Mechanisms of regulation of the spindle position checkpoint kinase, Kin4

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.Cataloged from PDF version of thesis.Includes bibliographical references.Most cells are polarized in that they are aware of spatial cues and can respond to these cues accordingly. One major aspect of cell function that is often responsive to these polarization cues is cell division. Cell division, the process of making two cells from one progenitor, requires equal distribution of the genetic material to the two progeny cells. When polarized cells divide, an additional constraint on the segregation of the genetic material is imposed, namely, cells must divide the genetic material along axes defined by polarization cues. In eukaryotes, this problem is generally solved by the positioning of the mitotic spindle according to these spatial cues. Defects in spindle positioning can lead to the generation of cells with incorrect organelle, genetic and molecular contents, fate and/or, spatial orientation. Cells have evolved feedback mechanisms that monitor defects in spindle positioning and delay the cell cycle in response to such defects. These mechanisms are best elucidated in the budding yeast, Saccharomyces cerevisiae. The protein kinase Kin4 inhibits the Mitotic Exit Network when the spindle is mis-positioned. How Kin4 is itself regulated and whether or how Kin4 responds to spindle mis-position is not known. The work presented in this thesis elucidates the regulation of Kin4. We identify a novel spindle position checkpoint component, PP2A-Rts 1, and show that it promotes checkpoint function by enabling proper Kin4 localization. We also identify domains and sequence determinants within Kin4 that control localization and function. We present a model of how the spindle position checkpoint senses spindle position and test this model for Kin4 function. We find that the generation of positive and negative mitotic exit regulatory zones allows the cell to sense and translate the spatial information of spindle position into a chemical cell cycle signal.by Leon Y. Chan.Ph.D

Advances in autophagy regulatory mechanisms

Autophagy plays a critical role in cell metabolism by degrading and recycling internal components when challenged with limited nutrients. This fundamental and conserved mechanism is based on a membrane trafficking pathway in which nascent autophagosomes engulf cytoplasmic cargo to form vesicles that transport their content to the lysosome for degradation. Based on this simple scheme, autophagy modulates cellular metabolism and cytoplasmic quality control to influence an unexpectedly wide range of normal mammalian physiology and pathophysiology. In this review, we summarise recent advancements in three broad areas of autophagy regulation. We discuss current models on how autophagosomes are initiated from endogenous membranes. We detail how the uncoordinated 51-like kinase (ULK) complex becomes activated downstream of mechanistic target of rapamycin complex 1 (MTORC1). Finally, we summarise the upstream signalling mechanisms that can sense amino acid availability leading to activation of MTORC1

Refactoring bacteriophage T7

Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11 515 base pairs (bp) of the 39 937 bp wild-type genome with 12 179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention

Increased Expression of PITX2 Transcription Factor Contributes to Ovarian Cancer Progression

BACKGROUND: Paired-like homeodomain 2 (PITX2) is a bicoid homeodomain transcription factor which plays an essential role in maintaining embryonic left-right asymmetry during vertebrate embryogenesis. However, emerging evidence suggests that the aberrant upregulation of PITX2 may be associated with tumor progression, yet the functional role that PITX2 plays in tumorigenesis remains unknown. PRINCIPAL FINDINGS: Using real-time quantitative RT-PCR (Q-PCR), Western blot and immunohistochemical (IHC) analyses, we demonstrated that PITX2 was frequently overexpressed in ovarian cancer samples and cell lines. Clinicopathological correlation showed that the upregulated PITX2 was significantly associated with high-grade (P = 0.023) and clear cell subtype (P = 0.011) using Q-PCR and high-grade (P<0.001) ovarian cancer by IHC analysis. Functionally, enforced expression of PITX2 could promote ovarian cancer cell proliferation, anchorage-independent growth ability, migration/invasion and tumor growth in xenograft model mice. Moreover, enforced expression of PITX2 elevated the cell cycle regulatory proteins such as Cyclin-D1 and C-myc. Conversely, RNAi mediated knockdown of PITX2 in PITX2-high expressing ovarian cancer cells had the opposite effect. CONCLUSION: Our findings suggest that the increased expression PITX2 is involved in ovarian cancer progression through promoting cell growth and cell migration/invasion. Thus, targeting PITX2 may serve as a potential therapeutic modality in the management of high-grade ovarian tumor.published_or_final_versio

A Deep Learning-Based Camera Approach for Vital Sign Monitoring Using Thermography Images for ICU Patients

Infrared thermography for camera-based skin temperature measurement is increasingly used in medical practice, e.g., to detect fevers and infections, such as recently in the COVID-19 pandemic. This contactless method is a promising technology to continuously monitor the vital signs of patients in clinical environments. In this study, we investigated both skin temperature trend measurement and the extraction of respiration-related chest movements to determine the respiratory rate using low-cost hardware in combination with advanced algorithms. In addition, the frequency of medical examinations or visits to the patients was extracted. We implemented a deep learning-based algorithm for real-time vital sign extraction from thermography images. A clinical trial was conducted to record data from patients on an intensive care unit. The YOLOv4-Tiny object detector was applied to extract image regions containing vital signs (head and chest). The infrared frames were manually labeled for evaluation. Validation was performed on a hold-out test dataset of 6 patients and revealed good detector performance (0.75 intersection over union, 0.94 mean average precision). An optical flow algorithm was used to extract the respiratory rate from the chest region. The results show a mean absolute error of 2.69 bpm. We observed a computational performance of 47 fps on an NVIDIA Jetson Xavier NX module for YOLOv4-Tiny, which proves real-time capability on an embedded GPU system. In conclusion, the proposed method can perform real-time vital sign extraction on a low-cost system-on-module and may thus be a useful method for future contactless vital sign measurements

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions