SCIENTIFIC PRESENTATION

Functionalised Diatom Biosilica as a Sustainable Platform for Probing Small RhoGTPase Activity

by: Ana Masara Ahmad Mokhtar*, Terence Lim Kek Siang, Saw Keat Chuan, Chien Fung Chong, Darerca Owen and Noorhayati Idros

Rho family small GTPases, such as Cdc42, Rac1, and RhoA, act as molecular switches controlling cytoskeletal dynamics, proliferation, and migration. Their dysregulation, particularly through effectors like p21-activated kinase 1 (PAK1), is linked to cancer, immune defects, and neurological disorders. Monitoring their activity is therefore essential in disease research. Current detection methods, including pull-downs and ELISA-like assays, rely heavily on antibodies. While effective, they are costly, time-consuming, and dependent on antibody quality. This has driven interest in alternative, low-cost platforms that are sustainable and scalable. Diatom biosilica offers such potential. These naturally occurring, porous silica structures provide high surface area, tunable chemistry, and optical properties. Their abundance and biocompatibility make them promising materials for biosensing applications. Here, we developed a biosilica-based pull-down assay using frustules from Skeletonema costatum. By functionalizing the surface with glutathione (GSH), we immobilized GST-tagged PAK1 GTPase-binding domain (GBD) to capture active small GTPases. We aimed to test whether this platform could discriminate between active and inactive forms of Cdc42, offering a cost-effective alternative to antibody-dependent methods.

Cacao Flavonoid Extracts and Their Impact on CDC42-Associated Cancer Signalling

by: Ayesha Fatima Ahmed, Mohamed Shazeli Che Zain, Ana Masara Ahmad Mokhtar*

The CDC42 protein is a crucial component of cell-signalling pathways. As a member of the Rho family of small GTPases, it plays a pivotal role in regulating cellular processes such as cytoskeletal reorganization, cell migration, and cell polarity. CDC42 functions as a molecular switch, alternating between an active GTP-bound state and an inactive GDP-bound state. Aberrant activation of CDC42 has been reported in cancers such as in breast and prostate cancers. Dysregulation of CDC42-related signalling pathways promotes uncontrolled cell division, resistance to apoptosis, increased angiogenesis, and a heightened propensity for metastasis. Most therapeutic approaches involve peptide-based agents, but these may cause side effects, highlighting the need for natural alternatives. In line with the Sustainable Development Goals (SDGs) 3, there is also a growing interest in strategies that transform agricultural waste into health-promoting resources. Therefore, this study investigates the potential of naturally derived compounds, specifically flavonoids, as therapeutic agents for modulating CDC42-related signalling pathways. We focus on flavonoids present in the rinds of Theobroma cacao (cocoa fruit), a rich source of antioxidants. However, only ~20% of the fruit is typically used, while the remaining ~80% is discarded as waste. Hence, this research explores the underlying molecular mechanisms through which flavonoid extracts may interact with and regulate the CDC42 signalling pathway. By doing so, we aim to establish a sustainable and health-oriented approach that utilizes cocoa fruit rinds to impact CDC42-related cancer progression.

Elucidating the Oncogenic Potential of RhoH Y38X Mutation in Cell Migration and Proliferation via The Deregulation of NFAT Signaling

by: Saw Keat Chuan, Nur Azzalia Kamaruzzaman, Ilie Fadzilah Hashim, Khoo Boon Yin, Syafinaz Amin Nordin, Saiful Effendi Syafruddin, Ana Masara Ahmad Mokhtar*

Award: AACR-oC-MY Travel award (Supported by Yayasan Sime Darby)

Belonging to the Rho family, RhoH is an atypical small Rho GTPase that does not undergo classical GDP/GTP cycling, unlike typical GTPases. As a result, RhoH is considered to be constitutively active. It is primarily expressed in hematopoietic cells, where it plays a critical role in regulating cell maturation, proliferation, migration, and survival. Deregulation of RhoH has been implicated in various cancers, particularly hematologic cancers such as lymphoma, leukemia, and myeloma. Notably, a novel RhoH Y38X mutation has been identified in patients with primary immunodeficiency (PID), who also suffer from persistent epidermodysplasia verruciformis (EV) infections, impaired T-cell function, and Burkitt lymphoma. Hence, the aim of this study was to investigate the role of the RhoH Y38X mutation in promoting oncogenic signaling in PID malignancies. To assess this, the pDEST12.2 FLAG-RhoH Y38X mutant was generated using site-directed mutagenesis and transfected into HEK293T cells. The proliferative and migratory abilities of the transfected cells were evaluated using the MTT assay and wound healing assay at 0, 24, 48, and 72-hour intervals. These effects were compared to cells transfected with pDEST12.2 FLAG-RhoH (wild-type) and a control group (GFP only). Interestingly, cells transfected with RhoH Y38X showed a lower rate of cell proliferation and migration compared to the wild-type, suggesting that RhoH deregulation may be influencing these processes, possibly through NFAT-induced signaling and the release of IFN-γ, IL-6, TNF-α, and IL-2. Moreover, low levels of RhoH were observed for the mutant compared to the wild-type, and in silico analysis revealed that the release of the aforementioned pro-inflammatory cytokines was affected. To validate these findings in vitro, an ELISA will be performed to quantify the effect of the mutation on IL-6 release upon activation of NFAT signaling. In conclusion, the data provide insight into the role of RhoH in regulating NFAT signaling in the context of PID-related malignancies.

Transcriptomic Analysis: Identifying the Oncogenic Signaling Involved in Cdc42 C81Y-Induced Primary Immunodeficiency Malignancy

by:  Nik Yasmin Umaira Hasnizan, Amira Suriaty Yaakop, Nurul Izza Ismail, Syafinaz Amin Nordin, Saiful Effendi Syafruddin, Ana Masara Ahmad Mokhtar*

Award: AACR-oC-MY Travel award (Supported by Yayasan Sime Darby)

Primary immunodeficiencies (PIDs) are a diverse group of congenital disorders, linking over 485 gene mutations. These conditions disrupt the normal function of immune cells, including their chemokine-mediated motility, receptor signalling, as well as their development and maturation. Individuals with PIDs have immune system dysregulation, often resulting in life-threatening conditions like cancer. Malignancy is the second most common cause of death in PID patients owing to mutations in genes encoding tumour-associated proteins. Among these genes, a mutated small Rho GPase Cdc42C81Y, has been implicated in the pathogenesis of PID malignancy as the patients show high CD8+ T-cells but low CD4+ T-cells are diagnosed with Hodgkin's lymphoma, signifying the involvement of Cdc42 as a nodal protein between the immune system and cancer. Interestingly, altered Cdc42 signalling is shown to promote some oncogenic pathways, including NF-B, PIK/Akt, and MAPK/ERK where this signalling is also involved in regulating immune system. Hence, this study aimed to investigate the potential signalling affected by Cdc42 C81Y. To test this, a transcriptomic analysis was done to profile the affected signalling and was validated using ELISA. These data would provide insights into the mechanism of crosstalk between PIDs and cancer, highlighting the potential of Cdc42 as therapeutic target for PID malignancy.

Oncogenic Crosstalk Between Cancer and Immunity: The Role of Cdc42 C81Y and RhoH Y38X Mutations in Primary Immunodeficiency Malignancies

by: Ana Masara Ahmad Mokhtar*

Award: Early Career Travel award, The Biochemical Society, United Kingdom (UK)

Primary immunodeficiencies (PIDs) are genetic disorders that impair immune function, increasing susceptibility to infections and malignancies. Mutations in small GTPases like Cdc42 and RhoH have been linked to PID-associated cancers, but their mechanisms remain poorly understood. Hence, this study examines the functional impact of Cdc42 C81Y and RhoH Y38X mutations on cell migration, proliferation, and cancer-related signaling. In silico modeling predicted impaired GTP/GDP cycling for both mutations, which will be validated via effector pull-down assays. The effects on cancer signaling, particularly NFAT, were assessed using in silico and transcriptomics analysis, while RhoH Y38X interaction with Rac1 will be analyzed via co-immunoprecipitation. Cell migration and proliferation were measured using scratch and MTT assays, and IL-6 release was evaluated through ELISA. Interestingly, Cdc42 C81Y was predicted to have impaired interaction with downstream effectors. Western blot analysis revealed high protein expression but reduced phosphorylation, leading to increased cell migration with moderate reductions in proliferation, suggesting enhanced invasiveness. Similarly, RhoH Y38X affected protein expression, resulting in Rac1-induced proliferation and migration, potentially via impaired NFAT signaling. Both mutations were predicted to alter cytokine release, such as IL-6, promoting immune dysregulation and migratory phenotypes. Conclusively, the Cdc42 C81Y and RhoH Y38X mutations disrupt immune cell behavior and promote malignancy in PID patients, highlighting the need for targeted therapies to restore immune function and prevent cancer progression.

Elucidating The Effect of Rhoh Y38x Mutation In Regulating Rac1-Induced Cell Proliferation and Migration

by:  Nurul Syafawani Binti Fizal, Ana Masara Ahmad Mokhtar*

Award: Student Member Bursaries, The Biochemical Society, United Kingdom (UK)

Primary immunodeficiencies (PIDs) are genetic disorders that impair immune function, increasing susceptibility to infections and malignancies. Mutations in small GTPases like Cdc42 and RhoH have been linked to PID-associated cancers, but their mechanisms remain poorly understood. Hence, this study examines the functional impact of Cdc42 C81Y and RhoH Y38X mutations on cell migration, proliferation, and cancer-related signaling. In silico modeling predicted impaired GTP/GDP cycling for both mutations, which will be validated via effector pull-down assays. The effects on cancer signaling, particularly NFAT, were assessed using in silico and transcriptomics analysis, while RhoH Y38X interaction with Rac1 will be analyzed via co-immunoprecipitation. Cell migration and proliferation were measured using scratch and MTT assays, and IL-6 release was evaluated through ELISA. Interestingly, Cdc42 C81Y was predicted to have impaired interaction with downstream effectors. Western blot analysis revealed high protein expression but reduced phosphorylation, leading to increased cell migration with moderate reductions in proliferation, suggesting enhanced invasiveness. Similarly, RhoH Y38X affected protein expression, resulting in Rac1-induced proliferation and migration, potentially via impaired NFAT signaling. Both mutations were predicted to alter cytokine release, such as IL-6, promoting immune dysregulation and migratory phenotypes. Conclusively, the Cdc42 C81Y and RhoH Y38X mutations disrupt immune cell behavior and promote malignancy in PID patients, highlighting the need for targeted therapies to restore immune function and prevent cancer progression.

The Potential Immunomodulatory Effects of Christia vespertilionis Leaf Extract (CVLE) on Immune Cell Proliferation via BAFF/Ras/PI3K/Akt Signalling

by: Muggunna Balasubramaniam, Ilie Fadzilah Hashim, Nurul Izza Ismail. Ana Masara Ahmad Mokhtar*

Award: Student Member Bursaries, The Biochemical Society, United Kingdom (UK)

Aberrant expression of B cell activating factor (BAFF) has been highly associated with autoimmune disease and lymphoid cancers, making BAFF a suitable candidate for potential therapeutic antagonism. While synthetic BAFF antagonists exist, a substantial percentage of individuals do not respond well to these treatments, justifying the need for alternative approaches with natural products from traditional plants. Christia vespertilionis (CV), commonly known as butterfly wing plant demonstrated various medicinal properties, though its immunomodulatory effects remain unexplored. Therefore, this study evaluates the potential immunomodulatory effect of aqueous CV leaf extract (CVLE) on BAFF-mediated immune cell proliferation. Cytotoxicity assay using CCK8 was used to determine the non-toxic working concentrations (0.125 – 2 mg/mL) of CVLE. The proliferative effects of CVLE were assessed in Ramos B cells and BAFF-stimulated THP-1 monocytes at 24, 48, and 72 h. Western blotting analysis determined the impact of CVLE on BAFF-related signalling, while ELISA was used to measure pro-inflammatory cytokines secretion (TNF- and IFN-γ) in BAFF-stimulated THP-1 monocytes. CVLE concentrations >1 mg/ml was observed to be toxic on normal NIH/3T3-L1 fibroblasts. Interestingly, 1 mg/mL CVLE shown to inhibit cell proliferation significantly in Ramos B cells and BAFF-stimulated THP-1 monocytes at 72 h, potentially by regulating BAFF/PI3K/Akt pathway. Treatment with 1 mg/mL CVLE also shown to reduce TNF- and IFN γ secretion in BAFF-stimulated THP-1 monocytes, indicating its potential to regulate pro inflammatory cytokine release. CVLE exhibits potent immunomodulatory effects by regulating pro-inflammatory cytokine secretion via BAFF/PI3K/Akt pathway. Collectively, these results indicate CVLE as a potential natural therapeutic approach for managing BAFF-related immunological disorders.

A dual role for Cdc42 C81Y in modulating immune function and cancer-related pathways

by: Chien Fung Chong, Fadzilah Hashim, Nurul Izza Ismail. Ana Masara Ahmad Mokhtar*

Award: Student Member Bursaries, The Biochemical Society, United Kingdom (UK)

Background: Cdc42 regulates cellular processes by cycling between active GTP-bound and inactive GDP-bound states. A C81Y mutation in its β4 segment is associated with primary immunodeficiency and Hodgkin's lymphoma. Evidence suggests this mutation perturbs Cdc42 structure-functions dynamics, promoting immune dysfunction and remodelling the tumour microenvironment (TME) to support malignancy. Objective: This study examines how C81Y affects Cdc42 protein expression, activation, and phosphorylation. We also investigate its oncogenic potential, focusing on cell proliferation and migration, alongside its influence on cytokine secretion relevant to inflammation and cancer. Methods: The activation profile of Cdc42 C81Y was analysed via molecular modelling and validated using effector pull-down. Western blotting assessed protein expression and phosphorylation relative to wild-type. Oncogenic potential was evaluated through MTT and wound-healing assays, while IL-6 and IL-10 secretion levels were quantified by ELISA. Results: Cdc42 C81Y favours the GDP-bound state and shows reduced affinity for effectors (PAK1, WASp, and N-WASp). Phospho-tyrosine levels were diminished, suggesting altered Cdc42-RhoGDI interaction and Cdc42 spatiotemporal signalling. Notably, IL-6 and IL-10 secretion was elevated. Although proliferation was mildly reduced, Cdc42 C81Y significantly enhanced directed cell migration. This aggressive behaviour likely results from altered downstream signalling, including MAPK/ERK axis and pro-inflammatory TME modulation. Conclusion: These findings uncover a mechanism by which Cdc42 C81Y alters activation and downstream signalling, contributing to immune dysregulation and tumour-promoting behaviour. Cdc42 emerges as critical mediator of cancer–immune crosstalk, with C81Y acting as a driver of pathogenic transformation.

Elucidating the Role of RhoH Y38X Mutation in Cell  Proliferation and Migration via Deregulation of NFAT Signaling

by: Saw Keat Chuan, Nur Azzalia Kamaruzzaman, Ilie Fadzilah Hashim, Khoo Boon Yin, Syafinaz Amin Nordin, Saiful Effendi Syafruddin, Ana Masara Ahmad Mokhtar*

Award: Student Member Bursaries, The Biochemical Society, United Kingdom (UK)

Belonging to the Rho family, RhoH is an atypical small Rho GTPase that does not undergo classical GDP/GTP cycling, unlike typical GTPases. As a result, RhoH is considered to be constitutively active. It is primarily expressed in hematopoietic cells, where it plays a critical role in regulating cell maturation, proliferation, migration, and survival. Deregulation of RhoH has been implicated in various cancers, particularly hematologic cancers such as lymphoma, leukemia, and myeloma. Notably, a novel RhoH Y38X mutation has been identified in patients with primary immunodeficiency (PID), who also suffer from persistent epidermodysplasia verruciformis (EV) infections, impaired T-cell function, and Burkitt lymphoma. Hence, the aim of this study was to investigate the role of the RhoH Y38X mutation in promoting oncogenic signaling in PID malignancies. To assess this, the pDEST12.2 FLAG-RhoH Y38X mutant was generated using site-directed mutagenesis and transfected into HEK293T cells. The proliferative and migratory abilities of the transfected cells were evaluated using the MTT assay and wound healing assay at 0, 24, 48, and 72-hour intervals. These effects were compared to cells transfected with pDEST12.2 FLAG-RhoH (wild-type) and a control group (GFP only). Interestingly, cells transfected with RhoH Y38X showed a lower rate of cell proliferation and migration compared to the wild-type, suggesting that RhoH deregulation may be influencing these processes, possibly through NFAT-induced signaling and the release of IFN-γ, IL-6, TNF-α, and IL-2. Moreover, low levels of RhoH were observed for the mutant compared to the wild-type, and in silico analysis revealed that the release of the aforementioned pro-inflammatory cytokines was affected. To validate these findings in vitro, an ELISA will be performed to quantify the effect of the mutation on IL-6 release upon activation of NFAT signaling. In conclusion, the data provide insight into the role of RhoH in regulating NFAT signaling in the context of PID-related malignancies.

Transcriptomic Analysis: Identifying the Oncogenic Signaling Involved in Cdc42 C81Y-Induced Primary Immunodeficiency Malignancy

by:  Nik Yasmin Umaira Hasnizan, Amira Suriaty Yaakop, Nurul Izza Ismail, Syafinaz Amin Nordin, Saiful Effendi Syafruddin, Ana Masara Ahmad Mokhtar*

Award: Student Member Bursaries, The Biochemical Society, United Kingdom (UK)

Primary immunodeficiencies (PIDs) are a diverse group of congenital disorders, linking over 485 gene mutations. These conditions disrupt the normal function of immune cells, including their chemokine-mediated motility, receptor signalling, as well as their development and maturation. Individuals with PIDs have immune system dysregulation, often resulting in life-threatening conditions like cancer. Malignancy is the second most common cause of death in PID patients owing to mutations in genes encoding tumour-associated proteins. Among these genes, a mutated small Rho GPase Cdc42C81Y, has been implicated in the pathogenesis of PID malignancy as the patients show high CD8+ T-cells but low CD4+ T-cells are diagnosed with Hodgkin's lymphoma, signifying the involvement of Cdc42 as a nodal protein between the immune system and cancer. Interestingly, altered Cdc42 signalling is shown to promote some oncogenic pathways, including NF-B, PIK/Akt, and MAPK/ERK where this signalling is also involved in regulating immune system. Hence, this study aimed to investigate the potential signalling affected by Cdc42 C81Y. To test this, a transcriptomic analysis was done to profile the affected signalling and was validated using ELISA. These data would provide insights into the mechanism of crosstalk between PIDs and cancer, highlighting the potential of Cdc42 as therapeutic target for PID malignancy.

The Potential Immunomodulatory Effects of Christia vespertilionis Leaf Extract (CVLE) on Immune Cell Proliferation via BAFF/Ras/PI3K/Akt Signalling

by: Fatin Nur Izzati Mohd Fadzil, Nur Azzalia Kamaruzzaman, Muaz Mohd Zaini Makhtar, Ana Masara Ahmad Mokhtar*

Microbial fuel cell (MFC) technology has emerged as a promising approach for sustainable electricity generation using electrogenic bacteria (EB) such as Bacillus subtilis (BS). The current approach utilizes a combination of EBs to enhance MFC performance. Nevertheless, despite its benefits in green energy production, there is still a lack of studies addressing the potential risks of this approach, particularly on human inflammatory response. Hence, this research aimed to assess the risk of MFC-treated dewatered sludge (DS) using BS in combination with another Bacillus sp., specifically Bacillus cereus (BC) and Bacillus tropicus (BT), against human inflammatory axis. The toxicity profiles of metabolites from selected EBs were predicted using ProTox 3.0 software. For in vitro analysis, we monitored the voltage output of MFC-treated DS with BS alone or in combination with BC and BT over 14 days. Samples taken on days 0 and 14 were assessed for cytotoxicity profiles using MTT assay on RAW 264.7 cells. We measured LPS-induced nitric oxide production and TNF-α levels using Griess Assay and ELISA, respectively. Twenty-six metabolites were secreted by the three Bacillus sp., with seven metabolites (Iturin A, Fengycin, Macrolactin A, Macrolactin B, Macrolactin F, Macrolactin W, and Menaquinone) predicted to cause immunotoxicity. The combination of BS and BC achieved the highest voltage output of 372 mV, while BS alone produced only 18.4 mV, indicating that combining EBs enhances electricity production. Cell viability for all MFC- treated DS (1, 0.25, and 0.5 mg/mL) was found to be greater than 80%. However, despite the non-toxic effects of MFC-treated DS on RAW 264.7 cells, these treated cells were unable to decrease LPS-induced nitric oxide and TNF-α production.

The Immunomodulatory Effects of Christia vespertilionis Leaf Extract (CVLE) on BAFF-mediated Immune Cell Proliferation

by: Muggunna Balasubramaniam, Ana Masara Ahmad Mokhtar*

Aberrant expression of B cell activating factor (BAFF) has been highly associated with autoimmune disease and lymphoid cancers, making a suitable candidate for potential therapeutic antagonism. While synthetic BAFF antagonists exist, a substantial percentage of individuals do not respond well to these treatments, justifying the need for alternative approaches  with natural products from traditional plants. Christia vespertilionis (CV), commonly known as butterfly wing plant demonstrated various medicinal properties, though its immunomodulatory effects remain unexplored. Therefore, this study evaluates the potential immunomodulatory effect of aqueous CV leaf extract (CVLE) on cancer cell proliferation via BAFF signalling. Cytotoxicity assay using CCK8 was used to determine the non-toxic working concentrations (0.125 – 2 mg/ml). Proliferative effects of CVLE were assessed in serum-starved Ramos cancer cells and BAFF-stimulated THP-1 monocytes at 24, 48, and 72 h. Western blotting analysis determined the impact of CVLE on BAFF-signalling, while ELISA was used to measure pro-inflammatory cytokines secretion (TNFa- and IFN-γ) in BAFF-stimulated THP-1 monocytes. CVLE concentrations >1mg/ml was observed to be toxic on NIH/3T3-L1 fibroblasts. 1 mg/ml CVLE inhibited cell proliferation significantly in Ramos cancer B cells and BAFF-stimulated THP-1 monocytes at 72 h . Western blot analysis revealed PI3K p85 downregulation in both cell types, while ELISA indicated reduced TNF- and IFN-γ secretion in BAFF-stimulated THP-1 monocytes. CVLE exhibits potent immunomodulatory effects by targeting BAFF signalling via PI3K/Akt pathway while regulating pro-inflammatory cytokine secretion. Collectively, these results indicate CVLE as a potential natural therapeutic approach for managing BAFF-related immunological disorders.

Assessing Current Antimicrobial Resistance Knowledge, Attitudes, And Practices Among Malaysian University Students

by: Muhammad Faiz bin Syarizan, Ana Masara Ahmad Mokhtar*, Nor Asniza Ishak, Amira Suriaty Yaakop, Nur Azzalia Kamaruzaman

As the world continues to respond to COVID-19, a more serious and hidden threat of antimicrobial resistance (AMR) lurks in the shadows, claiming hundreds of thousands of lives annually (approximately 700,000 deaths). Widespread and unnecessary antibiotic use has contributed to the emergence and spread of resistant pathogens. AMR remains a significant concern because pathogens causing resistant infections thrive in hospitals and medical facilities, putting all patients at risk, regardless of the severity of their medical conditions. This aligns with the World Health Organization (WHO), which has identified AMR as one of the top 10 threats to development and global public health. Therefore, this study aims to evaluate the knowledge, attitudes, and practices (KAP) related to the use of antibiotics and their resistance among university students in Malaysia. A cross-sectional, self-administered survey was conducted among university students, with a total of 228 students approached to participate in the study. Descriptive statistics and parametric tests were performed to assess KAP dimensions and identify subgroup differences. The results of the study show that university students' current knowledge significantly (p < 0.05) influences their attitudes and practices related to AMR and antibiotic consumption. This finding is expected to contribute to the improvement of the secondary school or pre-university curriculum, strengthening awareness about AMR and proper antibiotic consumption.

Mathematical modelling to determine the role of RhoH in regulating NFAT-induced pro-inflammatory cytokine production in T cells

by: Nurul Izza Ismail, Ana Masara Ahmad Mokhtar*

Cancer and immune-related disorders, including primary immunodeficiencies, systemic lupus erythematosus, and psoriasis, have been linked to altered RhoH protein expression. RhoH association with these diseases was predicted due to its potential involvement in regulating NFAT-induced pro-inflammatory cytokine productions, including IFN-γ, TNF-α, GM-CSF and IL-2. Interestingly, aberrant production of these cytokines has been observed in cancers and several immune-related diseases. Thus, mathematical modelling was employed to investigate the involvement of RhoH in regulating NFAT-induced pro-inflammatory cytokine production. The model was based on a qualitative description of the intracellular signalling leading to cytokines released from T cells obtained from the KEGG database. The model was encoded using CellML to produce models that are reproducible and FAIR. Ordinary differential equations (ODEs) were used to model signalling pathways' concentration variations over time. The generated mathematical model was expected to incorporate a comprehensive description of the involvement of RhoH in modulating T-cell intracellular pathways, focusing on the generation of pro-inflammatory cytokines, including IFN-γ, TNF-α, GM-CSF and IL-2; and this would later help to predict RhoH involvement in regulating T cell activity in cancer and immune-related diseases.