The Impact of Cyclin B1 on Tuberin Stabilization

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder caused by various mutations in either TSC1 or TSC2, genes that encode the proteins Hamartin and Tuberin respectively. Hamartomas (benign tumours), skin lesions, neurological symptoms, renal dysfunctions, and retinal malformations are often present in TSC patients with varying severity. Tuberin and Hamartin regulate protein synthesis through mTORC1 inhibition. Tuberin can also delay mitotic onset at the G2/M cell cycle transition by binding to Cyclin B1. Hamartin has been shown to stabilize Tuberin by inhibiting its ubiquitination by HERC1 and subsequent degradation. Preliminary data from our lab suggests that Cyclin B1 may also contribute to the stabilization of Tuberin levels during the G2/M transition. Phosphorylation status of the cytoplasmic retention sequence (CRS) of Cyclin B1 plays an important role in the formation of the Tuberin/Cyclin B1 complex. The unphosphorylated CRS form of Cyclin B1 (Cyclin B1 5xA) binds stronger to Tuberin compared to the phosphorylated form (Cyclin B1 5xE). My thesis investigates the role of Cyclin B1 in Tuberin stabilization and how CRS phosphorylation status impacts Tuberin/Cyclin B1 complex formation. HEK293-TSC1 null cells (IC2) will be transfected with varying concentrations of Cyclin 5xA DNA and the levels of the Tuberin protein will be quantified by Western blot techniques. The importance of each residue in the CRS region for the Tuberin/Cyclin B1 complex formation will also be evaluated using Immunoprecipitation studies. Understanding the role of Cyclin B1 in Tuberin stabilization will shed light on cell proliferation and growth mechanisms that underlie tumorigenic disorders

Chronic renal homograft function. Correlation with histology and lymphocyte antigen matching

Renal function was studied in twenty-nine of thirty-four surviving renal allograft recipients from an initial group of sixty-four patients two years after transplantation. Mean clearances of inulin and PAH were, respectively, greater than and equal to half the donors' initial predicted clearances. Minimum urine osmolality during water diuresis was greater, and maximum urine osmolality during hydropenia was less than normal, an effect attributable partly to enhanced solute load in a single transplanted kidney. Patients with compatible donor-recipient lymphocyte antigens demonstrated statistically better function than those with one or more incompatibilities, although there was a definite degree of overlap between the two groups. In contrast, little correlation could be demonstrated between the cumulative histopathology and renal clearances. Renal function in patients with compatible donors was statistically greater than half the donors' initial predicted function. Serial increase in renal clearances was documented in one patient with a compatible donor. Serial decreases were demonstrated in two patients with incompatible donors. These findings suggest that hypertrophy of the denervated, transplanted kidney occurs when immune reaction is minimal. © 1967

Inherited predisposition to spontaneous preterm delivery

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The Role of Tuberin in DNA Damage Repair During Cell Proliferation

The cell cycle contains DNA damage checkpoints that delay mitotic progression and allow for DNA repair before cell division. DNA damage can be caused by radiation, drugs, and other processes which lead to cellular mutations and carcinogenesis. The tumour suppressor protein p53 is activated in the presence of DNA damage. It induces apoptosis or cell cycle arrest which allows cells to repair themselves. Tuberin (TSC2), another tumour suppressor protein, regulates the G2/M transition in the cell cycle and negatively regulates protein synthesis and cell growth. Mutations in tuberin can lead to the multisystem autosomal dominant disease known as tuberous sclerosis (TSC). Previously, our lab has shown that Tuberin regulates mitotic onset through cellular localization of the G2/M Cyclin, Cyclin B1. My project focuses on the Tuberin/Cyclin B1 complex in relation to G2/M arrest and DNA damage repair. In this study, we will overexpress Tuberin-WT and Tuberin clinical mutants in NIH-3T3 (mouse) and U2OS (human) p53 wild type cells. Etoposide, a topoisomerase II drug, will be used to induce DNA damage. Cells will then be analyzed by flow cytometry, TUNEL assay, and western blot to assess their cell cycle profile, apoptotic levels, and protein expression. Using CRISPR-Cas9 technology, a NIH-3T3 null TSC2 cell line will be created to clarify the role of Tuberin during DNA repair. Preliminary results have determined that 4μM of etoposide treatment at 8 hours arrests 50% of NIH-3T3 cells at G2/M. This project will provide greater insight into DNA damage induced carcinogenesis, TSC, and other proliferative diseases

Applying CRISPR/Cas9 and fluorescent tools to dissect the role of Tuberin in cell cycle regulation

How cells regulate their growth and division involves a tightly controlled integration of many mechanisms. In cells, Tuberin (gene – TSC2) is a protein in the Tuberous Sclerosis Complex (TSC) that modulates cellular growth, size, and proliferation. Mutations in the proteins forming the TSC can cause Tuberous Sclerosis Complex, an autosomal dominant disorder characterized by multisystem pathologies that is often associated with benign hamartomas in the brain, kidney, lungs and skin. The focus of my research is to clarify the role of Tuberin in the regulation of cell size and proliferation at the G2/M cell cycle checkpoint. During late G2, Tuberin retains Cyclin B1 (gene – CCNB1), a mitotic cyclin, in the cytoplasm thereby prolonging mitotic onset. We constructed six TSC2 mutants that harbour clinically relevant mutations which are known to destabilize the TSC. Interestingly, these mutations fall within the Tuberin Cyclin B1 binding domain. Whether or not these mutations disrupt the regulation of the G2/M checkpoint is a key question of this project. This is studied by over-expressing the mutants with GFP tagged Cyclin B1 in Tuberin null cells. The resultant phenotypes are analyzed by flow cytometry, immunoprecipitation, and immunofluorescence. To aid in the temporal study of the cell cycle, I aim to validate successful CRISPR/Cas9-mediated knock-in of an iRFP tag within the TSC2 gene of HEK293 cells. This new cell line will be a powerful tool to dissect the roles of Tuberin in regulating cellular growth and division and can provide deep understanding of proliferative diseases like TSC and cancers

Dissipative Dynamics of Matter Wave Soliton in Nonlinear Optical Lattice

Dynamics and stability of solitons in two-dimensional (2D) Bose-Einstein condensates (BEC), with low-dimensional (1D) conservative plus dissipative nonlinear optical lattices are investigated. In the case of focusing media (with attractive atomic systems) the collapse of the wave packet is arrested by the dissipative periodic nonlinearity. The adiabatic variation of the background scattering length leads to metastable matter-wave solitons. When the atom feeding mechanism is used, a dissipative soliton can exist in focusing 2D media with 1D periodic nonlinearity. In the defocusing media (repulsive BEC case) with harmonic trap in one dimension and one dimensional nonlinear optical lattice in other direction, the stable soliton can exist. This prediction of variational approach is confirmed by the full numerical simulation of 2D Gross-Pitaevskii equation.Comment: 9 pages, 8 figure