Humans innately feel a desire for connection, but in its pursuit, some may inadvertently thwart the very connection they seek. One way this may happen is through forsaking a sense of autonomy in favor of relatedness using behaviors such as self- silencing (stifling the self to preserve a relationship) and emotional fusion (relying on validation from others). To better understand how these four variables—autonomy, relatedness, self-silencing and emotional fusion—function together, I used latent profile analysis to identify classes among US adults in long-term relationships (n = 1065). Separate classes were identified for men and women. I then used the Bolck-Croon-Hagenaars method to predict class membership using variables such as demographics, attachment styles, relational connection, depressive symptoms, and gender roles and ideologies. Results indicated four latent groups for women (stifled-59%, detached-29%, moderate-9%, and differentiated-3%) and four for men (stifled-33%, detached-50%, imbalanced-11%, and differentiated-6%). In all classes, autonomy and relatedness balanced each other out such that if autonomy was deficient, relatedness was also low. In most classes, autonomy and relatedness were inversely associated with self-silencing and emotional fusion so that those with high autonomy and relatedness were less likely to self-silence or engage in emotional fusion. Those with high autonomy and relatedness were also less likely to adhere to traditional gender roles and ideologies, less likely to experience depressive symptoms, less likely to be anxiously or avoidantly attached, and more likely to experience relational connection.

Resting-state fMRI (rsfMRI) has been widely used to map intrinsic brain network organization of the human brain both in health and in pathological conditions. However, the neural underpinnings and dynamic rules governing brain-wide rsfMRI coupling remain unclear. Filling this knowledge gap is of crucial importance, given our current inability to decode and reverse-engineer clinical signatures of aberrant connectivity into interpretable neurophysiological events that can help understand or diagnose brain disorders. Toward this goal, here we combined chemogenetics, rsfMRI, and in vivo electrophysiology in the mouse to investigate how regional manipulations of brain activity (i.e. neural inhibition, or excitation) causally contribute to whole-brain fMRI network organization. In a first set of proof of concept investigations, we empirically probed the widely held notion that neural inhibition of a cortical node would result in reduced fMRI coupling of the silenced area and its long-range terminals. Surprisingly, we found that chronic inhibition of the mouse medial prefrontal cortex (PFC) via viral overexpression of a potassium channel paradoxically increased fMRI connectivity between the inhibited area and its direct thalamo-cortical targets. Notably, acute chemogenetic inhibition of the PFC reproduced analogous patterns of fMRI overconnectivity. Using in vivo electrophysiology, we found that chemogenetic inhibition of the PFC enhances low frequency (0.1 - 4 Hz) oscillatory power via suppression of neural firing not phase-locked to slow rhythms, resulting in increased slow and δ band coherence between areas that exhibit fMRI overconnectivity. These results provide causal evidence that cortical inactivation can counterintuitively increase fMRI connectivity via enhanced, less-localized slow oscillatory processes, with important implications for neural modeling and interpretation of fMRI overconnectivity in brain disorders. Importantly, our observation that neural inhibition of the PFC results in fMRI overconnectivity allowed us to predict that neural activation of the same area might produce opposite results, i.e. fMRI underconnectivity and neural desynchronization. To test this hypothesis, we used chemogenetics to increase local excitatory-inhibitory (E/I) balance in the PFC. As predicted, chemogenetic stimulation of CamkII-expressing neurons, or inhibition of fast-spiking parvalbumin-expressing neurons, produced similar rsfMRI signatures of rsfMRI underconnectivity. Both manipulations produced analogous electrophysiological signatures characterized by increased firing activity and a robust LFP power shift towards higher (i.e. γ) frequencies, effectively reversing the corresponding neural signature observed in DREADD inhibition studies. Importantly, the same E/I affecting perturbations were also associated with socio-communicative deficits in behaving mice hence underscoring the behavioral relevance of the employed manipulations. These results show that excitatory/inhibitory balance critically biases brain-wide fMRI coupling, pointing at a possible unifying mechanistic link between E/I imbalance and rsfMRI connectivity disruption in developmental disorders. More broadly, these investigations reveal a set of fundamental rules linking regional brain activity to macroscale functional connectivity, offering opportunities to physiologically interpret rsfMRI signatures of functional dysconnectivity in human brain disorders.

Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23961号 / 農博第2510号 / 新制||農||1092(附属図書館) / 学位論文||R4||N5396(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 教授 日本 典秀 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM