occurred in a greater number in restricted animals than in the controls, suggesting that tumors were more advanced in the sleep-restricted animals.
For proteomics, histological sections were deparaffinized and subjected to protein extraction using the eFASP protocol for greater recovery of proteins present in the tissue. The isolated proteins were cleaved into peptides with trypsin, separated by high performance liquid chromatography (HPLC) and analyzed by mass spectrometry using an orbitrap mass analyzer.
Partial analysis indicated a set of approximately 2000 proteins. From that, some proteins were expressed only in the sleep-restricted group of animals and not in the control group. The opposite also occurred, with proteins present only in control group, suggesting that sleep restriction may interfere with usual metabolic pathways of cells. To ensure data robustness, the protein extraction process and mass spectrometry analysis was repeated with a larger number of samples and in replicates.
The present work is at statistical analysis phase of the second round of proteomics experiments. It will be possible to identify the differentially expressed proteins between the two groups in order to infer the role of sleep restriction in tumor development.

COMMITTEE MEMBERS:
Presidente - Interno ao Programa - 287.258.508-73 - FABIO MITSUO LIMA - UNIFESP
Membro Titular - Examinador(a) Interno ao Programa - 2605490 - SERGIO DAISHI SASAKI
Membro Titular - Examinador(a) Externo à Instituição - LUDMILLA THOMÉ DOMINGOS CHINEN
Membro Suplente - Examinador(a) Externo à Instituição - LUCIANA DE ANDRADE LUZ COSTA - UNIAN-SP